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The laws of physics establish the energetic efficiency of our movements . In some cases , like locomotion , the mechanics of the body dominate in determining the energetically optimal course of action . In other tasks , such as manipulation , energetic costs depend critically upon the variable properties of objects in the environment . Can the brain identify and follow energy-optimal motions when these motions require moving along unfamiliar trajectories ? What feedback information is required for such optimal behavior to occur ? To answer these questions , we asked participants to move their dominant hand between different positions while holding a virtual mechanical system with complex dynamics ( a planar double pendulum ) . In this task , trajectories of minimum kinetic energy were along curvilinear paths . Our findings demonstrate that participants were capable of finding the energy-optimal paths , but only when provided with veridical visual and haptic information pertaining to the object , lacking which the trajectories were executed along rectilinear paths .
One of the most established findings in planar multi-joint reaching movements is that hand trajectories tend to be executed along straight paths with a bell-shaped velocity profile [1–3] . Given that there are theoretically infinite paths that the hand could take for reaching from one point to another , the presence of this consistent feature in reaching movements has been used to suggest that the nervous system chooses this solution because it is “optimal” in some way . Mathematical optimization has been considered as an appealing principle to explain observed biological movements . Optimization requires an objective function , or cost , that includes the quantities being minimized . The choice of cost has received much attention in the study of neural information processing , in particular , by the motor system [4–8] . The components of an objective function generally fall into two main types: kinematic and dynamic . While the former relates only to the geometry of motion , the latter relates to the forces that cause the motion . Despite fundamental differences between the two types , objective functions consisting purely of one or the other have been similarly successful in predicting data obtained from unperturbed planar reaching movements . Adaptation studies have attempted to distinguish between kinematic and dynamic costs by introducing perturbations to these movements . It has been shown [9 , 10] that in the presence of kinematic perturbations , participants chose to move the hand along curved paths so as to produce visually straight trajectories . Similarly , under the dynamic perturbation caused by forces depending upon the velocity of the hand , subjects learned to recover straight hand trajectories through repeated practice of reaching movements [11] . More recently , to evaluate if mechanical energy costs play a role in motor learning , a custom force field was designed in a way that the path of minimum mechanical energy was substantially different from the straight path [12] . Under this situation , participants returned to straight line reaches even after they experienced moving along the energy optimal path . These studies suggest that the tendency to move the hand on a straight line in planar movements is strong and persistent , arising under a variety of dynamic and kinematic perturbations , and indicate that the kinematic costs are either necessary [13] or sufficient [14] components of the cost function . However , these previous studies were typically focused on unconstrained movements of the hand in free space and when a force field disturbed these movements . In the majority of earlier studies a cursor was used as a visual image of the system under control [11 , 12 , 15] . This representation makes all spatial directions visually equivalent and moving the cursor on a straight line appears to be an economical approach . Moreover , the haptic feedback ( i . e . the contact forces experienced during movements ) were predominantly in the form of force fields . In these experiments , there were no features in the visual scene or in the shape of the cursor that could be associated with the forces experienced by the subjects . We hypothesized that this dissociation between sensory modalities elicits a compensatory strategy where movements are channeled to restore the kinematics of the unperturbed hand motion . Conversely , congruence between feedback modalities , representing the action upon an identifiable external object is expected to result in energy efficient strategies . In this case , motor learning leads to a progressive optimization of the energetic costs of movements rather than a process towards recovering a straight invariant trajectory . Depending on the object's dynamics , the resulting trajectory may systematically and substantially deviate from the straight line . Therefore to test this prediction , we used an object manipulation task where there is a well-defined relation between the visual and haptic feedback .
Participants executed goal-directed reaching movements in the horizontal plane while holding the end point of a virtual planar double pendulum in the absence of gravitational effects . The choice of the double pendulum was motivated by the fact that the energy-optimal trajectories for moving this object were along curved paths , allowing us to tease apart the relative importance of kinematic and dynamic costs . The energy-optimal paths for moving this system were calculated as follows . The total energy for this system consists only of the kinetic term . The path of least kinetic energy between any two double pendulum configurations is a solution to a two-point boundary value problem—leaving the initial and final velocities as free variables- of the unforced system and it is generally curved in shape ( Fig 1 ) . However , this path is a purely geometric quantity and from a control perspective , it is not an admissible solution because the velocity requirements are not satisfied . To find an admissible solution we used optimal control theory with the only running cost of effort , defined as the force being applied to the object . Expectedly , the path of minimum energy and the effort optimal trajectory had similar shapes ( see S1 File ) . The energy ( mechanical work ) that is acquired by the object upon point-to-point maneuvers was calculated by E=∫0T|Fv|dt ( 1 ) where F represents the force applied to the object , v is the velocity of the hand and T is the movement time . The energy that was required to move along the straight path and the path of least energy between each pairs of targets is included in Fig 1 . These values are obtained from a minimum jerk trajectory with the movement duration of 1 sec . Participants were randomly divided in three groups . All received both visual and haptic feedback . Participants in Group 1 ( n = 8 ) received veridical visual and haptic information of the double pendulum . For Group 2 ( n = 8 ) , only the visual feedback was manipulated . Participants in this group were presented with a circular cursor representing the moving extremity of the pendulum . They did not see the linkage but the haptic feedback corresponded to the entire mechanism . Participants in Group 3 ( n = 8 ) could see the entire linkage while the haptic information was manipulated so as to emulate the isotropic inertia of a point mass . In this scenario , our hypothesis made an explicit prediction on the trajectory formation: If participants were provided with full vision of the manipulated object together with a congruent haptic feedback ( Group 1 ) , they would integrate the geometric structure with interaction forces to converge to the curvilinear paths of minimum energy . In contrast , if participants only received visual feedback of the endpoint ( Group 2 ) , or haptic feedback corresponding to point-mass dynamics ( Group 3 ) , then movements would be executed along rectilinear paths because of the lack of consonance between the sensing modalities . For each trial , movement initiation and termination were identified using 10% of peak velocity threshold . Participants in all the three groups started the experiment by moving along straight line trajectories . However , the trajectory divergence between Group 1 and the remaining groups started after the very first few trials . We found that with practice , all participants that received congruent visual and haptic feedback progressively moved towards producing curved trajectories that were similar to the path of minimum energy . This gradual adjustment suggests that the problem of finding the energetically optimal trajectory was solved via gradient descent beginning from the straight line trajectory typical of the freely moving hand , In contrast , all participants that were presented with incongruent feedback continued to move along rectilinear paths ( Fig 2 ) . We quantified the similarity of executed trajectories to both straight line and least energy paths using discrete Fréchet distance ( DFD ) [16] . The Fréchet distance between two curves is the minimum cord-length that is sufficient to join two points traversing each curve with arbitrary speeds without backtracking . Intuitively , imagine a dog walking along one curve and the dog’s owner walking along the other curve and they are connected by a leash . Both can change the speed and even stop at arbitrary positions with arbitrary durations but neither are allowed to move backwards . The Fréchet distance between the two curves is the length of the shortest leash that connects the man to the dog at all time . One-way ANOVAs on DFD from the straight path and DFD from the least energy path during the last block revealed a significant group effect on both distances . Dunnett’s post-hoc tests showed that Group 1 was significantly further from the straight path than the two other groups ( p <0 . 01 ) . Similarly Group 1 was significantly closer to the path of minimum energy compared to Group 2 ( p <0 . 01 ) . One feature in the result is that although the participants in Group 1 show greater curvature , they did not completely converge to the energy efficient path . We speculate that this may be due to the fact that participants in Group 1 did not have any explicit knowledge about the mechanical properties of the object and the geometric shapes of the effort optimal trajectories . They derived these trajectories solely based on the sensory information . Therefore , considering noise and model uncertainties in sensory transduction and neural computation , they were expected to move at larger distances from the paths of minimum energy and exhibit greater variability in their movements in comparison with participants in the remaining groups who moved along straight paths and had explicit kinematic plan for executing their movements .
Our results suggest that when learning novel dynamics , if the visual representation is a cursor or a shape that is indicative of isotropic dynamical structure , this impoverished representation provides a strong bias towards Euclidean representations of the configuration space , where all directions are equivalent and straight lines are the natural geometrical paths for joining two points . In this situation , participants experience a mismatch between expected and sensed forces under the assumption that they are moving the arm in free space . This mismatch between sensory information triggers a compensatory strategy where subjects fight the force field to recover the straight unperturbed trajectory . However , if subjects are provided with any visual information suggestive of an external object being manipulated , with non-Euclidean dynamical structure to begin with ( because of the non-isotropic , position dependent inertia tensor at the contact point ) , then they attribute the haptic feedback to the visual image and with practice try to develop a representation of the object’s configuration space . This harmony between sensing modalities promotes a control policy that requires less effort to perform the task . Work on remapping finger movements has highlighted that when subjects learn a novel task of manipulating a kinematic chain by continues finger motions , movement trajectories are formed along the geodesics ( i . e . , paths of minimum length ) corresponding to the geometrical structure of that object [17] . Subjects in Group 1 and 3 were both provided with the same visual feedback but at the end of the experiment they moved along different paths , each corresponding to the path of minimum energy of the object that they were manipulating ( double pendulum haptics vs point mass haptics ) . This result confirms that curved trajectories observed in Group 1 is not a solution to the kinematic problem but it is a progressive optimization of the energy exchanged with the object . Here , effort was defined as the force that subjects applied to the system to move it between target positions . Although we did not measure the metabolic effort ( i . e . the physiological energy cost ) , a recent study found that indeed the metabolic effort is reduced during force field adaptation [18] . The conclusions of previous studies on energy optimization in human motor control are mixed . Some studies suggest that the motor system is capable of minimizing the energetic costs of free limb movements both in arm reaching movements [19] as well as locomotion [20] , while other studies of learning novel dynamics suggest that the motor system does not take into account the energy when executing movements [12 , 14] . When moving a limb or manipulating an object the energy optimal solution depends on the mechanical properties rather than the visual representation . However , there is considerable amount of evidence that the movement control policy and consequently trajectory formation both in free reaching movements and in object manipulation depends remarkably on the visual feedback . Straight line trajectories are found typically in studies of free arm movements when the sight of the arm is obstructed and the subjects are presented with a cursor . However , it has been shown that trajectories of the free reaching movements of congenitally blind and even blind folded individuals to haptic targets are more curved than movements made by subjects to the same target positions under visual guidance [21 , 22] . Similarly , subjects performed curved motions when they were instructed to reach to physical targets with their arm rather than reaching with a cursor to a virtual target [19] . These studies suggest that in free limb movements , humans can flexibly alter movement behavior between geometric and energetic optimally depending on the feedback . In contrast to free movements , moving in a force field provides an additional challenge to the nervous system because in this case , the effort optimal trajectory not only depends on the mechanics of the body , but also on the dynamical properties of the field . It has been demonstrated that the representation of the dynamics of a manipulated object also depends on the visual representation [23] and that only specific and meaningful visual cues can promote proficient switching between different mechanical tasks [24] . Recent studies on learning novel dynamics reported that the motor system ignores energetic costs in favor of geometric optimality . However subjects in these studies were exposed to a force field with the representation of a cursor [12] or with no visual feedback [13] . In the latter study the visual feedback ( cursor ) was provided only at the beginning and at the end of each trial . Here , we showed that in object manipulation , the visual motion of the object resulting from an applied force is a critical piece of information for the brain to represent the dynamics . Given our finding that subjects learned to minimize the energy transfer with an object having anisotropic position-dependent inertial properties , we observe that the most common objects being transported by our hands have isotropic position-independent translational inertias . They are therefore characterized by straight-line kinetic energy geodesics when moving on the horizontal plane . Thus , we speculate that the tendency to perform straight-line planar movements of the hand may be a baseline behavior emerging from the experience of transporting such objects while optimizing the energy exchanged with them . It has been shown [19] that the arm trajectory in a 3D pointing movement is along the geodesic path that is obtained through the minimization of the kinetic energy on the configuration space of the arm . The Euler- Lagrange equations are equivalent to the equations of geodesic motion on a Riemannian manifold . We extended the computational model in [19] to learning novel dynamics , we showed that trajectory formation hinges on the consistency between feedbacks representing the system under control , and how feedback variations can lead to remarkably different behaviors . The demonstrated results provide insights into studies on adaptation , effort minimization and object manipulation by the human motor system .
Twenty four right-handed volunteers ( 12 female ) participated in the experiment . All participants were neurologically intact and had no prior knowledge of the experimental procedure . The study protocol was approved by Northwestern University’s Institutional Review Board and all the participants signed an informed consent form . Participants were positioned in front of a horizontal mirror and held the handle of a planar , two degree of freedom robotic manipulandum with their right hand . The mirror prevented the participant’s view of their hand and the robot . A LED monitor was positioned above the mirror with the same vertical distance as the distance between the robot and the mirror . This setup caused the visual information to appear at the same height as the hand . The display was calibrated so that the visual feedback of the hand was overlaid on its true position . Participants performed goal-directed reaching movements to three targets ( diameter = 3 cm ) . Targets were presented in a block structure , with randomized order within each block . After reaching to each target , participants maintained the position for 500 ms before the next target appeared . The experiment consisted of 10 blocks and in each block participants performed 48 reaching movement ( 16 reaches per target ) . Participants could rest between blocks . During all these reaching movements , the manipulandum was either connected to the endpoint of a virtual double pendulum with the mechanical properties that are listed in Table 1 or a virtual 15 kg point mass , by means of a virtual spring-damper ( K = 2200 N/m , B = 65 N . s/m ) . Position and velocity of the manipulandum handle were computed from instrumented encoders at the frequency of 1 kHz to provide haptic feedback of the forces resulting from moving the double pendulum or the point mass . The manipulandum was equipped with electric motors with the peak torque of 82 Nm . However , the maximum torque that the robot was asked to generate in the fastest recorded trial in this experiment was about 15 Nm . Data were recorded at the rate of 100 Hz . Participants were randomly divided into three groups of equal size ( n = 8 per group ) : Group 1 , where participants received both the visual and haptic feedback of the double pendulum . Group 2 , where participants received the visual feedback of the moving extremity of the pendulum that was held in their hand , in form of a circle ( diameter = 1 . 5 cm ) and the haptic feedback of the double pendulum . Group 3 , where participants received the visual feedback of the double pendulum with haptic feedback of the point mass . One of the participants in Group 3 revealed that he was familiar with the purpose of the study and was replaced by another participant .
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Recent studies have shown that when learning novel dynamics in the context of reaching movements , people often ignore energetic optimality in favor of Euclidean geometric optimality , preferring rectilinear paths over mechanically optimal trajectories . Although an explanation could be that sensory-motor coordination ignores energetic cost , another possibility is that different sensing modalities need to be in agreement before the brain will optimize energetic cost during motion . We provide evidence for this latter perspective , by showing that when provided congruency and consistency of visual and haptic feedback , participants take into account both geometric and mechanical properties of a manipulation task . However , when visual and haptic feedback are inconsistent , participants revert to the rectilinear paths seen in previous studies . We conclude from these observations , that when transporting an external object , sensory agreement between vision and touch guides the optimization of the kinetic energy exchanged during movement between the arm and the object .
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2016
|
Sensory Agreement Guides Kinetic Energy Optimization of Arm Movements during Object Manipulation
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This study is about the contribution of occupational therapy inside a rehabilitation group , and we focus on the autonomy of patients with disabilities due to leprosy . There are few studies on the use of assistive technology by leprosy patients; to our knowledge , none of them aim to have a subjective approach of care . Our purpose was to analyze the repercussions of assistive technology on autonomy of care of the self in patients with sequels of leprosy . A qualitative , descriptive exploratory study with a semi-structured interview and a field observation as a research method was conducted between November 2014 and February 2015 at a University Hospital in Rio de Janeiro . Eight patients from the service of Occupational Therapy were interviewed , and 44 hours of observation were performed . Interviews followed a semi-structured script and a field journal was used to take notes . Analysis was conducted by the hermeneutic approach . Costs were obtained after a global cost analysis of the fixed and variable expenses and direct and indirect costs to the manufactured products with an amount of 100 dollars . Results were grouped according to the following categories: contribution of the adapted devices for the care of the self and feelings and sensations provoked by the use of self-help devices . The reports revealed feelings , perceptions and meaningful contents about the social , familiar and individual dimensions , also the stigma coupled with leprosy . However , forms of re-signification were elaborated . Assistive technology empowers the subject to perform care of the self and promotes social inclusion .
The interest on the topic emerged after the implantation of the service of Occupational Therapy in a University Hospital in Rio de Janeiro , among the interdisciplinary team for Prevention , Physical and Surgical Rehabilitation in Leprosy . Once we started investigating the functional limitations of patients in their activities of daily living ( ADL ) , we noticed that these limitations could be reduced with assistive technology , by suggesting the use of adaptations that ease the occupational performance . With the purpose of achieving a better knowledge of the possibilities of helping the process of making in the human being as its impact on the subject , we first investigated the routine of the leprosy patients , to assess the tools they do or do not use during their ADLs which would require adaptations . This initial investigation in the practice of the Occupational Therapy motivated the elaboration of this research . Occupational Therapy presents resources , means or technologies to rescue the inclusion and an independent life . Assistive Technology is composed of resources and services that help providing or improving functional skills . It can be considered an indispensable tool to provide the inclusion and the integration of people with deficiency , with an amplification of the context of the society [1] . This technology is designed especially for people with disabilities with the aim of providing a framework for considering how products and environments can be designed to accommodate the broadest range of users [2] . Borg and Larson ( 2009 ) have observed that more attention has been given to the preventing role of assistive devices compared to the facilitating role . The focus of assistive devices facilitating functioning has been on mobility aspects of care of the self and domestic life , which might also facilitate activities and participation in other life areas such as work and employment . To achieve a better utilisation of assistive devices there should be a training of related professionals and staff on user-involved design [3] . The intersection between technology and subjectivity rescues the appreciation of the participation of the subject in the process of treatment and rehabilitation . The use of the assistive technology in the life of the subject influences the care of the self , that is the ability to manage their own life . This paper contributes to the theoretical and practical concepts of the health care practices , research and innovation in this area , also the changes in the teaching of the occupational therapy in both inter and multidisciplinary approaches .
This study employed a descriptive exploratory design , with a qualitative aspect , in which we used a semi-structured interview and field observation as research tools . The room for the conduction of the research was the ambulatory of Occupational Therapy created in November , 2012 , in a University Hospital placed at Ilha do Governador in Rio de Janeiro . The hospital provides assistance , teaching and research in several areas . The leprosy program has begun in 1990 and it has become a reference for diagnosis , treatment , surgeries and rehabilitation of leprosy . The interviews were conducted , taped in a digital recorder and fully transcripted by the authors . The interview script searched for the following aspects: report of the repercussion of the assistive technology in the autonomy of the subject's care of the self and instrumental aspects; value of the technology and subjective dimensions . Based on the Hermeneutic approach , we've established some wide questions to be answered by the patients , as follows below: The devices to be produced were determined after a motor and sensitive evaluation of the patient . During it , it was possible to detect their physical condition and to prevent risks associated with the absence of sensation . During the study , patients were asked about the difficulties in their regular activities and the importance given to such disability . All patients received at least one adapted utensil and were trained for the use of the following instruments: forks , knifes , spoons , mugs , wooden spoon , toothbrushes , shavers , pen and scissors . The work tools adapted were: screwdriver , monkey wrench and grip pliers . Orthoses for ulnar claw were also built . A total of 44 hours of observation were performed and registered in a field journal , that also contained the evaluation of the participants , production of the equipment and training of the adaptations . During the observational period , information about the professional care , relation aspects and process of selection of the patients for the interviews were gathered . During this period , we also took notes on the individuals' nonverbal language . The main objective of adaptations was to allow performing actions , especially the ones that the subject values , letting them to become more independent and fulfilled . The training process was also a moment to discuss the adhesion of the use of devices , and all patients were able to perform tasks in a new , adapted manner . Patients brought their own utensils from home to be adapted , such as flatwares and tools . This way , we respected patient's culture , did not change their lifestyle and also made them responsible for their own treatment . Materials for adaptations such as lining , foam , PVC , thermomoldable and others achieved a total cost of USD100 , and are shown in Fig 1 . This research was submitted and approved by the Human Research Ethics Committee from the Hospital Universitário da Universidade Federal Fluminense , with and acceptance letter from the coparticipant institution HUCFF/UFRJ , in the date of August 8th , 2014 , by under the number 774 . 178 , register CAAE: 30503914 . 5 . 0000 . 5243 according to the Resolution 466/12 of the National Health Council that controls researches with human beings . All participants have provided a signed informed consent .
After observation , 8 patients were selected . They were older than 15 years of age , with grade 2 disability[4] . They all accepted the use of the assistive technology . There were 13 patients in the service , but 5 were excluded due to sequels of other diseases and cognitive and/or mental disturbs ( Table 1 ) . The content of the interviews and the observations evidenced verbal and non-verbal expressions , emotional and meaningful contents regarding their social , family and individual dimensions and stigma related to leprosy . Unequal job opportunities , difficulties related to the physical limitation and the way patients became dependent on their family members were present in the reports and generated body expressions and gestures to be reviewed in the Discussion topic . The content of the research was divided into two categories: contribution of adapted devices for the care of the self; and feelings and sensations provoked by the use of the adapted instruments ( Table 2 ) .
The limitations acquired during ADL that were a topic for this research , such as grabbing , cutting , writing , screwing and performing other care of the self activities have led us to do more than build and train the devices . Such care practice approximated our group from the patients as we experienced the limitations and the search for ways to surpass them . Once we enter someone's routine , we must create an atmosphere of reception , approach and intimacy . Several gestures emerged together with the statements . “this finger ( index ) disrupts me , it is very small and I can't hold things” ( patient 4 ) . One must consider that anesthetic , amputated or with re-absorption hands are limited in their functionality . The patient above demonstrated how he performed actions , despite of his injured hands , by a reelaboration of his possibilities through adaptation as develops his own way of solving problems , such as “when the screw head is small , I place it in a magnet , which makes it easier to get…” ( Fig 2 ) . The contributions of adaptations for the patients' lives also aimed to prevent accidents , such as “…the knife sometimes cuts my hand . . I've never cut myself… . the adaptation is good… . the hilt is thicker…” ( patient 1 ) . The use of adaptations promotes a distribution of the prehension forces , enhances functionality and reduces the influence of the deficiency into an independent performance of functional activities . To be safe while performing an action was interpreted by the patients as a possibility for autonomy and care of the self . Patient 7 , who received a wider wooden spoon refers a safer sensation by being away from the steam of the pots . Even in the absence of pain , no patient wants to have an injury , which can be seen as sign of self-neglect . To facilitate self-care , patients received flatware and toothbrushes ( Fig 3 ) . Our results along with the search for the autonomy in the care of the self , establishes a connection with Foucault's care of the self idea . Foucault [5] affirms that “To take care of the self is to know oneself , one's soul , as soul-subject , the element that identifies to the divine . ” The adaptation of utensils provided a possibility of safety and independence during the recovery process , which are autonomy actions for the subject's care , frequently expressed as the examples “I' will be able to write again” ( patient 3 ) , “…now I can hold the bottle and easily drink water” ( patient 6 ) . Such reports connect themselves with the amplification of the subjective and bodily possibilities , as the adaptations can be considered an extension of the body and a facilitator of the restoration of the human making process . When the patients reported their perception of the use of the adapted utensil in their ADL , it was observed a re-elaboration of the making , with a new sense of the care of the self , as registered in the following sentences: “to be able to finish studying , to have a high school degree is a relief , a victory . ” ( patient 3 ) , “The more we can do without any help , the better” ( patient 7 ) . Therefore , to facilitate one's care of the self actions involves aspects such as: their way of living , choices , feelings , representations and other elements that act in the building of the way of being . It occurs an ethical implication of respecting the autonomy of the subject , rescue their dignity and providing social interactions . When observing the frequent expressions of joy and satisfaction facing the rescue of the ADL , it is observed an aesthetics of existence , which , according to Foucault[6] aims for the constitution of one's self as the artesian of the beauty of one's own life . This aesthetic dimension shows that the taste for the new form of living , the satisfaction for the autonomy , brings up the promotion of health and quality of life in a context of treatment and rehabilitation . A patient's report was “Do you know how it feels to ask someone who doesn't want to help ? ” ( patient 3 ) followed by “This adaptation increased my self-steem” ( patient 5 ) . “It brings happiness and joy” ( patient 1 ) , and another one affirmed “We feel safer inside” ( patient 7 ) . Foucault [6] affirms that the hermeneutics analysis is a joint point between the body and the history . It evidences the body as marked by the history , and denotes the need to interpret the interpretation of the routine activities incorporated . In the field of the hermeneutics analysis there are the corporeality and its historical representations , throughout the subject's sensibility . The unique reports show the need for acceptance and integration in the society , which they have been denied due to the body sequels and social stigma historically produced . “We just feel equal , right ? …other people , the normal ones , then I become normal” ( patient 1 ) . “How come someone else can cut and I can't ? ” ( patient 5 ) , “I used to watch people sitting at a table holding knives and forks , and I felt powerless , you know ? . . Now I don't , when I cut , I also participate” , “I was sad because I couldn't do anything” ( patient 1 ) ( Fig 4 ) . Subject is submitted to mercy of distinct factors linked to someone else by control and dependence; and tied to his own identity by a conscience or self-knowledge . Despite one's individual perception of unique and owner of its own destiny , we also have a tendency to totality and fullfillness [7] . Social indifference leads to a feeling of sadness , described as a psychic suffering , which must be carefully identified and discussed when working with such clients . That is due to an affective sequel between the bodily representation and the subject's history . Once we faced such situation , we could understand it and help the subject when developing its own the care of the self . The assistive technology was applied to help achieving autonomy , social participation and a will to live , reducing one's suffering . Once we discussed the differences , emotion emerged along with a search for comprehension , as the following quotes affirm: “Doctor , you don't know what it feels like ! ! ” ( patient 2 ) and indignation and sadness looks manifested . This reflexion leads us to believe that technical knowledge is not enough to comprehend the complexity , one must also listen , welcome and follow the process of care of the subject . Some overcome sentences manifested as “It's like we are reborn , learning it all again” ( patient 3 ) , “I couldn't get anything with this hand , but now I can” ( patient 4 ) , “…it's a good exercise to learn what I couldn't do before” ( patient 7 ) . That evidences the desire , the possibility to make choices and avoid placing oneself as a victim as reinventing through the limits and possibilities of life . These are evidences for relearning and reconstruction of the making ( Fig 5 ) . It is fundamental , during this period , to deal the patient's resistance , to avoid them from keeping in the process and to maximally enjoy the opportunities for a nurturing and interactive relearning . Such changing moment was observed through manifestations of joy , satisfaction and surprise throughout the possibilities . Also gestures and lines of conquer emerged , accompanied by hopeful smiles and looks , provided in a comprehensive and conscious manner by the assistive technology . Although the sensation of happiness was manifested , feelings of anger and indignation were also observed . Objective and subjective limitations appeared in a few reports: “This is not good to use , but a lot of people need it . ” ( patient 8 ) “I become so angry that I call names , sometimes” “…I don't eat to avoid disturbing others” ( patient 2 ) . Sometimes movements could not be performed even in the use of the adapted devices , so frustration appeared . This topic was also discussed in our sessions , without giving up the new possibilities . To live with pleasure and the lack of it , love and hate are indispensable parts of the human learning process , and can't be removed from the health care . The use of self help devices causes social awkwardness and can attract as much attention as the deformities caused by leprosy . This is probably one of the reasons of the low adhesion of the use of adapted devices when in public . Some patients affirm the use of adapted equipment at a restaurant as an award . Others demonstrate shame and report to be more observed in the use of the adaptations . “I don't use it outside , people look at it” ( patient 2 ) . The truth is that society does not easily tolerate differences . One will always search for the appropriate behavior , the desire for uniformness . This problematic was also discussed , the adhesion of the use of the orthoses and adaptations , with a focus on the adapted action , and how this subjectively affects the person . The hermeneutics analysis points the need for the interpretation to constantly interpret itself , especially the re-signification and re-codification of the messages and signs . The sign begins to hold the contradictions , oppositions , tensions , negativity and positivity required to play a dialectic of interpretation [8] . Hermeneutic procedure allows to “speak again” and provides a wide possibility to new destiny on the speech . When a patient with injured hands sadly says “Everything we do is more difficult” ( patient 4 ) and follows that by “I won't need to ask for help to open a bottle anymore” ( patient 7 ) it is important to allow them to listen how they can give a new meaning to this impotence and tiredness towards their difficulties . The professional must recognize this endless building process . As Paulo Freire says [9] , “The unfinished or inconclusive is part of the vital experience of the human being . Where there's life , there is something unfinished” , it can be realized that this impulses self knowledge and the appearance of latent desires , such as fantasies , dreams and other subconscious manifestations . This is a perennial , never conclusive movement . We don't walk only forward , but we can walk back , go deeper , walk in any direction . The same occurs for the interpretations , they need to relate through a complex net of symbols . It has been a priority in our research to guarantee this environment of welcome and rebuilding . The main findings of our research appeared in the rediscovered feelings during the use of the self-help devices , through expressions of overcome or frustration during the training sessions , smiles , moments of silence , and strengthening of the therapeutic bonding between patient and health care professional . Assistive technology is a worthy therapeutic instrument in the care of people with special needs . It must be involved in a context of a symbolic net that involve the body , he function , social role and the society when dealing with the inclusion , among other existential and relational aspects . The lack of more information on the use of the assistive technology in leprosy has led us to continue this research . The number of cases is high , and new forms of sensitive and effective care must be searched , to change the profile of the rehabilitation offered , providing forms to rescue the autonomy and independence . To affirm that the patient is autonomous , free and conscious of their choices , facing the embarrassing economic and social moments that difficult the access to information , it is fundamental to equal the relation of power between patient and health care professional . Aspects as one's desires , potentials and rights must be valued . When analyzing the contribution of the devices in the routines and care of the self , feelings and sensations provoked , it was verified how important it is to provide instruments to the patients that allow the care of the self and how much this interferes in the preservation of the autonomy and social inclusion of the individual . The use of the devices does not represent a high cost for one's treatment , but has an impact in the rescue of the abilities lost with the evolution of the leprosy disabilities , which should be funded as research . We must consider through gestures and speaking that the body of the patient with leprosy expresses scars of a collective history of suffering . Each subject express its own issues towards body and symptoms , but the conditions at the moment can not be ignored . The perception that the assistive technology represents a tool with a changing power for the rescue of these patients' identity leads to believe in new possibilities of care .
|
This study is about occupational therapy and we focus on the autonomy of patients with disabilities due to leprosy . Our purpose was to analyze the repercussions of assistive technology on the autonomy of care of the self in patients with sequels of leprosy We performed a qualitative , descriptive , exploratory study between November 2014 and February 2015 at a university hospital in Rio de Janeiro , and interviewed eight patients . Analysis was conducted by the hermeneutic approach . The following categories were studied: contribution of the adapted devices for the care of the self and feelings and sensations provoked by the use of self-help devices . The reports revealed feelings , perceptions and meaningful contents about the social , familiar and individual dimensions , also the stigma coupled with leprosy . Assistive technology is an approach with a powering potential that provides tools for the subject's care of the self and promotes social inclusion .
|
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"Abstract",
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"Methods",
"Results",
"Discussion"
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2016
|
The Use of Assistive Technology to Promote Care of the Self and Social Inclusion in Patients with Sequels of Leprosy
|
CULLIN3 ( CUL3 ) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases ( called CRL3s ) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes . Here , we report that in the model plant Arabidopsis thaliana , CUL3 regulates plant growth and development , not only during embryogenesis but also at post-embryonic stages . First , we show that CUL3 modulates the emission of ethylene , a gaseous plant hormone that is an important growth regulator . A CUL3 hypomorphic mutant accumulates ACS5 , the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response . Second , we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway . In particular , we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number . This phenotype is suppressed by ethylene-insensitive or resistant mutations . Finally , we identify a function of CUL3 in distal root patterning , by a mechanism that is independent of ethylene . Thus , our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells .
Regulation of protein stability through the ubiquitin proteasome system ( UPS ) is now considered as a major mechanism underlying many cellular and organismal processes , such as cell division , DNA repair , quality control of newly produced proteins , developmental and immune defense pathways , and in plants , light and phytohormone signal transduction [1]–[3] . Degradation via the UPS is a two-step process: the protein is first tagged by covalent attachment of ubiquitin and subsequently degraded by a multicatalytic protease complex called the 26S proteasome . The transfer of ubiquitin to a target protein substrate requires an ubiquitin protein-ligase ( E3 ) . E3 enzymes act to specify the substrates and thus they play a key role in the ubiquitylation reaction . Several hundred different E3s have been identified in metazoan and plant genomes , based on specific , commonly shared structural motifs . However , the most intensively studied subclasses of E3s are those of the cullin-RING ligase ( CRL ) superfamily , which form multi-protein complexes [4] . CRL E3s can be viewed as two functional modules brought together by the CULLIN proteins , acting as molecular scaffolds . The first module forms the catalytic centre and is composed of a RING finger domain protein and an ubiquitin conjugating enzyme ( E2 ) . The second module can be considered as the substrate recognition module , in which a specific protein physically interacts with the target substrate . A series of recent reports has shed light on the molecular composition and function of the CUL3-based CRL E3s ( reviewed in [5] ) . Certain ‘Bric a brac , Tramtrack and Broad Complex/Pox virus and Zinc finger’ ( BTB/POZ ) domain proteins function as substrate specific receptors in Schizosaccharomyces pombe and Caenorhabditis elegans [6]–[9] . These BTB domain proteins bind CUL3 , via the BTB domain and direct substrate specificity through an independent protein-protein interaction domain . The best-documented substrate for the CUL3-BTB ligases thus far , is the nematode MEI-1 protein , which regulates the meiosis-to-mitosis transition of fertilized embryos [6] , [8]–[9] . In mammals , CUL3 function is essential and its loss-of-function in mouse produces an arrest during early embryogenesis [10] . Recent data have also implicated vertebrate CUL3 in cell cycle regulation [11] and signal transduction pathways , such as the Wnt-beta-catenin pathway [12] . In contrast to metazoans , the function of the CUL3 orthologs is not essential in budding and fission yeasts [7] , [13] . The Arabidopsis genome encodes two CUL3-related proteins , called CUL3A and CUL3B [14] . Disruption of both genes causes embryo lethality [15]–[17] , indicating that CRL3s play important functions during early steps of plant development . Moreover a genomic search revealed the existence of about 80 BTB-domain proteins in Arabidopsis belonging to different families depending on additional protein domains , either upstream or downstream of the BTB-domain , such as the meprin and TRAF homology ( MATH ) domain , the Armadillo repeats ( ARM ) and the tetratricopeptide repeats ( TPR ) [15]–[16] , [18]–[19] . Protein interaction studies in yeast suggested that CUL3A and CUL3B may form many different CRL3 complexes , but their nature and substrates are still poorly documented in plants . ETO1 , an Arabidopsis BTB-domain protein , controls the stability of ACS5 , a member of the 1-aminocyclo-propane-1-carboxylic acid synthases ( ACS ) that catalyse a rate-limiting step in ethylene biosynthesis [20] . Moreover , ETO1 was found to directly interact with CUL3A , which prompted the authors to propose ACS5 as the first reported substrate for a plant CUL3-based E3 . However ETO1 is also able to inhibit ACS5 activity without CUL3 , indicating a higher complexity in this regulation [20] . Supporting a function of CRLs in ethylene biosynthesis is the fact that cycles of ( de ) neddylation seem to play an important role in this process , because Arabidopsis RNAi lines in which the expression of two NEDD8-related proteins , RUB1 and RUB2 , are reduced exhibit a triple response and overproduce ethylene [21] . To better characterize the function of CRL3 E3s in Arabidopsis , we identified a hypomorphic mutation in CUL3A , which , when combined with the cul3b null mutation strongly impairs overall CRL3 functions . Here we report a molecular and genetic characterization of this line , with a focus on ethylene biosynthesis and primary root growth .
Previously it was shown that the combined disruption of both Arabidopsis CUL3A and CUL3B genes causes embryo lethality [15]–[17] . To further investigate the function of CUL3 in plants , we searched for additional Arabidopsis T-DNA insertion lines . One line was of particular interest as the T-DNA was inserted at the very end of the CUL3A coding sequence . This mutant allele , called cul3a-3 was further characterised . The T-DNA insertion creates a mutation , in which the last two amino acids of the protein are replaced by an eight-residue peptide ( Figure 1A ) . The cul3a-3 mutant line produces a lower abundant truncated transcript ( Figure 1B ) . Interestingly , the CUL3A protein detected by a specific anti-peptide antibody [18] was not only less abundant , but was also hyper-rubylated ( Figure 1C ) , suggesting that the truncated CUL3A protein is less prone to de-rubylation . It is noteworthy that cycles of rubylation/de-rubylation are important for CRLs activity ( reviewed in [22] ) . Homozygous cul3a-3 mutant plants are fertile and do not show morphological defects under normal growth conditions . As CUL3A and CUL3B genes are functionally redundant , we generated a double mutant using the previously characterized cul3b-1 knockout line [17] . The double homozygous cul3a-3 cul3b-1 mutant , hereafter called cul3 hypomorph ( cul3hyp ) , was viable , but exhibited several developmental defects . Approximately 10% of homozygous cul3hyp seedlings displayed altered cotyledon phenotypes ( Table 1 ) . Some seedlings exhibited a single cotyledon while others , at a lower frequency , had three cotyledons ( Figure 1D ) ; seedlings with partially or totally fused cotyledons were also observed ( not shown ) . In addition , the vascular patterning of cotyledons was often abnormal ( Figure 1E ) . In particular , we observed interrupted and freely ending veins . In less than 1% of these seedlings we observed other abnormalities , such as root-less seedlings ( not shown ) . The absence of root meristem was previously revealed in some of the Arabidopsis cul3a-1 cul3b-1 double null mutants that could complete their embryonic development [17] . The subset of cul3hyp seedlings that had normal organ patterning displayed epinastically curled cotyledons and shorter roots when grown in the light ( Figure 1F ) . At a latter stage of development , the most striking phenotype was a reduced rosette size and a delay in flowering ( Figure 1G ) . Consistently , a slight delay in flowering was observed in single cul3a-1 loss-of-function mutant [18] . Overall , our data indicate that Arabidopsis CUL3A and CUL3B are important for plant growth and development , both during embryogenesis and at post-embryonic stages . Because ETO1 is involved in the ethylene biosynthetic pathway and physically interacts with Arabidopsis CUL3A [20] , most likely through its BTB domain , we investigated whether the cul3hyp mutant is affected in ethylene-mediated processes . In accordance with this speculation , etiolated cul3hyp mutants displayed a typical triple response in the absence of ethylene , which is characterized by short hypocotyls , short roots , and exaggerated apical hooks ( Figure 2 ) . The phenotype was similar to that of eto1-1 , though less severe than the constitutive triple response1 ( ctr1-1 ) mutant . It is noteworthy that the single cul3a-3 mutant displayed a weak triple response . Moreover , when germinated in the presence 5 µM ACC , the cul3hyp mutant was still responsive to ethylene in a root elongation assay ( Figure S1 ) . To better characterize whether the CUL3A and CUL3B genes are involved in the control of ethylene biosynthesis or might also play functions further downstream in the signalling cascade , we first used a pharmacological approach . We found that treatment with 2 µM aminoethoxyvinyl glycine ( AVG ) , which inhibits ACC synthase and hence ethylene biosynthesis [23] , significantly reversed the cul3hyp triple response ( Figure S1 ) . However , AVG has toxic effects and it inhibited root elongation of wild-type plants even at low concentrations ( not shown ) . Thus , we undertook a genetic approach and generated triple mutant combinations with ethylene-insensitive or resistant mutants in the ethylene-signalling cascade . In all triple mutants , etr1-1 cul3hyp , ein2-1 cul3hyp and ein3-1 cul3hyp , the triple response observed in the cul3hyp hypomorph was significantly , but not entirely suppressed ( Figure 2 ) . We conclude that the triple response phenotype of cul3hyp can be mostly explained by a function of CUL3A and CUL3B upstream of ethylene perception . However , the fact that both hypocotyl and root length were slightly reduced in all three triple mutants compared to their corresponding single ethylene insensitive or resistant mutants indicates that Arabidopsis CUL3 genes act also at other levels , which are ethylene independent . We generated an eto1-1 cul3hyp triple mutant and found only a slight additive effect on the triple response regarding root growth ( Figure S1 ) . These data indicate that the triple response observed in cul3hyp is mainly attributed to a defect in the CUL3ETO1 E3 ligase , but does not exclude the possibility that the two ETO1-related proteins , EOL1 and/or EOL2 [20] may also play some minor roles in this process . As a next step , we measured ethylene emission between day 3 and day 4 in the etiolated mutant seedlings . Consistently , the cul3hyp mutant accumulated two to three-fold more ethylene gas than did the wild type control ( Figure 3A ) , but significantly less than eto1 seedlings . To further confirm that the triple response observed in the etiolated seedlings is the consequence of CUL3A/B knockdown , we transformed the cul3hyp double mutant with a binary vector spanning a CUL3A genomic fragment [18] . Several transformants were recovered , which suppressed the triple response ( not shown ) as well as ethylene overproduction ( Figure 3A ) . Because the triple response of the eto1-1 cul3hyp triple mutant was slightly more severe than in the eto1-1 single mutant ( Figure S1 ) , we measured ethylene production in the triple eto1-1 cul3hyp mutant ( Figure 3A ) . There was an increase of about 3-fold in ethylene production as compared to the eto1-1 parent , suggesting that CUL3A/B controls ethylene production by both ETO1-dependent and independent mechanisms . There was also an increase of about 3-fold in ethylene production in the etr1-1 cul3hyp mutant compared to etr1-1 ( Figure 3A ) . Likewise , ethylene production was increased in the ein3-1 cul3hyp triple mutant compared to the cul3hypparent , despite the fact that ein3-1 does not overproduce ethylene . This data indicates that CUL3A/B acts additively with the autoinhibition control of ethylene biosynthesis . ETO1 directly interacts with ACS5 to inhibit its activity , but also mediates ACS5 26S proteasome-dependent degradation , most likely via CUL3A/B [20] . Moreover , the cin5-3 mutation , which disrupts the ACS5 gene [24] , significantly reduces ethylene production in eto1-1 and partially suppresses its constitutive triple response [25] . Thus , we speculated that the constitutive triple response observed in CUL3A/B knockdown was mainly the consequence of ACS5 protein stabilisation . To address this issue , we first introgressed a transgenic line expressing a Dex-inducible myc-tagged ACS5 [25] into the cul3hyp double mutant background . Due to partial silencing of the ACS5 reporter construct in the cul3 hypomorph , we could not compare directly the myc-ACS5 protein accumulation in cul3hyp and wild-type backgrounds at identical concentrations of dexamethasone ( Dex ) . However , by increasing Dex levels , we could normalize the expression of myc-ACS5 in cul3hyp and compare myc-tagged ACS5 protein half-lives in both genetic backgrounds ( Figure 3B ) . After Dex-induction and subsequent removal , seedlings were incubated in presence of cycloheximide , which blocks de novo protein synthesis , and the myc-tagged ACS5 protein levels were then determined by immunoblot analysis . Whereas ACS5 protein in the wild type background had a very short half-life of ±15 min as previously reported [25] , there was no decrease in the level of ACS5 protein after 1 hour . Thus , Arabidopsis CUL3A and CUL3B are involved in the turnover of the ACS5 isoform . Furthermore , we produced the cin5-3 cul3hyp triple mutant and found that cin5-3 suppresses partially the triple response of cul3hyp ( Figure 3C ) . Overall , we conclude that ACS5 is a primary target of CUL3A/B and ETO1 in seedlings , but other ACSs , in particular of the type-2 class , are most likely also degraded by this E3 ligase . The cul3hyp mutant exhibits a shorter root ( Figure 1F ) and CUL3A/B genes are essential during embryogenesis for proper patterning of the hypophyseal lineage , important founders of the future root meristem [17] . Furthermore , the role of ethylene on primary root growth was recently emphasized by several reports [26]–[29] . To characterize the function of CUL3A/B during root development , the work focused on primary root growth . At 11 days post-germination , the elongation of cul3hyp primary root showed a reduction of about 80% as compared to wild type ( Figure 4A and Figure S2 ) . The root growth defect was similar to ctr1 . ein2-1 and ein3-1 ( not shown ) significantly suppressed this phenotype , but these mutations were unable to restore wild type root growth . Thus , CUL3A and CUL3B regulate primary root growth via both ethylene-dependent and independent mechanisms . To better characterise this phenotype , we measured the length of cells in the cortex in the differentiation zone , because it was found that ethylene stimulates auxin biosynthesis and its basipetal transport to the root elongation zone , where auxin inhibits cell elongation [27] , [29] . Consistent with such a scenario , we observed a 50% reduction in length of these cells in eto1-1 ( Figure 4B ) . However to our surprise , cortical cell length in the differentiation zone was only marginally affected in the cul3hyp mutant . This suggests that the mechanism by which ethylene inhibits root growth in a CUL3-deficient mutant background is different from that reported for eto1 mutants and ACC-treated wild-type plants [27] , [29] . Root growth depends on cell elongation and on cell production rates in the root apical meristem . Therefore we investigated whether CUL3A/B knockdown affects the meristem size and/or activity . Strikingly , we observed that in cul3hyp the meristem size and cell number was reduced compared to wild-type plants ( Figure 4C–D ) . Interestingly , in the ctr1-1 mutant , there was also a significant reduction in both meristem size and cell number . Conversely , in eto1-1 , the root meristem size and cell number were even slightly increased in comparison to wild-type plants , suggesting that ethylene overproduction in this mutant can have opposite effects in the root , negatively affecting cell expansion in the elongation zone but positively affecting root meristem size . Importantly , the meristem phenotype of cul3hyp was suppressed by the ein2-1 and ein3-1 mutations and therefore is dependent on ethylene signalling . We investigated whether this phenotype in cul3hyp was the consequence of reduced cell cycle activity . To this end we introduced the pCYCB1;1::GUS reporter construct [30] into cul3hyp mutant background . The Destruction box of plant B-type mitotic cyclins targets proteins for degradation after mitosis [31] and thus the pCYCB1;1::GUS reporter is a suitable marker to identify cells in G2-to-early M phase . The number of CycB;1:GUS expressing cells was not significantly reduced in the root apical meristem of cul3hyp compared to wild-type plants ( Figure 4E ) . Thus , the most likely explanation of the ethylene-dependent inhibition of root growth is that cells in the cul3hyp mutant prematurely exit the meristem and make the transition to cell expansion . To get more insights into the mechanism ( s ) by with CUL3A/B regulate root growth , we determined the expression pattern of both genes in Arabidopsis roots by using promoter-GUS fusions . After short GUS staining , we observed the strongest histochemical pCUL3A::GUS localization in the stele , but also in the distal part of the root ( Figure 5A ) . A similar expression pattern was also observed for the pCUL3B::GUS reporter , although the signal was weaker in the stele . In the cul3hyp mutant , this region revealed clear defects of cell division patterns in the quiescent center ( QC ) , other cells of the root stem cell niche and the columella root cap ( Figure 5B–E ) . Starch granule staining , which marks only differentiated columella cells , showed premature differentiation of the columella root cap initials ( Figure 5B ) . To investigate whether this defect in root meristem patterning is associated with mis-specification of the QC or columella cells , we examined the expression of different markers in the cul3hyp mutant background . In cul3hyp plants expressing QC-specific marker QC46 ( Figure 5B ) or endodermis and QC marker pSCR::H2BYFP ( Figure 5C ) , we could identify signal in cells of the QC region , even when their morphology or cell number was altered . However , when we used columella-specific markers ( Figure 5D–E ) and in particular Q1630 , which is only expressed in layers C1 and C2 in wild type ( n = 21 ) , we observed a different pattern in the cul3hyp mutant plants . The marker was in general expressed in additional columella cell layers ( 50% , n = 32 ) or showed a patchy distribution ( 22% , n = 32 ) . It was recently reported that ethylene modulates cell division in the QC , which can eventually lead to additional columella cell layers [26] . Therefore we investigated whether the phenotype observed in cul3hyp is dependent on ethylene signalling . However , this was not the case as defects of cell division in the QC and columella remained in the ein3-1 cul3hyp triple mutant ( Figure 5B ) . Moreover , contrarily to the report of Ortega-Martinez et al . [26] , we did not observe deregulated QC cell divisions in the eto1-1 mutant . Thus , we conclude that the knockdown of CUL3A/B function in Arabidopsis disturbs distal root patterning by a mechanism that is ethylene independent . Auxin is involved in distal pattern formation of Arabidopsis roots [32] . To investigate whether auxin signalling is affected in cul3hyp , we introduced the DR5rev-GFP reporter construct into the cul3hyp mutant and monitored GFP expression in the root tip ( Figure 6A ) . The spatial distribution of DR5 expression in the mutant was more narrow and also reduced in intensity in comparison to wild type roots ( Figure 6A ) , indicating that auxin signalling is reduced in the distal part of cul3hyp roots . PIN-FORMED ( PIN ) proteins are rate-limiting factors catalysing polar auxin transport [33]–[34] . These proteins are crucial for auxin distribution and as such provide positional information to coordinate plant development . Because auxin signalling was reduced in the cul3hyp distal part of the root , one possibility is that the auxin gradient is disturbed in this mutant . Thus , we introduced into the cul3hyp mutant background different PIN::PIN-GFP reporters , consisting of their endogenous promoters and translational fusions between PIN1 , PIN2 and PIN7 proteins and GFP . Whereas PIN1 and PIN2 expression patterns and levels were similar to wild type in cul3hyp ( Figure 6B–C ) , we observed a higher expression level of PIN7 in columella cells ( Figure 6D ) . The ratio of the GFP signal between the stele and the columella tissues was four-to-five times higher in cul3hyp roots in comparison to wild type ( Figure 6E ) . PIN gene expression is regulated at the transcriptional , but also post-transcriptional levels [35] . Thus we performed quantitative RT-PCR assays on PIN7 gene expression on isolated wild type and cul3hyp root tips . In contrast to the PIN7 protein accumulation in columella cells , the PIN7 transcript level in cul3hyp was slightly reduced in comparison to wild type ( Figure 7F ) . Our data indicate that CUL3A/B knockdown induces in the highest CUL3 expression domain PIN7 accumulation , most likely by a post-transcriptional mechanism .
ACSs are rate-limiting enzymes in ethylene biosynthesis ( reviewed in [36] ) . Arabidopsis has nine ACSs , which are subdivided into three different types [37] . ETO1 , a BTB domain-containing protein interacts with type-2 ACS proteins and mediates the degradation of at least one of them , ACS5 [20] . Because ETO1 also interacts in a yeast two-hybrid assay with Arabidopsis CUL3A , it was concluded that ACS5 becomes ubiquitylated by a CUL3ETO1 E3 ligase and subsequently degraded by the 26S proteasome [20] , [37] . Our data indicate that ethylene production is induced in the cul3hyp mutant , which is consistent with such a scenario . In addition , we found that the half-life of ACS5 is prolonged in the cul3hyp mutant providing a molecular evidence for the involvement of CUL3A/B in the turnover of ACS5 . However it is noteworthy that cul3hyp is a weak ethylene overproducer in comparison to eto1-1 , cul3hyp produces about six-fold less ethylene than does eto1-1 . Thus , it is possible that this difference in ethylene production is the consequence of a longer ACS5 half-life in eto1 in comparison to cul3hyp mutant . This is also consistent with the fact that cul3hyp still keeps some CRL3 activity , whereas eto1-1 is a null allele . However , we also cannot rule out that ETO1 , which is still present in the cul3hyp mutant background , inhibits ACS5 protein activity by a process independent of protein degradation . Moreover , BTB-containing proteins themselves are also substrates for these CUL3 complexes [8] , [38] , which may even lead to a higher ETO1 protein accumulation in the cul3hyp mutant . This data would be consistent with the finding that ETO1 overexpression in Arabidopsis reduces kinetin-induced ethylene production and most importantly that ETO1 inhibits ACS5 activity via a direct interaction [20] . Arabidopsis ETO1 is part of a small gene subfamily containing two other closely related BTB-domain proteins , called EOL1 and EOL2 [20] . Genetic interactions showed only minor additive effects on the triple response morphology in eto1-1 cul3hyp triple mutant compared to eto1-1 , but a strong increase in ethylene production was observed . This suggests that EOL1 and EOL2 are also involved in ethylene biosynthesis , though ETO1 is the main CUL3 receptor in this process . Although it was previously shown that the single eol1 and eol2 mutants grow like wild type in the dark [16] , recent data demonstrated that both EOL1 and EOL2 negatively regulate ethylene biosynthesis by directing type-2 ACS proteins for degradation [39] . Is ACS5 the only ACS target of CUL3A/B in Arabidopsis ? Based on our data we can say that ACS5 is a primary target , because ACS5 loss-of-function in cin5-3 significantly suppressed the triple response during early seedling development ( Figure 3C ) and the root growth inhibition ( Figure 4A ) of cul3hyp . Nevertheless , cin5-3 does not entirely revert the cul3hyp mutant; thus , it is likely that other ACSs are also targeted by CUL3A/BETO1 ( Figure 7A ) . As for ETO1 , EOL1 and EOL2 only interact with type-2 ACSs [37] , [39] , whereas type-1 and type-3 ACSs , which are also degraded by the 26S proteasome [40]–[41] , are most likely recognized by another Arabidopsis E3 ligase . It was previously found that ethylene-insensitive or resistant mutants , such as etr1-1 , ein2-1 and ein4-1 produce increased amounts of ethylene [42]–[44] , whereas ctr1-1 does not [45] . This suggested a negative feedback mechanism from ethylene perception and signal transduction to ethylene biosynthesis [43] . The finding that the cul3hyp mutation synergistically enhances ethylene production in etr1-1 indicates that CUL3A/B and ETR1 act in parallel and most likely independently in the regulation of ethylene biosynthesis ( Figure 7A ) . This is consistent with previous studies showing that ETO1 also acts synergistically with etr1 to enhance ethylene production [46] . Arabidopsis primary root growth is reduced in a concentration-dependent manner when plants are exposed to ACC or to exogenously applied ethylene and this process is the consequence of down regulation of cell elongation ( [47] and references therein ) . Recent results demonstrated that ACC treatment of wild-type plants positively regulates auxin biosynthesis and distribution in Arabidopsis roots [27]–[29] . Based on different approaches , a model was proposed in which ethylene-stimulated auxin is subsequently basipetally transported to the elongation zone where it inhibits cell elongation [27] , [29] . In the present report we provide evidence that the ethylene pathway also acts on root development at a different level ( Figure 7B ) . We show that the inhibition of CUL3A/B activity impairs primary root growth in an ethylene-dependent manner by reduction of root meristem size and cell number . In contrast to eto1-1 or ACC-treated plants [27] , [29] , [47] , no significant effect was observed on cell elongation in the cul3hyp mutant . However , we found a similar root phenotype in ctr1-1 and the double ebf1-1 ebf2-1 [48] mutant backgrounds ( this work and data not shown ) , both accumulating the transcription factor EIN3 leading to a constitutive ethylene response . It is noteworthy that the ctr1-1 mutation affects both cell elongation and root meristem size , indicating that ethylene signalling is involved in both mechanisms . Because the number of mitotic cells in the cul3hyp root meristem was not significantly different from wild type , we conclude that the reduced meristem size is the consequence of the premature transition of cells from the meristem to the cell elongation zone , rather than caused by differences in cell division activity . An intriguing observation is that in eto1-1 , which overproduces ethylene , or in ACC-treated roots ( [27] and our data ) , no decrease on the root meristem size was observed . This suggests that despite the fact that ethylene is a volatile gas , depending on its sites of production and/or perception , ethylene can induce different local responses . Hence , this fits with the evidence that ACS genes in Arabidopsis display distinct expression patterns during plant development [49] . The strong CUL3A and CUL3B expression in the distal part of the root and their involvement in distal root patterning suggest that CUL3A/B proteins control the division and organisation of the stem cell niche and columella root cap cells . By which mechanism ( s ) do CUL3A/B genes maintain QC and root cap organisation ? It was recently reported that ethylene modulates cell division in the stem cell niche [26] . Such an effect of ethylene would be consistent with the role of CUL3A/B in ethylene biosynthesis ( see above ) . However , several lines of evidence argue against this possibility . First , 1 µM ACC treatment of wild type roots revealed no abnormalities in columella cell differentiation and/or tissue organisation [27] . Second , such a phenotype was not observed in the eto1-1 null mutant . Third and most importantly , the ethylene insensitive mutation ein3-1 did not suppress cell division defects in the QC and in columella cells . A possible explanation for the discrepancies between our data and [26] is that the two eto1-11 and eto1-12 alleles used in their studies , which are point mutations , are acting in a dominant negative way to inhibit CUL3A/B activity . Overall , even if a subtle effect of ethylene on stem cell division is not excluded , our data point to an ethylene-independent role of CUL3A/B in organizing stem cell niche and columella ( Figure 7B ) . Interestingly , we noticed that the DR5::GFP signal in the cul3hyp root tip was weaker and more restricted than in wild type roots , suggesting altered auxin signalling . In addition , we also observed a higher PIN7 protein accumulation in cul3hyp columella cells . One way to connect these observations is a scenario in which PIN7 missexpression leads to the depletion of intracellular auxin from the root tip , resulting in reduced auxin signalling and as a consequence patterning defects . Further experiments need to confirm such a model . Moreover , because PIN7 accumulation in columella cells is under post-transcriptional control and because CUL3A/B genes are strongly expressed in these cells , it will be interesting to address whether the PIN7 protein is a direct target of a still unknown CRL3 complex . Finally , a broader connection between CUL3A/B and auxin may exist , since several developmental abnormalities in the cul3hyp mutant , such as altered number of cotyledons , or defects in root and venation patterning and embryogenesis ( this work and [17] ) are reminiscent of auxin transport or signalling defects .
The Arabidopsis cul3a-3 ( SALK 012973 ) mutant line has been identified using the web assisted program: http://signal . salk . edu/cgi-bin/tdnaexpress . The insertion site was confirmed by sequencing the T-DNA flanking srquences . The precise location of the T-DNA in the cul3a-3 mutant has been determined by sequencing , showing an insertion after nucleotide 2191 in the last exon of CUL3A . The last 8 nucleotides ( TAGCCTAA ) of CUL3A are replaced by 26 nucleotides from the left border of the T-DNA ( ACATACGGTATCATATTGTGGTGTAA ) leading to the addition of 8 amino acids ( HIRYHIVV ) to CUL3A protein sequence . All Arabidopsis thaliana lines used are in the Columbia background except for the Q1630 reporter line , which is in the C24 background . The transgenic and mutant lines have been described elsewhere: ein3-1 [50] , etr1-1 [51] , eto1-1 [43] , ctr1-1 [45] , ein2-1 [43] , cin5-3 [24] , proPIN1-PIN1-GFP and proPIN2-PIN2:GFP [52] , proPIN7-PIN7:GFP [53] , CYCB1-GUSDB [54] , Q1630 [55] and PET111 [56] . The CUL3A- and CUL3B-promoter GUS transgenic lines are described respectively in [18] , [19] . Arabidopsis thaliana seeds were sterilized with chloral gas or with ethanol method , plated on 1/2 MS medium ( 1/2 MS salts [Gibco-BRL , Cleveland , OH] , pH 5 . 8 , 1% sucrose , and 0 . 8% agar ) , stored 2 to 3 days at 4°C in the dark , and then transferred to a plant growth room ( 21/25°C , 16-h photoperiod ) . For the triple response assay , surface-sterilized seeds were germinated in the dark on 1/2 MS medium . Plates with seeds were cold-treated at 4°C for 2 to 3 days , exposed to light at room temperature for 2 to 4 h to improve germination , then wrapped with aluminum foil and incubated at 22°C for 3 days in the dark . A minimum of 15 seedlings were scored per mutant by pulling them out of the growth medium , stretching them flat on the surface of another agar plate , taking in pictures , and then quantifying root and hypocotyl lengths using ImageJ ( National Institutes of Health; http://rsb . info . nih . gov/ij ) . For seeds propagation , plants were grown to maturity at 22°C under 16-h photoperiod . For Northern–blot analysis RNA was extracted from plant material using the Trizol reagent ( Invitrogen , Paisley , UK ) . RNA gel blot analysis was performed with 20 µg of total RNA per lane . Northern blot procedure is described in [31] . 32P-labelled probes were synthesized with the Prime-a-Gene random prime labelling kit ( Promega Corporation , Madison , WI , 8USA ) using a 900-bp CUL3A cDNA fragment amplified with two gene-specific primers ( c3as4 5′-ATGGATTTGGGTGAATCTGT-3′ and c3aS5 5′-CTCGGGGTGACTGCCATA-3′ ) . For qRT-PCR assays , RNA was extracted from 10 days old root tips ( 1cm of the root starting from the root tip ) using the kit NucleoSpin RNA XS ( Macherey Nagel ) . 1 µg of total RNA were reverse transcribed with High Capacity cDNA Reverse Transcription kit TM ( Applied Biosystems ) . PCR was performed using gene-specific primers in a total volume of 15 µL SYBR Green Master mix ( Roche ) on a Lightcycler LC480 apparatus ( Roche ) according to the manufacturer's instructions . The TIP4l and At4g26410 genes were used as internal controls . The relative expression level of PIN7 gene in cul3hyp plants was compared with Col-0 control plants using GenEx Pro 4 . 3 . 5 . software ( MultiD Analyses ) after normalization using the At4G26410 cDNA level and averaging over three replicates . Primer list . PIN7:TGGGCTCTTGTTGCTTTCA and TCACCCAAACTGAACATTGC TIP4l: GTGAAAACTGTTGGAGAGAAGCAA and CAACTGGATACCCTTTCGCA AT4G26410: GAGCTGAAGTGGCTTCAATGAC and GGTCCGACATACCCATGATCC Transgenic lines used were dex-inducible myc-tagged ACS5 [25] . The coding region of ACS5 was amplified from cDNA of wild type and fused to a 6× myc cassette , and cloned into the binary GVG vector pTA7002 [57] . Plants were transformed with the plasmids by the floral dip method [58] , and transformants were selected on MS medium containing hygromycin . T2 seedlings were grown on MS medium containing 10 nM dex and screened for lines that expressed the myc-tagged proteins at low levels in an inducible manner . Tissues for protein analyses were ground in a denaturing buffer [59] followed by boiling for 5 min . After centrifugation , 20 µg of total protein extracts were fractionated on a 10% SDS-PAGE gel and blotted onto Immobilon-P membrane ( Millipore , Bedford , MA , USA ) . The immunoreactive proteins were detected using peroxidase-conjugated goat anti-rabbit antibodies ( Dianova , Chalfont St Giles , Bucks , UK ) and ECL Western blot analysis system from Amersham . Immunoblots were performed using a 5000-fold dilution of anti-Myc monoclonal antibodies from mouse ( clone MYC-1A1 Euromedex ) and a 5000-fold dilution of peroxidase-conjugated goat anti-mouse IgG ( Molecular Probes ) . Signals were detected by film ( within the linear range of detection ) using the enhanced chemiluminescence protein gel blot analysis system ( Amersham Biosciences ) . The blot was stained subsequently with Comassie blue to control the loading . For ACS5 turnover assays , wild type and cul3hyp transgenic seedlings harbouring DEX-inducible myc-ACS5 were grown on MS medium for 8 days at 22°C . Twenty ml of liquid MS medium containing different DEX concentrations ( see Figure 3 ) were poured onto the plates where seedlings were growing and incubated for 4 hours . Seedlings were incubated for different times in 1M cycloheximide after three washes of liquid MS medium . Total proteins were extracted and used for immunoblot analysis . Seeds were sterilized using chlorine gas sterilization ( 100 mL bleach +3 mL HCl for ∼4 hours ) , and seeds were aliquoted in 0 . 4% top agar to 3 mL MS 1% sucrose media in 22-mL gas chromotography vials . Vials were maintained sterile using autoclaved aluminum foil , for 5 days in 4°C . Vials were incubated in the light for 4–6 hours , then capped and incubated at 22°C dark for 4 days . The vials were capped in the dark at day 3 and incubated to day 4 . The accumulated ethylene was measured by gas chromatography as described in [24] . All genotypes are represented by 3 repetitions of 2–3 vials each . Ethylene measured in each vial was then divided by number of seedlings in the vial . Root seedlings were photographed and their lengths were measured with ImageJ . At least 15 seedlings were processed , and at least three independent experiments were performed , giving the same statistically significant results . Root meristem lengths and epidermal cell lengths were measured on root mounted in chloral hydrate . Images were captured with a Zeiss Axioskop microscope ( Carl Zeiss , New York , NY ) equipped with a Nikon DXM1200 digital camera ( Nikon Instruments Europe , Badhoevedorp , The Netherlands ) . The number of root meristematic cells was obtained by counting cortical cells showing no sign of vacuolisation . Root meristem length was assessed as the distance between the quiescent center and the first cell with a vacuole . ImageJ was used also for measurements of the length of root cortical cells . At least 10 seedlings were processed in at least three independent experiments giving similar results . Histochemical GUS staining analyses of the CUL3A/B promoter , the CYCB-GUS and QC reporter lines were done as described in [60] . For the confocal microscopy , roots were visualized using a Leica ( Wetzlar , Germany ) MZ FLIII fluorescence stereomicroscope equipped with GFP and YFP filters . Propidium iodide ( 10 µg/mL in distilled water ) was used to stain the cell walls of living root cells ( red signal ) . For quantification of PIN7:GFP signal fluorescence ImageJ program was used . The ratio of GFP signal intensity between the stele and the columella was calculated for the wild type and cul3hyp roots . Approximately 15 seedlings/images were examined , in three independent experiments giving similar results .
|
Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules in all eukaryotic organisms . This protein degradation pathway has a large number of components , including hundreds of ubiquitin protein ligases ( E3s ) that are predicted to have regulatory roles in cell homeostasis , cell cycle control , and development . Recent research revealed the molecular composition of CULLIN3 ( CUL3 ) -based E3 ligases , which are essential enzymes in both metazoans and plants . Here , we report that in the model plant A . thaliana , CUL3 modulates the emission of ethylene , a gaseous plant hormone that controls a variety of processes such as fruit ripening and stress response . In particular , we provide evidence that CUL3 regulates root growth by a novel ethylene-dependant pathway . Thus , we showed that CUL3 knockdown inhibits primary root growth by reducing the root meristem size . Finally , we also identified a function of CUL3 in distal root patterning . Indeed , CUL3 function is required for normal division and organisation of the root stem cell niche and columella root cap cells . Overall , our results show that Arabidopsis CUL3 is essential for plant growth and development , not only during embryogenesis but also at post-embryonic stages .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"physiology/cell",
"signaling",
"cell",
"biology",
"plant",
"biology/plant",
"growth",
"and",
"development",
"plant",
"biology"
] |
2009
|
Arabidopsis CULLIN3 Genes Regulate Primary Root Growth and Patterning by Ethylene-Dependent and -Independent Mechanisms
|
Children born from filarial infected mothers are comparatively more susceptible to filarial infection than the children born to uninfected mothers . But the mechanism of such increased susceptibility to infection in early childhood is not exactly known . Several studies have shown the association of active filarial infection with T cell hypo-responsiveness which is mediated by regulatory T cells ( Tregs ) . Since the Tregs develop in the thymus from CD4+ CD25hi thymocytes at an early stage of the human fetus , it can be hypothesized that the maternal infection during pregnancy affects the development of Tregs in children at birth as well as early childhood . Hence the present study was designed to test the hypothesis by selecting a cohort of pregnant mothers and children born to them subsequently in a filarial endemic area of Odisha , India . A total number of 49 pregnant mothers and children born to them subsequently have been followed up ( mean duration 4 . 4 years ) in an area where the microfilariae ( Mf ) rate has come down to <1% after institution of 10 rounds of annual mass drug administration ( MDA ) . The infection status of mother , cord and children were assessed through detection of microfilariae ( Mf ) and circulating filarial antigen ( CFA ) . Expression of Tregs cells were measured by flow cytometry . The levels of IL-10 were evaluated by using commercially available ELISA kit . A significantly high level of IL-10 and Tregs have been observed in children born to infected mother compared to children of uninfected mother at the time of birth as well as during early childhood . Moreover a positive correlation between Tregs and IL-10 has been observed among the children born to infected mother . From these observations we predict that early priming of the fetal immune system by filarial antigens modulate the development of Tregs , which ultimately scale up the production of IL-10 in neonates and creates a milieu for high rate of acquisition of infection in children born to infected mothers . The mechanism of susceptibility and implication of the results in global elimination programme of filariasis has been discussed .
Lymphatic filariasis ( LF ) is a major cause of chronic morbidity in the tropics and sub tropics . According to a recent estimate more than 1 . 4 billion people across the world are at the risk of infection [1] . To eliminate LF globally by 2020 , WHO has introduced annual mass drug administration ( MDA ) in different endemic countries since one and half decades . But studies have shown that the infection remains highly prevalent among children below five years of age even after several rounds of MDA [2–4] . Here question arises what makes these children more susceptible to infection even though infection levels have come down below threshold in these endemic areas . It is known that besides host genetics and environmental factors , maternal filarial infection plays some role to increase the susceptibility and outcome of the disease . Since pregnancy and early childhood are critical periods during which the inherited immune system of a child is shaped by the environment , the disease outcome in older age is possibly determined both in in-utero and at birth [5] . But it is not exactly known how in-utero exposures to parasite antigens affect immune responses and ultimately the outcome of disease in early childhood . The mechanism of such effects deserves to be explored since our previous findings suggest supervised therapy before pregnancy can reduce the infection rate among children [6] . Induction of regulatory T cells ( Tregs ) by pathogen is regarded as one of the mechanism of immune evasion in human . It is known that T cell hyporesponsiveness is associated with the active filarial infection , which is partly mediated by regulatory T cells [7] . The immune suppressive capacities of Tregs are due to production of down regulatory cytokines to inhibit inflammatory responses and facilitate the parasite survival [8 , 9] . Moreover a highly skewed Th2-type cytokine pattern , with a prominent role for the regulatory cytokine interleukin-10 ( IL-10 ) has also been marked in neonates born to helminth-infected mothers [10] . In case of patent filarial infection the state of immune hyporesponsiveness has been observed to be associated with decreased proliferative responses and increased anti-inflammatory cytokines such as IL-10 and TGF-β [8 , 11] . As the Tregs develop in the thymus at an early stage of the human fetal development from CD4+CD25hithymocytes [12] , the question arises that whether maternal infection during pregnancy affects the development of Tregs in children during their early life . Here we have made an attempt to find out the answer by evaluating the infection status , level of Tregs and regulatory cytokine IL-10 in a cohort of children born to filarial infected and non infected mothers .
Healthy pregnant women and their offspring born in Khurda District Headquarter Hospital of Odisha , India were enrolled in this mother-child cohort study . The study has received the approval from human ethical committee of the institute with a clause to obtain informed verbal consent from the research participants . The purpose of this research study has been explained in detail to all enrolled mothers in local language in presence of an unbiased witness of the community like Auxiliary Nurse Midwife ( ANM ) / Accredited Social Health Activist ( ASHA ) / Anganwadi Workers ( AWW ) . All participants have given face to face oral consent for themselves and their children without a sign consent form to participate over the entire period of study . The name and detailed address of each consent participant has been recorded in data sheet both at the time of enrolment and during follow-up . The oral consent was preferable because ( i ) the project involves no risk while giving service to the public and benefits to the ongoing LF elimination programme and ( ii ) linguistic or literacy demands of the written format which requires signature or thumb impression . This is a cohort study conducted in District Headquarter Hospital of Khurda , Odisha , India , known to be endemic for filarial ( Wuchereria bancrofti ) infections . The district has experienced 10 rounds of MDA with > 85% coverage since 2004 and reported 0 . 34% Mf in 2013 against 12% in 2004 . The pregnant mothers admitted in the hospital for delivery during 2009–2011 without any complications , free from other chronic diseases and belongs to this region have been selected for the study . The pregnant mothers and their subsequently born children enrolled in the study live in 8 adjacent villages . The mother’s age , parity status , levels of formal education , clinical history of filariasis and history of drug consumption in MDA were recorded after enrollment . None of the mothers had signs/symptoms of clinical filariasis at the time of admission . All enrolled mothers have affirmed consumption of anti-filarials distributed during the annual MDA before pregnancy but not during pregnancy since the drugs are not recommended during pregnancy . At the time of delivery blood samples were collected from both mother and cord aseptically and aliquot in different sized tubes to avoid the chance of mislabeling . Serum was separated after centrifugation and stored at—70°C until further use . Enrolled mothers having healthy full-term children were followed up in a house-to-house visit in the year 2014–15 . During follow up along with detailed clinical history 1ml of venous blood sample was collected aseptically from each enrolled mothers and her children . On the basis of the availability of the baseline immunological parameters 49 mother-child pairs were identified for follow-up out of 158 mother-newborn pairs enrolled during 2009–2011 . Amongst those 49 children , 28 are within 2–4 years of age and 21 within 4–7 years of age . Infection status of the mother-cord pair at the time of delivery and mother- child during follow-up was determined by diagnosing the presence of microfilaria and/or circulating filarial antigen in the peripheral blood collected at night between 20:30 to 22:30 . The Mf ( W . bancrofti ) was determined by microscopy by examining the Giemsa stained thick blood smear and CFA was evaluated in serum samples using commercially available Og4C3 antigen detection assay kit ( Trop BioMed , Townsville , Australia ) following the manufacturer’s instructions . The identification of Tregs ( CD4+ and CD25hi T-cells ) was determined by using fluorescently labeled antibodies specific to surface markers ( CD4 and CD25 ) . Briefly , 50 μl of heparinized blood collected from mother , cord and children were incubated in dark with 10 μl of anti-human CD4-FITC ( BD-Bioscience ) , anti-human CD25-PE ( BD-Bioscience ) for 30 minutes at 4°C followed by addition of 2ml of lysing solution and incubation for 10 minutes at room temperature . The samples were then centrifuged at 250 X g for 10 mins and cell pellets were washed twice with 2 ml of sheath fluid ( BD Bioscience ) . Finally the cell pellets were re-suspended in 0 . 5 ml of sheath fluid and subjected to flow cytometric analysis . Data were acquired by using BD FACS calibur flow cytometer and analyzed using cellquest pro software . The gating strategy for Tregs ( CD4+CD25+ hi ) cells is displayed in Fig 1 . The level of IL-10 was determined using IL-10 assay kit ( Sigma Aldrich , USA ) according to the instructions supplied by the manufacturer . Briefly , 100 μl of plasma and standards were added to each well of the antibody coated ELISA plate . The plate was sealed and incubated overnight at 4°C with gentle shaking followed by ( i ) 4 x wash with wash buffer and incubation with 100 μl of biotinylated detection antibody for 1 hour at room temperature , ( ii ) 4 x wash and incubation with 100 μl of HRP—streptavidin conjugate for 45 minutes at room temperature and ( iv ) 4x wash and incubation with 100 μl of colorimetric TMB reagent for 30 minutes . Finally50 μl of stop solution of 0 . 2M H2SO4was added and read in ELISA reader at 450 nm . The statistical analysis was performed using GraphPad Prism software ( version 4 ) . Mann-Whitney test was used to analyze the difference between two groups of unpaired data and Wilcoxon signed rank test for paired data . Fisher's exact test was used to compare the difference of proportions between two groups . Kruskal-Wallis test with the addition of Dunn test was used to analyze the difference between more than two independent groups . The associations between Tregs and IL-10 levels were analyzed using Pearson’s correlation analysis . The level of significance was set at 0 . 05 .
The summary of the enrolment and follow up of participants is depicted in Fig 2 . A total number of 179 pregnant women admitted to hospital for delivery from July 2009 to July 2011 were evaluated for inclusion in this study . Twenty one ( 11 . 7% ) of them was excluded because of complication during delivery or infant death or unwillingness . Finally 158 mother-new born pairs were enrolled for the study . At the time of enrolment 11 . 8% of the mother were microfilariae positive ( 3–210 per 60μl blood ) , whereas 44 . 5% of pregnant mothers were CFA positive ( GM: 1925 , range: 630–16596 ) . Interestingly , 24 . 5% of infected mothers have shown transplacental transfer of filarial antigen to their cord , while none of the cord blood from CFA negative mother was CFA positive . Similarly the cord blood of neither CFA +ve nor CFA–ve mother was positive for Mf . During the study period total 109 mother-child pairs have been dropped because they are either non traceable , decline to participate , death of the children , moved out of study area or non availability of immunological parameter . Finally 49 pregnant mothers and their subsequently born children have been followed up during 2014-15 . The mean duration of follow-up was 4 . 4 years ( range , 2–7 years ) . The characteristics of follow-up mothers and children have been described in Table 1 . Amongst 49 follow up mothers 28 were CFA positive and 21 were CFA negative at the time of recruitment . Of the total 28 CFA positive mothers , only 3 were Mf positive at the time of enrollment . All of the study participants were living in rural areas and majority of them ( 83 . 3% ) were house wives by occupation with primary level of school education ( 77 . 5% ) . Except filarial infection status , no difference was noticed in terms of age in years , multiparity status and educational level among the CFA +ve and CFA-ve mothers during follow-up . Amongst the CFA positive ( n = 28 ) follow-up mothers , 18 mothers are still harbouring filarial infection ( CFA +ve but Mf–ve ) without any clinical symptoms of filariasis , 4 mothers have cleared CFA but have developed acute symptoms of filariasis ( episodic attack of fever associated with inflammation of lymph nodes and lymphatics of legs/arms ) and 6 mothers have cleared CFA without development of any clinical symptoms of filariasis . Whereas none of the CFA negative mothers had acquired filarial infection or developed any clinical sign/symptoms of filariasis . Out of 28 children born to the infected mothers , 12 ( 42 . 8% ) children have acquired filarial infection and become CFA positive . In contrast one of the children ( 1/21 , 4 . 7% ) born to the uninfected mothers has acquired filarial infection and become CFA positive . ( OR = 15 , 95% CI: 1 . 75–127 . 9 , Z = 2 . 47 , p = 0 . 013 ) . Amongst the infected children 7 children were in the 2–4 years of age and 6 children were in 5–7 years of age . Out of the 12 CFA positive children 5 were from mothers who continued to be CFA positive where as 7 were from mothers those cleared CFA . While analyzing the infection status of cord of these 28 children it was observed that 21 . 4% ( 6/28 ) of them were CFA positive . Amongst those 6 cords positive children only 2 have become CFA positive during follow up . Statistically no significant difference ( p = 0 . 67 ) was observed in acquiring infection among children born from CFA +ve mothers having CFA +ve ( 2/6 , 33 . 3% ) and CFA-ve ( 10/22 , 45 . 4% ) cord at the time of delivery . Interestingly none of the cord from uninfected mother was CFA positive at the time of enrollment . Also none of the children born to either infected or uninfected mother have detectable microfilariae and/or with any clinical signs/symptoms of filariasis . Besides presence of CFA no difference was observed in age , gender in children born to infected and uninfected mother . Based on the presence/absence of CFA in mothers and children during follow-up , the children of CFA positive mothers have been divided into 4 sub-groups i . e . group I: both mother and child are CFA positive ( M+Ch+ , n = 5 ) , group II: mother positive but child negative for CFA ( M+Ch- , n = 13 ) , group III: mother negative but child positive ( M- Ch+ , n = 7 ) and group IV: both mother and child negative for CFA ( M- Ch- , n = 3 ) . The expression of Tregs in infected mother–cord pairs was significantly high as compared to mother-cord pairs of uninfected mother ( mother: p = 0 . 016 , cord: p<0 . 001 ) . Similarly Tregs cell expression was significantly high ( p < 0 . 0001 ) in children born to enrolled CFA positive group of mothers in comparison to children born to enrolled CFA negative group of mothers ( Fig 3A ) . Further we have observed a decreasing trend in the level of Tregs in children born to both infected and uninfected mother as compared to the cord blood ( p <0 . 0001 for CFA+ve and p < 0 . 0001 for CFA-ve ) . To evaluate the impact of maternal infection on development of Tregs in children during their early childhood , we have analyzed the Tregs in mothers as well as children born to two groups i . e . CFA positive and CFA negative group during follow up . Irrespective of the CFA status of mother at the time of follow-up , Tregs cells were significantly high ( p = 0 . 01 ) in mothers who were CFA positive at the time of enrollment compared to enrolled CFA negative mothers ( Fig 3B ) . But no significant difference ( p = 0 . 14 ) in Tregs cell expression was observed among mothers of four different subgroups belonging to CFA positive group . Whereas significantly high Tregs cell expression was observed between these four subgroups of mothers compared to CFA–ve group mothers ( M+Ch-vs M-Ch-: p<0 . 001 , M+Ch+ vs M-Ch-: p = 0 . 0008 , M-Ch+ vs M-Ch-:p = 0 . 0001 , M-Ch-vs M-Ch-:P = 0 . 01 ) . Children born to four sub-groups of CFA positive mothers showed significant ( p = 0 . 01 ) difference among themselves . Further , children born to these four subgroups of mothers had higher levels of Tregs expression than children born toM-Ch-of CFA -ve mother ( ( M+Ch-vsM-Ch–:p<0 . 0001 , M+Ch+ vs M-Ch-:p = 0 . 0008 , M-Ch+ vs M-Ch-: p = 0 . 0003 , M-Ch-Vs M-Ch-:p = 0 . 02 ) . On the other hand one of the children born to CFA–ve mother has acquired filarial infection during follow-up . with 0 . 15% of Tregs expression . We have quantitatively assessed the level of IL-10 , the hallmark cytokine for regulatory response , in plasma of cord blood as well as children born to infected and uninfected mothers to evaluate role differentiated T helper cell subsets in filarial infection . At the time of enrollment level of IL-10 was significantly higher in mother as well as cord blood of CFA positive mothers as compared to cord and mother of CFA -ve group ( mother: p<0 . 0001 , cord: p<0 . 0001 ) as shown in Fig 4A . Further a decreasing trend in level of IL-10 has been marked in children compared to cord ( p < 0 . 001 for CFA+ve and p = 0 . 007 for CFA-ve group ) . Similarly during follow up significantly higher level of IL-10 was observed in CFA +ve mother as well as their children in comparison to CFA–ve mothers and their children ( p<0 . 0001for mothers , p<0 . 0001 for children ) . However when the comparisons were made between the four subgroups of mothers as well as children belonging to the CFA +ve mothers , no significant difference was observed in IL-10 level ( p = 0 . 07 for mothers , p = 0 . 5 for children ) among them ( Fig 4B ) . But IL-10 level in the subgroup of enrolled CFA positive mothers was significantly higher compared to enrolled CFA negative mothers during follow up ( M+Ch- vs M-Ch–: p = 0 . 002 , M+Ch+ vs . M-Ch- p:0 . 002 , M-Ch+ vs . M-Ch-: P = 0 . 013 , M-Ch-vs . M-Ch-: P = 0 . 017 ) . More than that IL-10 level was significantly higher in children born to all four sub-groups of CFA positive mothers than born to CFA negative mother as evident in Fig 4B ( M+Ch- vs M-Ch–: p = 0 . 001 , M+Ch+ vs M-Ch-: p = 0 . 002 , M-Ch+ vs M-Ch-: p = 0 . 002 , M-Ch- vs M-Ch- p = 0 . 03 ) . One child born to CFA–ve mothers acquired infection and having IL-10 level of 9 pg/ml . To find out the effect of Tregs cells on IL-10 secretion in infected and uninfected mother as well as their children , a correlation was made between percentage of CD4+CD25hi cell expressions and IL-10 level during the follow up . As shown in Fig 5A and 5B , no significant correlation was observed between Treg and IL-10 of enrolled CFA+ve ( p = 0 . 38 , r2 = 0 . 029 ) and CFA–ve mother ( p = 0 . 91 , r2 = 0 . 012 ) during follow up . However when we differentiate the CFA+ve and CFA-ve group of enrolled CFA+ve mother during follow-up , a highly significant positive correlation was observed among the CFA +ve mothers ( p = 0 . 0008 , r2 = 0 . 518; Fig 5C ) in contrast to CFA-ve mothers ( p = 0 . 47 , r2 = 0 . 066 , Fig 5D ) . From Fig 6A , it is evident that a significant positive correlation ( p< 0 . 0001 , r2 = 0 . 6987 ) exists between IL-10 level and Tregs in children of infected mothers . In contrast no correlation was marked between IL-10 level and Tregs in children born to CFA negative mothers ( Fig 6B , p = 0 . 8541 , r2 = 0 . 001 ) . Moreover there was no difference in Treg and IL-10 correlation between CFA+ve and CFA-ve children born to enrolled CFA+ve mothers ( CFA+ve: p = 0 . 01 , r2 = 0 . 445; CFA-ve: p<0 . 001 , r2 = 0 . 7732 ) as shown in Fig 6C and 6D ) .
The current study reveals that maternal W bancrofti infection during pregnancy up- regulates the production of Tregs and IL10 in offspring from infancy to early childhood and children born to infected mothers are at greater risk of acquiring filarial infection than children born to uninfected mothers . Further , evaluation of cord blood response and their correlation with infection status of children born to infected mothers suggests that in-utero sensitization rather than transplacental transfer of filarial antigen leads to increased susceptibility to filarial infection after birth . Presence of parasitic infections during pregnancy is known to influence the immune system of an unborn child directly , through transfer of parasites or antigens across the placenta . As a consequence the neonates born to the infected mother become more susceptible to infection [2 , 13 , 14] . We have observed filarial antigen in 21 . 4% ( 6/28 ) of the cord blood of infected mothers but 42 . 8% of children born to them have acquired filarial infection which is double the figure of antigenemia of cord blood . This finding is supported by the previous work which showed that prenatal filarial specific immune tolerance as a consequence of active maternal filariasis increased the risk of infection during the first 7 years after birth [15] . Further , out of the 6 CFA positive cord only two children have acquired infection during follow up indicating no significant difference in acquiring infection between children born with CFA positive and CFA negative cord ( from CFA positive mother ) . Therefore , it is speculated that early priming due to in-utero exposure rather than the transplacental transfer of filarial antigen is playing the key role towards disease susceptibility . Conversely , children born to infected mother but have not acquired infection during the childhood even though living in same endemic area may be due to heterogeneity in exposure to infective larvae , co-infection with malaria and geo-helminths that could bias T cell cytokine response and differences in genetic makeup [15] . Initially it was thought that filarial infections profoundly suppress the T-cell-proliferative and IFN-γ responses because of Th2 bias . Though these infections undoubtedly elicit Th2 cells , but recent studies show expansion of regulatory T-cell population while maintaining the hyporesponsive state in filariasis [16] . Epidemiological evidence suggests that in-utero sensitization results down-regulated responses among the offspring , on encountering the homologous antigen which may be due to bias in the fetal and neonatal immune response towards the development T regulatory cell . Moreover filaria-associated Tregs has been demonstrated to modulate the T and B cell proliferation and polarized cytokine production by effector T cells in microfilaraemics [7 , 14] . In the present study , significantly high level of IL-10 and Tregs cell from infancy to early childhood signifies their role towards the disease susceptibility . Recently some other studies have revealed that modulated/ regulated T cell responses associated with patent filarial infection reflects expansion of Tregs that include both Tregs induced in peripheral circulation and the thymus-derived Tregs [11 , 17] . Further filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep Th1 and Th17 in check [18] . In the present study significantly high expression of Tregs in children born to infected mothers is at par with the findings of others and the high level of CD4+CD25hi cells in cord blood of infected mothers supports our hypothesis that regulation of Tregs cells start from the time of early priming during pregnancy . Contribution of such regulatory network towards hyporesponsiveness has been well documented during an in vitro study in newborns of filaria infected mothers [19] . Further , studies have shown that in utero stimulation with helminth-derived antigens divert fetal immunity towards Th2 responses and/or lead to anergy or tolerance [20 , 21] . Since Treg cells produce regulatory cytokine IL-10 that modulates the entire repertoire ( Th1/Th2/Th17 ) of CD4+ effector cell responses indiscriminately in filariasis that limits the Th1 response [22 , 23] , we have evaluated the level of IL-10 in two groups of children to draw the functional relationship with Tregs . In our earlier study , cord blood from filarial infected mother exhibited decreased production of IFN-γ ( Th1 ) response and increased production of IL-10 ( Th2 ) indicating that immune responses have already been skewed towards Th2 type of response at the time of birth [24] . In addition the high level of T- regulatory cells and increased production of IL-10 in cord blood of infected mothers could down regulate inflammatory responses and create a susceptible environment for the parasite to grow . Similar to our observation , in a cross sectional study conducted in Kenya by Malhotra and others have found that maternal filarial infection increases childhood susceptibility to W . bancrofti and skews filaria-specific immunity toward a Th2-type cytokine response [25] . In the present study significantly high level of IL-10 in children born to infected mothers in comparison to children born to uninfected mother emphasizes that in utero sensitization down regulate the immune response in children since the time of birth . Though some of the children born to infected mother are free from infection during follow up yet they maintain high level of Treg and IL-10 which is in agreement with the previous work that shows that helminth-specific T cell immunity acquired in utero is maintained until at least 10 to14 months of age in the absence of infection [26] . In case of CFA-ve group of mothers who are still CFA-ve within the follow-up groups could be then classified as endemic normal as there is no record of infection prior to the survey . The low level of IL-10 in this group suggests that endemic normals have specific immune profile preventing filarial infection . Though the focus on this immunomodulation during helminth infections has been on IL-10 , yet contributions of natural T regulatory cells ( nTregs ) appear to be significant [27 , 28] in this context of our study . A recent study in India has shown that frequencies of regulatory T cell markers were higher in asymptomatic microfilaremics and/or circulating filarial antigen positive subjects than in patients with chronic pathology . It also suggests a more prominent regulatory role of IL-10 producing Tregs [7 , 17] . Though in the present study it was not possible to measure the IL-10 producing Tregs still a strong association of Tregs and IL-10 was observed in children born to filarial infected mother during their early childhood . It was also observed that expression of Treg and production of IL-10 in CFA+ve and CFA-ve children born to CFA+ve mothers do not differ from each other . This indicates that in utero priming determines the Treg and IL-10 production independent of acquisition of infection in later life . These findings supports the notion that immunologic memory established by priming of prenatal T cells with antigens that pregnant women encounters the infection that persists from gestation to childhood . This might be the cause of high incidence of infection among the younger age children ( 2–4 years old ) in this cohort as observed by others [3 , 15 , 25 , 26] . From this we can speculate that increased level of Tregs and high production of IL-10 initiates a cascade of hyporesponsive mechanism in children from the time of birth that down regulates the inflammatory responses and lead to a Th2 type of response so as to make them susceptible for parasite survival and ultimately determines the disease outcome in children . The obvious limitation of our study is small sample size corresponding to both children born to infected and uninfected mothers . Albeit by drawing correlation we can interpret that Tregs in offspring from filarial infected mothers influence the IL-10 production as described in adults . When analyzing regulatory T cells , the measurement of transcription factor FoxP3 , CD49b and LAG-3 markers for Treg and Tr1 cell population and intracellular FACS antibodies such as IL-10 in IL-10-producing Tregs were not possible due to poor resource which might have been useful to analyze the functional relationships between their number and mechanism of action .
In conclusion we can state that maternal filarial infection during pregnancy increases the susceptibility of children to infection by immune priming through expression of Tregs as well as regulatory cytokine IL-10 . The high incidence of infection among the younger children even after 10 rounds ( 2014 ) of MDA in this area is due to high rate of Mf among pregnant women during 2009 . While the cause of high Mf rate among the pregnant women might be due to low compliance because of social customs or back to back pregnancy . Hence the present findings relates to a greater impact on mass treatment programs aimed at elimination of transmission of W bancofti infection . To prevent the prenatal immune priming and tolerance supervised therapy can be introduced at the child bearing age of the women , so that they can be free from infection by the time of pregnancy and , thus , decrease the risk of infection during childhood . Implementation of such strategy will help the programme in achieving the target of global elimination of LF by 2020 .
|
Lymphatic filariasis caused by thread like filarial worms involves asymptomatic to acute and/or disfiguring chronic conditions like lymphoedema , elephantiasis and scrotal swelling . Infection occurs when filarial parasites are transmitted to humans through mosquitoes . Adult worms lodge in the lymphatic system and disrupt the immune system that causes the disease . Nonetheless the infection if present during pregnancy , it affects the immune system of the unborn child in such a way that they become more susceptible to infection . But how the immune system of a fetus is affected by the maternal filarial infection is not known . Since regulatory T cells are responsible for development of hyporesponsiveness , a condition that supports the active filarial infection , and develops in thymus at an early stage of the human fetal development , we hypothesized that maternal filarial infection might be affecting the development of Tregs cell . Because Tregs secret IL-10 , a regulatory cytokine , we have also measured its level in children born to infected and uninfected mother and correlate it with Tregs . We have observed a significantly high as well as a positive correlation between Tregs and IL-10 levels in children born to infected mother than the children of uninfected mother at the time of birth as well as early childhood indicating that Tregs and IL-10 contribute to immune modulation during pregnancy . Since ongoing MDA excludes pregnant mothers and children below 2 years of age , hence implementation of supervised therapy at the time of adolescent through MDA may help the programme in achieving the target of global elimination of LF by 2020 .
|
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2016
|
Maternal Filarial Infection Influences the Development of Regulatory T Cells in Children from Infancy to Early Childhood
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This report describes a real-time PCR ( Q-PCR ) strategy to quantify Trypanosoma cruzi ( T . cruzi ) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence . The Q-PCR has a detection limit of 0 . 1 and 0 . 01 parasites/mL , with a dynamic range of 106 and 107 for Silvio X10 cl1 ( T . cruzi I ) and Cl Brener stocks ( T . cruzi IIe ) , respectively , an efficiency of 99% , and a coefficient of determination ( R2 ) of 0 . 998 . In order to express accurately the parasitic loads: ( 1 ) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; ( 2 ) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and ( 3 ) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol ( Lg-PCR ) . The Q-PCR strategy was applied ( 1 ) to estimate basal parasite loads in 43 pediatric Chagas disease patients , ( 2 ) to follow-up 38 of them receiving treatment with benznidazole , and ( 3 ) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression . All together , the high analytical sensitivity of the Q-PCR strategy , the low levels of intra- and inter-assay variations , as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients .
Infection with the parasite Trypanosoma cruzi ( T . cruzi ) remains a major concern in 21 endemic countries of America , with an estimated prevalence of almost 8 million infected people [1] . The infection may be acquired mainly through the triatomid insect vector , blood transfusion or the trans-placental route . Furthermore , in areas under vector control , cases of congenital and transfusional transmission are relatively emerging [2] . This parasitic disease shows a variable clinical course , which ranges from asymptomatic cases , to severe chronic stages characterized by low parasitaemia and cardiac and/or gastrointestinal disorders [1] . Individuals from different endemic regions are infected with distinct parasite populations that may play a role in pathogenesis , clinical forms and severity of the disease [3] . Parasite populations are classified into two main phylogenetic lineages , T . cruzi I ( TcI ) and T . cruzi II ( TcII ) [4]; the later composed by five subdivisions designated as TcIIa to TcIIe [5] . Current chemotherapies based on the nitrofuran nifurtimox , and the nitroimidazole benznidazole , are unsatisfactory since these compounds are almost exclusively effective in recent infections and frequently have toxic side effects [6] . In this context , the development of novel drugs is necessary [6] . After etiological treatment ( tmt ) , the criterion of cure relies on serological conversion to negative of the anti- T . cruzi antibody response [2] , but in patients initiating therapy at the indeterminate phase , seroconversion usually occurs several years after treatment , requiring long-term follow-up [7] . Moreover , parasitological response to treatment is usually monitored by means of traditional methods such as Strout , hemoculture or xenodiagnoses , which lack sensitivity , and therefore are also inadequate for these purposes [2] . In this context , quantitative real-time PCR ( Q-PCR ) has the potential to become a novel parasitological tool for prompt evaluation of trypanocidal treatment . As a target for amplification , the nuclear satellite DNA , represented in 104 to 105 copies in the parasite genome is highly conserved [8]–[11] and therefore may provide accurate Q-PCR based measurements . Proper Q-PCR performance also requires high quality DNA extraction procedures from blood samples , which in most cases are collected in guanidine hydrochloride and EDTA buffer ( GEB ) [12] . The co-purification of trace PCR inhibitors may not impede amplification but may reduce its efficiency resulting in erroneous quantification of the parasitic load . Accordingly , we aimed to develop a satellite-DNA based Q-PCR strategy for accurate quantification of T . cruzi loads in peripheral blood samples along with an adequate DNA extraction protocol . The following features have been regarded: Finally , we applied this Q-PCR strategy to: ( 1 ) calculate the basal T . cruzi loads in blood specimens collected from Chagas disease pediatric patients , ( 2 ) follow-up their parasitological response to treatment with benznidazole , and ( 3 ) monitor T . cruzi recrudescence and parasitological response to treatment in chronic Chagas heart disease patients undergoing heart-transplantation and receiving immunosuppressive therapy .
This study was conducted according to the principles expressed in the Declaration of Helsinki . The study was approved by the Institutional Review Boards of the “Ricardo Gutierrez” Children's Hospital and of the Fundacion “Rene Favaloro” . All patients or responsible adults provided written informed consent for the collection of samples and subsequent analysis . T . cruzi epimastigotes were grown in liver infusion tryptose ( LIT ) medium containing 10% calf serum at 27–28°C . The parasites were harvested and stored at −70°C . T . cruzi DNA was purified after Phenol-Chloroform extraction and ethanol precipitation . Reference T . cruzi stocks used as controls were: TcI ( Silvio X10 cl1 , SN3 , HA , Pal V2-2 , Pav 00 , G ) ; TcIIa ( CanIII ) ; TcIIb: ( Tu18 , JG , Gilmar , Y , Basileu , Mas cl1 ) ; TcIIc: ( M5631 , Cu-Tom-229 , Cu-Yaya-211 ) , TcIId ( Mn cl2 , Bug 2148 cl1 , SO3 cl5 , PAH 265 , Tev 41 ) , TcIIe ( Cl Brener , Tul 77 , Tep 6 , Tep 7 , MC ) . They were kindly provided by Patricio Diosque ( Universidad Nacional de Salta , Salta , Argentina ) , Andrea M . Macedo ( University Federal of Minas Gerais , Belo Horizonte , Brasil ) , and Michel Tibayrenc ( UR62 “Genetics of Infectious Diseases” , IRD Centre , Montpellier , France ) . Some T . cruzi I strains were provided by Omar Triana Chavez ( University of Antioquia , Medellín , Colombia ) . Argentinean T . cruzi IIc isolates were provided by Ricardo Gurtler ( Universidad de Buenos Aires , Argentina ) . All these protocols were approved by the ethical committees of the corresponding institutions and written informed consents were required from each patient or a responsible adult . Ten mL or 2 mL blood samples collected from T . cruzi infected adults or infants , respectively , were immediately mixed with one volume of 2× lysis buffer containing 6 M guanidine hydrochloride ( Sigma , St Louis , USA ) and 200 mM EDTA , pH 8 . 0 ( GE ) [12] . QIAmp DNA Mini Kit ( Qiagen , Valencia , CA ) based extraction was carried out from 400 µl of GEB and eluted with 200 µl of water according to the manufacturer's instructions using the Blood and Body Fluid Spin Protocol , with slight modifications . Briefly , since blood samples were initially mixed with one volume of GE lysis buffer , treatment with proteinase K and “AL” lysis buffer ( which contains guanidine hydrocloride ) were omitted . The following steps were carried out following the manufacturer's instructions . Phenol-chloroform based DNA extraction was carried out from 100 µl of GEB aliquots and resuspended in 50 µl water as reported [15] . Cl Brener epimastigotes were added to non-infected human blood to result in a concentration of 105 p/mL of reconstituted blood and immediately mixed with one volume of 2× lysis GE buffer . The resulting GEB was serially diluted 10-fold with non-infected GEB to cover a range between 105 and 0 . 001 parasite equivalents/mL . Total DNA was purified using the QiAmp DNA Mini Kit based extraction method , as above described . An MJR-Opticon II device ( Promega , USA ) was used for amplification and detection . The 20 µL reaction tube contained 0 . 5 µM of novel primers Sat Fw ( 5′-GCAGTCGGCKGATCGTTTTCG-3′ ) and Sat Rv ( 5′-TTCAGRGTTGTTTGGTGTCCAGTG-3′ ) , 3 mM MgCl2 , 250 µM of each dNTP , 0 . 5 U of Platinum Taq polymerase , ( Invitrogen , Life Technologies , USA ) SYBR Green ( Invitrogen , Life Technologies , USA ) at a final concentration of 0 . 5× and 2 µL of sample DNA . After 5 min of pre-incubation at 95°C , PCR amplification was carried out for 40 cycles ( 94°C for 10 s , 65°C for 10 s and 72°C for 10 s ) . The plate was read at 72°C at the end of each cycle . A linearized p-ZErO plasmid containing a sequence of Arabidopsis thaliana was used as a heterologous internal standard ( IS ) . All clinical samples were co-extracted with 200 pg of recombinant plasmid , which was assumed as 1 arbitrary unit ( AU ) of IS . This amount of IS input was chosen because the amplicons are detected at approximately the Cycle threshold ( Ct ) number 20 , the mid point of the dynamic range of the PCR . For each Q-PCR test , the IS was added to 400 µl of GEB lysate , immediately before the DNA extraction procedure . The standard calibration curve for the IS was carried out using the same reconstituted blood samples as for the T . cruzi calibration curve: those samples containing 105 , 104 and 103 p/mL were spiked with 2 AU of IS , those containing 100 , 10 and 1 p/mL were spiked with 0 . 2 AU of IS , and those containing 0 . 1 , 0 . 01 and 0 . 001 p/mL were spiked with 0 . 02 AU of IS . Two non-infected blood samples with and without IS DNA were used as negative controls of the extraction procedure . The IS was quantified using 1 µM of primers , IS Fw ( 5′-AACCGTCATG GAACAGCACGTAC-3′ ) and IS Rv ( 5′-CTAGAACATTGGCTCCCGCAACA-3′ ) . All other PCR reagents and cycling conditions were identical to those used for T . cruzi Q-PCR . Post-treatment follow-up of parasitological response to benznidazole in pediatric and heart transplanted patients was conducted by means of kDNA-PCR as previously reported [14] , [16] . In order to analyze the variability in the number of satellite sequences detected by Q-PCR , comparative quantification was performed using as a normalizer the single copy ribosomal protein P2α gene ( GenBank accession number XM_800089 ) . Assays for quantification of P2α gene were performed using 1 µM of primers P2α Fw ( 5′-ATGTCCATGAAGTACCTCGCC-3′ ) and P2α Rv ( 5′-GCGAATTCTTACGCGCCCTCCGCCACG-3′ ) . All other PCR reagents were used at the same concentrations as for T . cruzi Q-PCR . After 5 min of pre-incubation at 95°C , PCR amplification was carried out for 40 cycles ( 94°C for 10 s , 60°C for 15 s and 72°C for 10 s ) . The plate was read at 72°C at the end of each cycle . Since TcI and TcIIa parasites have a lower number of satellite sequences than TcIIb/c/d/e parasites , we have developed a method to distinguish between both groups according to the melting temperatures ( Tm ) of their corresponding amplicons to enable more precise parasitic load assessments . The identification of the type of satellite sequence was performed using 0 . 5 µM of primers TcZ1 ( 5′-CGAGCTCTTGCCCACACGGGTGCT-3′ ) and Sat Rv ( 5′-TTCAGRGTTGTTTGGTGTCC AGTG-3′ ) . All other PCR reagents were used at the same concentrations as for T . cruzi Q-PCR . The PCR conditions consisted of an initial denaturation at 95°C for 5 min , followed by 40 cycles of 94°C for 10 s , 65°C for 10 s and 72°C for 10 s with fluorescence acquisition at 81 . 5°C and a final step of 2 min at 72°C . Amplification was immediately followed by a melt program with an initial denaturation of 5 s at 95°C and then a stepwise temperature increase of 0 . 1°C /s from 72–90°C . Since satellite DNA is arranged in tandem repeats , if more than 0 . 05 parasites ( equivalent to approximately 10 p/mL ) are loaded in the reaction tube , a satellite sequence dimer is amplified giving place to a melting temperature peak typically above 86°C for both lineage groups . Satellite sequences were obtained by direct sequencing of satellite DNA amplicons obtained with TcZ1 and TcZ2 primers ( GenBank Accession numbers EU728662-EU728667 ) . Sequence alignment was conducted using MEGA version 4 [17] . The parasite load in the clinical sample was normalized respect to the standard curve according to ( 1 ) the efficiency of the DNA extraction procedure measured by the amplification of the IS , and ( 2 ) the parasite lineage group . The following equation was used: Np/mL = ( Np/well×LF / AU ) / V , where Np/mL is the number of parasites per millilitre of blood , Np/well is the number of parasites per well , LF is the lineage factor , AU are the arbitrary units of IS quantified and V is the volume of extracted DNA sample used per reaction .
The analytical sensitivity of the Q-PCR was tested by using serial dilutions of purified T . cruzi DNAs from TcI ( Silvio X10 cl1 ) and TcIIe ( Cl Brener ) reference stocks . The detection limits were 2 fg and 0 . 2 fg DNA per reaction tube with a dynamic range of 107 and 108 for Silvio X10 cl1 and Cl Brener stocks , respectively ( data not shown ) . These detection limits correspond to 0 . 01 and 0 . 001 parasite genomic equivalents considering that one parasite cell harbors approximately 200 fg of DNA . Furthermore , we tested the operational parameters of Q-PCR in reconstituted - blood samples spiked with known quantities of Cl Brener and Silvio X10 cl1 cultured epimastigote cells . The dynamic range of Q-PCR performed with samples reconstituted with Silvio X-10 was 0 . 1–105 p/mL and with those spiked with Cl Brener was 0 . 01–105 p/mL ( Figure 1 ) . The reproducibility of the Q-PCR assay at 100 p/mL and 1 p/mL was estimated by testing each reconstituted sample 15 times in the same PCR run . The coefficients of variation of the Ct values were 1 . 27% and 2 . 30% , respectively for Cl Brener and 1 . 60% and 5 . 42% , respectively for Silvio X10 cl1 . The reproducibility of the DNA extraction was also characterised: aliquots from the same GEB-reconstituted samples containing 10 Cl Brener p/mL were processed in twenty independent DNA purification experiments . For each of the 20 DNA lysates , the p/mL were measured in triplicate PCR runs and a mean value was calculated . The coefficient of variation of the Ct values among the 20 mean values was 1 . 69% . In order to evaluate if the differences in the detection limits of the Q-PCR obtained from Cl Brener or Silvio X10 cl1 samples were related to a different copy number of satellite repeats in their respective genomes , the amounts of satellite sequences , relative to the single copy gene encoding the ribosomal-P2α protein , were estimated for parasite stocks belonging to the 6 lineages ( Table 1 ) . The stocks belonging to TcIIb/d/e lineages showed similar amounts of satellite repeats ( Table 1A ) , the M5631 stock ( TcIIc ) harbored 2-fold less repeats than the aforementioned stocks ( Table 1A ) , whereas Can III stock ( TcIIa ) and six stocks belonging to T . cruzi I from Argentina , Colombia and Brazil , harbored a 10-fold lower number of satellite repeats ( Table 1A and 1B ) . This is in agreement with the analytical sensitivities obtained with purified DNA and reconstituted samples of the reference stocks . Two groups of lineages , T . cruzi I/IIa ( group I ) and T . cruzi II ( IIb , c , d , e ) ( group II ) were clearly distinguished due to the differential melting temperatures of their corresponding satellite sequence amplicons , above or below 85°C , respectively ( Figure 2 ) . The Lg-PCR was validated using a panel of 25 characterised stocks ( Table 2 ) : five stocks belonging to TcI , one to TcIIa , six to TcIIb , three to TcIIc , five to TcIId and the remaining five to TcIIe . The 99% confidence interval of the melting temperatures depicted by the T . cruzi strains from group I ( mean: 85 . 5°C; CI 99% 85 . 2–85 . 8 ) and II ( mean: 84 . 4°C; CI 99% 84 . 2–84 . 7 ) did not overlap . This distinction allows a more accurate parasite load quantification by incorporating a correction factor ( Lineage Factor LF ) , according to the number of target sequences , when the parasitic load is calculated . We extracted 55 GEB samples from Chagas disease patients with the QIAmp DNA Mini Kit adapted protocol as well as with a Ph-Chl based method . Using the Ph-Chl based method , we detected traces of PCR inhibitors in 16 ( 29% ) samples , whereas for the commercial kit we did not detect PCR inhibitors in any of the samples . The presence of PCR inhibitors was assessed by ( 1 ) the low yield of IS amplification , giving rise to less than 0 . 1 AU of the IS and ( 2 ) the improved yield of IS amplification when the DNA lysate was diluted 1/100 prior to the Q-PCR run . Out of the 16 samples that presented traces of PCR inhibitors when extracted with the Ph-Chl based method , 4 were negative for T . cruzi by both extraction methods , 3 rendered similar parasite loads by both methods , 8 rendered higher parasite loads when extracted with the commercial kit , and one rendered a positive result only when extracted with the commercial kit . For the 39 samples extracted by Ph:Chl that did not contain PCR inhibitors , similar results of T . cruzi quantification were obtained with both methods of extraction . On the basis of these data , the QIAmp DNA Mini Kit extraction protocol was selected for Q-PCR in clinical samples . A panel of human blood samples was analyzed using the Q-PCR strategy , namely samples from seropositive pediatric patients and chronic Chagas heart disease adults presenting clinical reactivation after heart transplantation . Table 3 describes the necessary steps for calculating the parasitic loads , considering the lineage factor ( LF ) , the AU of the IS and the input volume of DNA sample . For example , in case Tx1c , the Q-PCR alone quantified 0 . 39 p/mL , but the AU of the IS was 0 . 43 , and the LF was 10 ( T . cruzi I ) , giving a final result of 9 . 07 p/mL , which is 23 times higher than if the quantification had been made based only on the Q-PCR crude measurement . In case Pd6 , a pediatric patient with very low parasitemia , the lineage could not be determined since the low concentration of amplicons gave rise to non-reliable melting temperature peaks and therefore the parasitic load could only be expressed within a range of 10-fold . In cases Tx1a and Tx2c , also with low levels of parasitemia , the lineage was presumed from previous data obtained from genotyped samples of the same patients , because it was demonstrated that the T . cruzi lineages were persistent during recrudescence leading to clinical reactivation in these patients [14] . Cases Pd2a and Tx2b presented high parasitic loads , 512 and 468 p/mL , respectively . In both of them , dimers of the satellite repeats were preferentially amplified when Lg-PCR was performed , giving melting temperatures above 86°C ( Table 3 ) . Thus , when the non-corrected Q-PCR results are above 10 p/mL , the DNA lysate should be diluted to approximately 1 p/mL before performing Lg-PCR . The reproducibility of the whole Q-PCR assay was evaluated in 5 clinical samples from different Chagas disease patients covering a range of 3 logarithms of parasitic loads ( 0 . 06 to 70 . 29 p/mL , Table 4 ) . For each peripheral blood sample ( A to E , Table 4 ) , the entire protocol was carried out in 5 independent replicates . The coefficients of variation of the Ct values and p/mL for the T . cruzi Q-PCRs ranged from 1 . 30 to 5 . 49% and from 25 . 19 to 137 , 95% , respectively , and those for the IS-PCRs ranged from 0 . 66 to 2 . 28% and from 9 . 56 to 29 . 51% , respectively ( Table 4 ) . The final parasitic load measurements were inversely correlated with their coefficients of variation , which ranged from 32 . 43 to 114 . 20% ( Table 4 ) . The pre-treatment parasitic loads were assessed in 43 children with Chagas disease . Basal parasitic loads ranged from 640 to 0 . 01 p/mL , and were correlated to the patients' ages at the time of diagnosis ( coefficient of Spearman: −0 . 5832 , P<0 . 05 ) ( Figure 3A ) . Thirty eight of these patients were monitored by Q-PCR during 60 days of etiological treatment with benznidazole . Parasitic loads were determined at time of diagnosis ( t1 ) in 38 cases , at 7 ( t2 ) , 30 ( t3 ) and 60 ( t4 ) days of treatment in 31 cases . Figure 3B and Table 3 show the parasitic response of these patients during treatment monitoring . Q-PCR results at t2 were negative in 24 out of 31 patients ( 77% ) , at t3 in 27 out of 31 patients ( 87% ) and at t4 in 29 out of 31 patients ( 94% ) . One of the Q-PCR positive patients at t4 was a 7 year-old boy whose parasite load declined from 8 . 28 p/mL at t1 ( Pd1a ) to 0 . 1 p/mL at t2 ( Pd1b ) , relapsing to 0 . 63 p/mL at t3 ( Pd1c ) and 1 . 16 p/mL at t4 ( Pd1d ) ( Figure 3B , red triangle , Table 3 ) . The other Q-PCR positive patient at t4 was a 3 month old infant who displayed detectable parasitic loads in the three analysed samples; 512 p/mL at t1 ( Pd2a ) , 0 . 43 p/mL at t3 ( Pd2b ) and 0 . 75 p/mL at t4 ( Pd2c ) ( Figure 3B , green triangle , Table 3 ) . Both patients were followed-up by kDNA-PCR with persistently positive results at 6 , 12 and 18 months post-tmt suggesting treatment failure ( data not shown ) . Stored peripheral blood samples from three heart transplanted patients infected with different parasite lineages and presenting different patterns of clinical reactivation were retrospectively analyzed using the Q-PCR strategy ( Figure 4 ) . Case Tx1 presented positive Q-PCR results ( 0 . 22 p/mL ) prior to heart transplantation . Clinical reactivation was diagnosed at day 78 post-Tx by a skin chagoma biopsy and a positive Strout result . The parasite population was characterized as group I by Lg-PCR . Case Tx2 did not present positive PCR results before heart Tx , but parasitemia became detectable 7 days post-Tx ( 2 . 66 p/mL ) . Clinical reactivation was diagnosed at 92 days post-Tx due to positive Strout findings . Upon etiological tmt , the parasitic load decreased reaching undetectable levels at 21 days post-tmt . The T . cruzi population was identified as group II . Case Tx3 presented a parasite load of 0 . 02 p/mL 30 days before Tx , showing a 10-fold increase ( 0 . 2 p/mL ) 7 days after Tx . The parasite load continued to rise until clinical reactivation was diagnosed 38 days after Tx based on positive Strout findings . Accordingly , the patient was treated with benznidazole and the parasite load dropped rapidly , with negative Q-PCR findings at 14 days post-tmt , persisting PCR negative for 53 days . However , 70 days after tmt Q-PCR monitoring revealed detectable loads ( 0 . 01 p/mL ) ( Figure 4 ) . The bloodstream T . cruzi population was identified as group II .
Herein we report a highly sensitive , reproducible , accurate and rapid real-time Q-PCR strategy for quantification of the T . cruzi parasitic load in human blood samples . Indeed , this is a first attempt to develop a real-time Q-PCR strategy for reliable T . cruzi quantification , because it incorporates: ( 1 ) a commercial kit for sample processing which minimizes carry-over of PCR inhibitors and standardizes the yield and quality of DNA extraction , ( 2 ) a closed-tube single-round PCR reaction which minimizes carry-over contamination , ( 3 ) an appropriate internal quality control and ( 4 ) a correction of the parasitic load , according to the variation in the number of target sequences between the different lineage groups . This is a key step towards the standardization and validation of in-house Q-PCR tests for application to routine laboratory practice . Although studies showing the advantages of real-time PCR for screening and quantification of T . cruzi have recently appeared , none have implemented procedures to normalize the DNA extraction yield and the representativity of the PCR target according to the lineage group . Some studies [18]–[20] have chosen as an internal control a host DNA sequence , but the human DNA content in blood can be highly variable , especially in immunosupressed patients . In this work , the addition of a standardized amount of a plasmid containing a heterologous sequence , allows normalization of the DNA extraction yields and detection of false negatives due to inhibition under any clinical situation . Regarding the selected molecular target of amplification , Elias et al . [8] , [11] and Vargas et al . [10] demonstrated that satellite DNA is 4 to 9 times more abundant in TcIIb/d/e than in TcI stocks . Herein , we have extended this analysis to all 6 T . cruzi lineages , describing for the first time the satellite repeats of T . cruzi IIa , IIc and IId representative stocks ( GenBank Accession numbers EU728662-EU728667 ) . Indeed , we detected a 5 to 10-fold variation in the satellite DNA content between group I ( TcI/IIa ) and group II ( TcIIb/c/d/e ) parasite stocks ( Table 1 ) . Therefore , this variability must be taken into account in order to calculate accurately the parasitic loads . Accordingly , we have also designed a highly sensitive real-time PCR procedure ( Lg-PCR ) to distinguish T . cruzi group I ( with lower satellite sequence copy number and higher melting temperature ) from group II lineages ( with higher satellite sequences copy number and lower melting temperature ) . All the analyzed stocks rendered only one temperature melting peak , including hybrid stocks like Cl Brener although harboring both types of satellite sequences . This can be explained by the fact that Cl Brener ( Tc IIe ) harbors ten times more type II than type I satellite repeats [10] and due to exponential amplification , only the predominant sequence type is detected . The high analytical sensitivity of Lg-PCR makes it useful for direct lineage group characterization in biological samples that may not be typed using other typing methods [16] , [21] , [22] . Alternatively , a recently reported multiplex PCR strategy might be useful for typing T . cruzi groups in clinical specimens [23] when a Q-PCR test targeted to satellite sequences is carried out . Sample processing and DNA extraction must also be optimized for reliable quantification . In this direction , we adapted a commercial kit , based on silica-membrane technology ( QIAmp DNA Mini Kit ) for processing GEB samples . Phenol-chloroform DNA extraction is a cost-effective method for qualitative PCR , but traces of PCR inhibitors may be co-purified [24] , [25] . These interfering substances may not impede Q-PCR amplification , but could affect its efficiency leading to inaccurate results . In fact , we detected traces of PCR inhibitors in 29% of the samples extracted with Ph-Chl . When present , these inhibitors underestimated the parasite loads in 67% of the positive samples , although they did not seriously affect the positivity of the PCR . These results suggest that Ph-Chl based extraction of GEB samples is not suitable for Q-PCR but can be used for qualitative purposes . In this report , we applied the Q-PCR strategy to blood samples collected from patients under different clinical scenarios . Its wide dynamic range allowed direct measurements in cases with high parasitic loads such as immunosuppressed Chagas disease patients and congenitally infected newborns , as well as in cases with low parasitemias , such as patients at the indeterminate phase or under etiological treatment . In this sense , the coefficients of variation of the Q-PCR measurements obtained from clinical samples were similar to those obtained from reconstituted blood samples ( Table 4 ) . When applied to newborns , infants and children with T . cruzi infection , Q-PCR estimated their basal parasitic loads in a vast range , between 0 . 01 and 640 p/mL of blood ( Figure 3A ) . The highest parasitic loads observed in the younger pediatric population are in agreement with the results obtained by conventional parasitological and kDNA-PCR analysis [15] , [26] . Moreover , the lower parasitic loads detected in the older pediatric patients reflect their evolution to the indeterminate phase of congenital infection [15] . Furthermore , we were able to follow-up their parasitological responses to treatment with benznidazole ( Figure 3B ) with a favorable outcome in 94 . 7% ( 36/38 ) cases . It is worth to note that at t3 , under 30 days of tmt , 4 patients still showed detectable parasitic loads . At the end of tmt ( t4 ) 2 of them became Q-PCR negative and remained negative during 18 months of post-tmt follow-up . This observation emphasizes the importance of a treatment regimen of 60 days . Interviews with the mothers of the two patients who persisted Q-PCR positive at t4 revealed the non-adherence in one of them ( Pd2 , Table 3 and Figure 3B , green triangle ) , whereas in the other case ( Pd1 , Table 3 and Figure 3B , red triangle ) persistence of parasitemia indicated lack of parasitological response to benznidazole . These cases demonstrate the usefulness of the Q-PCR assay as surrogate marker for early detection of treatment failure . Two pediatric patients ( Figure 3B , pink and yellow squares ) presented an increase of their parasitic loads from t1 to t2 , showing a favorable parasitological response to treatment in the samples collected at t3 and t4 , fact that was confirmed by means of kDNA-PCR in the successive post-tmt controls ( data not shown ) . The lower parasitic loads detected at t1 , before initiation of tmt , compared with t2 , might be due to natural fluctuations of the parasitemia in chronic Chagas disease patients [27] . In this context , it is important to analyze serial blood samples to be able to observe an increasing or decreasing tendency in the parasitic loads in chronic patients under treatment . The Q-PCR test was also used for early detection of T . cruzi reactivation after heart transplantation . This was visualized through the increment of the parasitic loads in all patients who presented clinical manifestations of reactivation [14] . In case Tx1 , the patient is infected with T . cruzi I , and the number of parasites increased from 0 . 22 p/mL ( 5 days pre-Tx ) to 9 . 07 p/mL ( 78 days post-Tx ) when the patient presented signs and symptoms of skin reactivation and patent parasitemia [14] . In the other two tested cases , Tx2 and Tx3 , who were infected with group II populations , the parasitic loads increments were notably higher ( Table 3 and Figure 4 ) . In Tx3 , treatment with benznidazole after reactivation achieved transitory parasitological response because samples collected at 44 and 70 days after tmt were Q-PCR positive ( Figure 4 ) . Parasite relapse was confirmed by means of kDNA-PCR in successive samples until a second episode of clinical reactivation was diagnosed [14] . In Tx cases , the Q-PCR allowed to detect parasitic load increase , previous to diagnosis of reactivation , as well as to follow-up parasitological response during treatment with benznidazole . All together , the high analytical sensitivity of the Q-PCR strategy , the low levels of intra- and inter-assay variation , as well as the accuracy provided by the Lg-PCR correction , promotes this method as a key laboratory tool to follow-up patients under etiological treatment or at risk of clinical reactivation . This will be of particular significance for future drug trials in which an early assessment of efficacy or failure is mandatory [28] .
|
Infection with the parasite Trypanosoma cruzi ( T . cruzi ) , causing American trypanosomiasis or Chagas disease , remains a major public health concern in 21 endemic countries of America , with an estimated prevalence of 8 million infected people . Chagas disease shows a variable clinical course , ranging from asymptomatic to chronic stages with low parasitaemias , whose severest form is heart disease . Diagnosis at the asymptomatic and chronic stages is based on serological detection of anti-T . cruzi antibodies , because conventional parasitological methods lack sensitivity . Current chemotherapies are more effective in recent infections than in the chronic adult population . The criterion of cure relies on serological conversion to negative , which may occur only years after treatment , requiring long-term follow-up . In this context , we aimed to develop a real-time PCR assay targeted to repetitive sequences of T . cruzi for sensitive quantitation of parasitic load in peripheral blood of infected patients . It was applied to monitor treatment response of infected children , allowing rapid evaluation of drug efficacy as well as detection of treatment failure . It was also used for early diagnosis of chagasic reactivation in end-stage heart disease patients who received immunosuppressive drugs after cardiac transplantation . This laboratory strategy may constitute a novel parasitological tool for prompt and sensitive evaluation of anti-parasitic treatment of Chagas disease .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"infectious",
"diseases/neglected",
"tropical",
"diseases",
"pediatrics",
"and",
"child",
"health",
"surgery/transplantation",
"microbiology/parasitology",
"infectious",
"diseases/protozoal",
"infections"
] |
2009
|
Accurate Real-Time PCR Strategy for Monitoring Bloodstream Parasitic Loads in Chagas Disease Patients
|
Since miltefosine monotherapy against visceral leishmaniasis ( VL ) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures , single dose liposomal amphotericin B is now advocated as a treatment option of choice . Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential . Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness . Given the possible clinical implications of these findings for the current field situation , the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy . Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine . In hamsters infected with L . infantum , the combination resulted in cumulative efficacy in reducing parasite burdens in the liver , spleen and bone marrow . Selected resistant lines against the single drugs did not display cross-resistance . When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination , either in vitro or in vivo , no significant susceptibility decrease towards either drug was noted . These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L . donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance .
Depending on the geographical location , visceral leishmaniasis ( VL ) can either be caused by Leishmania donovani ( East Africa and Indian subcontinent ) or L . infantum ( Mediterranean basin , central Asia and Latin America ) [1] . Although both species are closely related and belong to the same complex ( L . donovani complex ) , transmission of L . donovani is mostly anthroponotic while the domestic dog serves as the main reservoir of L . infantum . Various treatment recommendations for VL have been proposed in the past and up to the early 2000’s most cases were treated with antimonials ( Sb ) [2] . However , the spread of Sb-resistant parasites in the Indian subcontinent has enforced a shift in therapeutic modalities [3] . Given its limited toxic effects , oral administration and reasonable price , the introduction of miltefosine ( MIL ) in 2002 as a novel antileishmanial agent looked very promising . As a result , the drug was even presented as first-line therapy in 2005 in India , Bangladesh and Nepal in the frame of the Kala-azar elimination program aimed at reducing the disease burden to less than 1/1000 by 2015 [4] . Unfortunately , recent studies in the Indian subcontinent demonstrated an increasing number of MIL-treatment failures hence limiting its further use in monotherapy [5] and leading to the recommendation of a single dose of liposomal amphotericin B ( L-AmB ) as treatment option of choice [6 , 7] . However , the requirement for temperature-controlled cold-chain facilities to transport and preserve L-AmB restricts its widespread use . In regions without cold-chain assurance , the short-term combination of MIL and paromomycin ( PMM ) has been suggested as alternative [8] . The development of MIL-resistance in monotherapy can be anticipated as a major limitation given its long elimination half-life and long treatment course [9] . Although no decreased MIL-susceptibility could yet be demonstrated in vitro in isolates of L . donovani derived from treatment failures in the Indian subcontinent , the first actual MIL-resistant clinical Leishmania isolates did already surface [10–12] . Rapid development of laboratory-induced PMM-resistance has been demonstrated [11 , 13–15] which also proved to be associated with a potential fitness gain [16 , 17] paving a way to rapid emergence and spread of PMM-resistance upon its routine use in the field . Although drug resistance would theoretically arise slower for drug combinations , it remains essential to assess the potential effect of introducing PMM-MIL combination therapy on a large scale , given the above mentioned limitations of PMM- or MIL-monotherapy . This laboratory study assessed the interaction between MIL and PMM in vitro and in vivo , the occurrence of cross-resistance and the development of resistance upon repeated drug exposure cycles to this drug combination .
The use of laboratory rodents was carried out in strict accordance to all mandatory guidelines ( EU directives , including the Revised Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes that came into force on 01/01/2013 , and the declaration of Helsinki in its latest version ) and was approved by the ethical committee of the University of Antwerp , Belgium [UA-ECD 2011–77 ( 17-02-2012 ) ] . Female Swiss mice ( 20–25 g ) and female golden hamsters ( 80–100 g ) were purchased from Janvier ( France ) . Food for laboratory rodents ( Carfil , Arendonk , Belgium ) and drinking water were available ad libitum . Hamsters were kept in quarantine for at least 5 days before infection and were randomly allocated to experimental units of 5 animals each , with the exception of the groups ( n = 3 ) for the selection of resistance . The L . infantum laboratory strain ITMAP263 ( MHOM/MA/67/ITMAP263 ) was routinely cultivated in Syrian golden hamsters . To infect naive hamsters , ex vivo amastigotes were purified from the spleen of heavily infected donor hamsters , as previously described [14] . Infection inoculates containing 2 x 107 amastigotes/100 μL phosphate buffered saline ( PBS ) were used to infect hamsters by intracardial injection under isoflurane inhalation anaesthesia . The general condition and body weight of infected animals were monitored daily to evaluate the course of infection . Upon each drug resistance selection cycle , promastigote back-transformation was performed to allow in vitro expansion of surviving parasites for in vitro susceptibility determination purposes . Promastigotes of the L . infantum clinical isolate LEM3323 ( MHOM/FR/96/LEM3323 ) were obtained from the ‘Centre National de Référence des Leishmania ( CNRL ) ’ ( Dr . L . Lachaud ) and derived from a French HIV patient . Promastigotes were maintained in HOMEM promastigote medium supplemented with 10% inactivated fetal calf serum ( Invitrogen , Ghent , Belgium ) . To facilitate promastigote back-transformation from infected tissues during in vivo selection of resistance , 20% spent promastigote medium was added and the concentration of inactivated fetal calf serum was augmented to 20% [14] . Both MIL ( MW = 407 . 57 ) and PMM-sulphate ( MW = 713 . 71 ) were purchased from Sigma ( Diegem , Belgium ) . For the in vitro work , stock solutions of 20 mM were prepared in PBS ( MIL ) or distilled water ( PMM ) . For the treatment of infected animals , MIL and PMM were formulated in distilled water respectively at 20 mg/mL and 150 mg/mL . In vitro amastigote susceptibilities were determined as previously described [18] . To determine the drug susceptibility of intracellular amastigotes , primary peritoneal macrophages were harvested from starch-stimulated female Swiss mice . Forty-eight hours later , cells were infected with metacyclic promastigotes at an infection ration of 2:1 ( LEM3323 ) or 20:1 ( ITMAP236 ) . After 24h , the medium was changed to remove potential extracellular promastigotes . The plates were incubated at 37°C in 5% CO2 atmosphere for another 5 days in presence of 2-fold drug dilutions before staining with Giemsa and microscopic reading . The normalized fixed ratio isobologram method [19] was used to evaluate the efficacy of the combination of PMM and MIL against the L . infantum clinical isolate ( LEM3323 ) in vitro at their IC50 level . Intracellular amastigotes were exposed to 2-fold dilution series of the drug combinations in fixed drug ratios ( 5:0 , 4:1 , 3:2 , 2:3 , 1:4 , 0:5 ) . Top concentrations of MIL and PMM were selected based on their known susceptibility profiles with the intention to center the IC50 of each respective drug in the middle of a seven-point two-fold drug dilution series . For MIL , a top concentration of 5 μM was used while for PMM 500 μM was selected . As in the standard susceptibility assay , drug exposure lasted for 5 days where after the plates were stained with Giemsa and the susceptibility ( IC50 ) towards each drug was determined for each combination ratio . Fractional inhibitory concentrations ( FICs ) were determined by dividing the IC50 of the drug in combination by the IC50 of the drug alone and were used to construct the isobologram . The sum of FICs ( ∑FICs ) ( FIC MIL + FIC PMM ) was determined for each fixed-ratio solution and the mean ∑FIC was used to classify the nature of the drug interaction ( Fig 1 ) . Interactions are classified synergistic when the mean ∑FIC < 0 . 5 , indifferent when the mean ∑FIC ranges between 0 . 5 and 4 and antagonistic as the mean ∑FIC > 4 . Before defining the appropriate treatment regimen for the selection of drug resistance in vivo , the efficacy of the envisaged treatment regimens either combined or alone was evaluated . Hamsters were infected with ex vivo ITMAP263 amastigotes as described above . At 21 days post-infection ( dpi ) , animals were treated with either MIL , PMM or the combinations , as listed in Table 1 . Due to toxicity , the combination 350 PMM/kg and 40 mg MIL/kg was not included . At 35 dpi , the animals were sacrificed to assess parasite burdens in liver , spleen and bone marrow . Upon fixation in methanol and Giemsa-staining , the liver and spleen imprints and bone marrow smears were evaluated microscopically by counting the number of amastigotes associated with 500 macrophage nuclei . To evaluate drug efficacy , the percentage reduction compared to the untreated control group was calculated . To check whether drug resistance would arise upon PMM-MIL combination , resistance was selected in vitro in the LEM3323 isolate as described earlier [13] . The highest combined drug concentrations were 500 μM PMM and 40 μM MIL . The drug susceptibility was evaluated in vitro after each selection cycle . The selection process was performed for five successive cycles or until resistance towards PMM ( IC50 >150μM ) or MIL ( IC50 >15μM ) was reached [20] . Resistance against the MIL-PMM combination therapy was also selected in vivo , as described earlier [14] . Infected hamsters were repeatedly exposed to sub-curative and sub-toxic doses of the drug combination ( i . e . oral administration of MIL at 20 mg/kg and intraperitoneal injection of 350 mg PMM/kg for 2 days ) . Infected animals were treated for 2 days starting from 21 dpi and closely monitored for treatment relapse . When treatment relapse was suspected , a liver biopsy was taken to quantify the infection burden and to enable susceptibility testing of the drug-exposed parasites upon promastigote back-transformation . Maximum two subsequent treatment cycles were performed in the same animal before ex vivo amastigotes were harvested and transferred to a next naïve animal . Subsequent selection rounds were terminated either when drug susceptibility values indicated a drug-resistant phenotype ( IC50 MIL >15μM and IC50 PMM >150μM ) or after a maximum of 5 successive treatment/relapse cycles .
For the construction of the isobologram ( Fig 1 ) and determination of drug interaction on the LEM3323 isolate by the applied fixed-ratio isobologram method , four independent assays were performed , each in duplicate . the mean ∑FIC was 1 . 1 ± 0 . 3 at the IC50 level with ∑FICs ranging between 0 . 94 and 1 . 17 , indicating an indifferent interaction between MIL and PMM . Results of the in vivo efficacy study are represented in Table 1 . Combination of PMM-MIL for 5 days showed a superior efficacy compared to monotherapy with PMM or MIL at either treatment regimen used . MIL reached higher efficacies in clearing the splenic parasite burden , whereas PMM seemed more efficacious against the hepatic parasite burden . Combination of both drugs resulted in splenic and hepatic reductions of >99% and >98% in the bone marrow at the highest dosage scheme . Shortening the duration of the combination therapy from five to two days resulted in a significant efficacy drop with reductions of <60% in the liver and spleen and only 32 . 5% in the bone marrow . Results of the in vitro resistance selection are presented in Table 2 . Despite a shift in promastigote back-transformation following exposure to the PMM-MIL combination , allowing collection of surviving promastigotes exposed to increasing drug concentrations , no differences were observed between the susceptibility results of the wild-type strain and the strain that was repeatedly exposed PMM and MIL . Also no cross-resistance could be detected in the MIL and PMM resistant lines . Five subsequent treatment/relapse cycles were conducted and despite recurrent relapses , no resistant phenotype could be observed for either MIL or PMM ( Table 3 ) . In contrast , exposure to PMM alone resulted in a significant decrease in susceptibility against PMM without altered MIL resistance profile .
In response to the recent development and spread of clinical Sb-resistance in L . donovani , other drugs such as MIL , PMM and AmB have been proposed for VL treatment . Nowadays , a single dose of liposomal AmB has been recommended as first-line treatment in the Indian subcontinent [6] , however , despite the initiative from Gilead of donating a large batch of liposomal AmB , the need for temperature-controlled settings still severely limits its widespread use . Moreover and comparable to MIL , the first cases of AmB unresponsiveness have recently been reported [22 , 23] . In those endemic areas with large-scale Sb-resistance and an increasing number of MIL and AmB treatment failures , a switch from monotherapy towards combination therapies has been advised whereby the short-term combination of PMM and MIL was already shown to be a safe and efficacious alternative [24] . However , concerns regarding drug resistance have already been raised for both PMM and MIL monotherapy [10–14 , 17 , 22 , 25] . Given these concerns a better understanding of the possible implications of large scale implementation of PMM-MIL combination therapy seems essential . Therefore , the present study aimed at evaluating the efficacy of PMM-MIL combination therapy on L . infantum both in vitro and in the in vivo VL hamster model . Additionally , repeated simultaneous exposure to high doses of PMM and MIL was performed in vivo to evaluate the likelihood of PMM- or MIL-resistance development upon combination therapy . Given the difficult adaptation of clinical isolates maintained in vitro for some time , the in vivo adapted laboratory strain MHOM/MA/67/ITMAP263 was used for the selection of drug resistance in vivo . For the in vitro work , the clinical isolate LEM3323 was used as this was the only strain so far for which resistance towards both MIL and PMM could already been demonstrated upon monotherapy in vitro [11] . The clear difference in intrinsic susceptibility between both strains is in line with inter-strain variability reported for different parasite species and clinical isolates [26–30] . Although the selection of MIL resistance on amastigotes proved to be very challenging in vitro , it could be hypothesized that the enhanced fitness profile associated with PMM resistance could facilitate development of resistance towards other drugs [17] . Our susceptibility analyses did not reveal any direct cross-resistance between PMM and MIL in the individual resistant lines . Despite the fairly rapid selection of PMM resistance upon monotherapy both in vitro and in vivo [11 , 14] , no decrease in drug susceptibility could be observed when PMM was administered in combination with MIL either in vitro or in vivo . This contrasts with the findings of Garcia-Hernandez et al . , who were able to select resistance to MIL-PMM combinations on L . donovani promastigotes [31] . This discrepancy is undoubtedly linked to the stage-dependent outcome of the selection procedure for PMM where selection on amastigotes proved to result in parasites with a susceptible promastigote phenotype [13] , indicating that the induced mechanisms of resistance are probably different . Contrary to our previous in vivo resistance selection experiments during which the parasites were exposed to monotherapy of PMM at 350 mg/kg/day x 5 days or to MIL at 20 mg/kg/day x 5 days [14] , the dose regimen in the present study ( PMM at 350 mg/kg/day and MIL at 20 mg/kg/day x 2 days ) proved to be poorly effective with only 59 . 3% reduction of parasite burden in the liver , 56 . 1% in the spleen and 32 . 5% in the bone marrow ( Table 1 ) . Comparable to the field situation where treatment duration was reduced from 28 days MIL at 100 mg/kg/day [32] or 21 days PMM at 11 mg/kg/day [33] to 10 days combination therapy ( MIL 100 mg/kg/day + PMM 15 mg/kg/day ) [8] , we also opted to reduce the treatment duration by about half in the current experimental design . While this resulted in better efficacy in man [8] , this approach was clearly inferior to drug monotherapy in the Syrian golden hamster model . On the contrary , combining sub-curative doses of both drugs while maintaining the same treatment duration as in monotherapy resulted in an enhanced efficacy , which is in agreement with previous reports [34] . The relatively moderate in vivo activity of PMM at near-toxic doses ( 350 mg/kg/day ) reported here ( 85 . 1% reduction in liver , 74 . 4% in spleen and 84 . 5% in bone marrow ) was already reported previously [14 , 35] . Unlike the respective monotherapies , the combination therapy resulted in equally efficient reductions of splenic and hepatic parasite burdens . Although the latter combined dosing schedules showed an excellent efficacy , their high efficacy would severely delay the onset of relapse and complicate repeated relapse/treatment cycles . The in vitro interaction between PMM and MIL on the recent clinical L . infantum isolate LEM3323 seemed to be indifferent , corroborating observations made for L . donovani [35] . Although both a MIL- and a PMM resistant phenotype could be generated in vitro within 5 drug selection cycles [13 , 21] , no shift in susceptibility was observed upon in vitro drug combination exposure ( Table 2 ) . Similar to MIL resistance selection on other strains , a shift in promastigote back-transformation could be observed , indicating that parasites were able to resist elevated drug concentrations [11] . In case of MIL monotherapy , this observation could not be linked to phenotypic parasite changes in drug susceptibility or changes in treatment outcome [36] , leaving its clinical relevance still debatable . Despite several concerns in the past , the results of this study support the view that MIL-PMM combination therapy can be an effective and appealing choice of treatment in view of the fact that the anticipated rapid development of PMM resistance appears to be delayed in combination treatment with MIL .
|
Liposomal amphotericin B is presently being used as first-line treatment option against visceral leishmaniasis in the Indian subcontinent . However , the need for temperature-controlled transport and storage limits its widespread use in rural areas . Previous studies already suggested that paromomycin-miltefosine combination therapy could be a valuable alternative , side passing some of the disadvantages associated with monotherapy , such as development of drug resistance . As the first reports of miltefosine resistant clinical isolates have already surfaced and paromomycin resistance could be easily induced under laboratory conditions , it remains essential to assess the risk of developing resistance against both drugs upon combination therapy . This study evaluated the efficacy of combined therapy against a Leishmania species closely related to the agent found in the Indian subcontinent , using both in vitro and in vivo models with the aim to select multidrug-resistant species by simultaneous exposure to paromomycin and miltefosine . The combination of both drugs in the hamster model resulted in a cumulative efficacy but did not lead to a significant susceptibility decrease , indicating that paromomycin-miltefosine combination therapy may represent a safe and affordable treatment option for visceral leishmaniasis .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"methods",
"Results",
"Discussion"
] |
[
"medicine",
"and",
"health",
"sciences",
"immune",
"physiology",
"spleen",
"immunology",
"microbiology",
"vertebrates",
"animals",
"mammals",
"protozoan",
"life",
"cycles",
"parasitic",
"protozoans",
"developmental",
"biology",
"protozoans",
"pharmaceutics",
"leishmania",
"promastigotes",
"hamsters",
"life",
"cycles",
"leishmania",
"donovani",
"amastigotes",
"immune",
"system",
"rodents",
"leishmania",
"infantum",
"physiology",
"bone",
"marrow",
"biology",
"and",
"life",
"sciences",
"protozoology",
"drug",
"therapy",
"amniotes",
"organisms"
] |
2017
|
Combined treatment of miltefosine and paromomycin delays the onset of experimental drug resistance in Leishmania infantum
|
The dynamics of the late stages of the HIV-1 life cycle are poorly documented . Viral replication dynamics are typically measured in populations of infected cells , but asynchrony that is introduced during the early steps of HIV-1 replication complicates the measurement of the progression of subsequent steps and can mask replication dynamics and their variation in individual infected cells . We established microscopy-based methods to dynamically measure HIV-1-encoded reporter gene and antiviral gene expression in individual infected cells . We coupled these measurements with conventional analyses to quantify delays in the HIV-1 replication cycle imposed by the biphasic nature of HIV-1 gene expression and by the assembly-inhibiting property of the matrix domain of Gag . We further related the dynamics of restriction factor ( APOBEC3G ) removal to the dynamics of HIV-1 replication in individual cells . These studies provide a timeline for key events in the HIV-1 replication cycle , and reveal that the interval between the onset of early and late HIV-1 gene expression is only ~3h , but matrix causes a ~6–12h delay in the generation of extracellular virions . Interestingly , matrix delays particle assembly to a time at which APOBEC3G has largely been removed from the cell . Thus , a need to prepare infected cells to be efficient producers of infectious HIV-1 may provide an impetus for programmed delays in HIV-1 virion genesis . Our findings also emphasize the significant heterogeneity in the length of the HIV-1 replication cycle in homogenous cell populations and suggest that a typical infected cell generates new virions for only a few hours at the end of a 48h lifespan . Therefore , small changes in the lifespan of infected cells might have a large effect on viral yield in a single cycle and the overall clinical course in infected individuals .
The HIV-1 replication cycle consists of several discrete , sequentially occurring processes , involving numerous viral and host cell components . For the early steps in HIV-1 replication , there is a reasonably good appreciation of the kinetics with which individual steps occur . Viral entry occurs over about <1–3h after exposure of cells to virus [1–5] , reverse transcription occurs over the ensuing 6 to 48 hours [5–10] and integration takes place about 5 hours after the completion of reverse transcription [6 , 7] . The dynamics of the early steps in HIV-1 replication , particularly entry and reverse transcription , appear to be cell type dependent , and the rather large variability in published estimates of these dynamics may be due to variation in receptor and intracellular dNTP levels . In contrast , the dynamics of the various steps of the post-integration phases of the viral life cycle , e . g . the relative timing of the onset of early versus late gene expression , and the timing of particle assembly/release relative to viral gene expression are comparatively poorly documented . A challenge in determining the dynamics of HIV-1 replication is its inherent asynchrony in populations of cells , which can obscure the underlying dynamics in individual cells . Nevertheless , time-of-addition experiments indicate that resistance to transcription inhibitors is acquired at ~35h after infection [11 , 12] . Interestingly , the overall time taken to complete an HIV-1 replication cycle is broadly similar to the lifespan of an infected cell in its natural environment [13] . Approaches in which mathematical modeling was coupled with measurements of viral RNA in the blood of patients beginning anti-retroviral treatment or undergoing plasmapheresis have provided estimates for the lifetime of infected cells and the generation time of HIV-1 in a natural setting [14–18] . Successive refinements of these models led to the conclusions that that the viral generation time and the average lifetime of infected cells were about 48 hr [13] . The fact that the lifespan of an infected cell in vivo is somewhat similar to time taken to complete an HIV-1 life cycle suggests the lifespan of the cell should limit burst size . Given that cells are at risk of death through cytopathic effects and/or lysis from cytotoxic immune cells from the time at which they begin to express viral proteins , one might expect viral variants to be selected that minimize the time interval between the onset of viral protein expression and release of infectious virions . In other words , there should exist a selection pressure to optimize each of the late ( post transcriptional ) phases of viral replication cycle for speed [19] . It is intuitively surprising , therefore , that at least two features of the HIV-1 replication cycle appear to extend the time interval between onset of viral protein expression and the generation of infectious viral particles . First , to express nine genes from a highly compact genome with a single promoter , HIV-1 employs extensive alternative splicing , with unspliced , singly spliced , and multiply-spliced mRNAs providing all the necessary templates for translation of viral proteins [20] . Because incompletely spliced mRNAs do not efficiently exit the nucleus , the so-called ‘early’ HIV-1 proteins are products of fully spliced mRNAs . The viral Rev protein is one such early protein that shuttles between the nucleus and the cytoplasm , moving intron-containing viral mRNAs from nucleus to cytoplasm [20–25] via the CRM1 nuclear export pathway [26–29] . The Rev-dependence of ‘late’ genes ( Gag , Pol , Env , Vif , Vpr and Vpu ) predicts that their expression should be delayed so that viral gene expression is divided temporally into two phases . However , there is a paucity of data on the dynamic structure of early versus late gene expression during HIV-1 replication in the context of a 48h replication cycle . Early studies of mRNA production using northern blot or semi-quantitative PCR analyses of populations of infected cells [25 , 30] suggest that late , incompletely spliced transcripts are expressed ~0 to 12h after early completely spliced transcripts . Thus , the magnitude and significance of the delay in the replication cycle imposed by the biphasic nature of HIV-1 gene expression is unclear . Another delay in the late stages of the HIV-1 replication may occur during particle assembly . The binding of Gag to cell membranes , a necessary step in particle assembly , is driven by the N-terminal matrix ( MA ) domain of Gag , via a basic patch and a myristate group linked to the N-terminal glycine residue [31 , 32] . However , MA-membrane binding is inhibited by cellular tRNAs [33 , 34] and via a myristoyl-switch mechanism in which the N-terminal myristate is sequestered in a hydrophobic pocket in the MA globular head when Gag is monomeric [35–37] . Thus , mutations in MA or , more dramatically , deletion of the MA globular head , can induce increased membrane binding and increased levels of virion release [38–40] , particularly at the low Gag concentrations at which WT Gag is usually cytoplasmic [41 , 42] . Since Gag concentration in infected cells is expected to be low initially , and to progressively increase with time , these findings suggest that HIV-1 Gag actively delays its own assembly into particles . Because this property should lengthen the viral replication cycle , there are likely to be important functional reasons why virion assembly is inhibited or delayed in the context of wild-type HIV-1 . A possible impetus for the acquisition of programmed delays in the HIV-1 life cycle may be the apparent need for HIV-1 to manipulate the cellular environment in advance of virion generation . In particular , cells express a number of molecules that can inhibit viral replication [43 , 44] . To allow particle assembly to proceed prematurely in the presence of high levels of such molecules may be futile , or even deleterious to overall virus replication . For example , APOBEC3G ( A3G ) is a constitutively expressed antiretroviral protein that is packaged into virions and catalyzes lethal hypermutation of nascent viral DNA during reverse transcription [45] . However , human A3G is bound by the HIV-1 accessory protein Vif , a late gene product , that recruits a ubiquitin ligase complex to drive A3G degradation [45–48] . The timing of the removal or downregulation of A3G , and other proteins that exert antiviral activity , relative to viral replication is unknown , but A3G depletion must follow late gene expression . In this study we attempted to quantify ( i ) The delay in HIV-1 replication imposed by the biphasic nature of HIV-1 gene expression , ( ii ) the delay in particle assembly that is attributable to MA and ( iii ) the dynamics of restriction factor removal ( using A3G as a specific example ) relative to viral gene expression and particle release . To ameliorate the difficulties associated with measuring dynamics in asynchronously infected cells , we utilized a combination of techniques , including quantitative fluorescence microscopy of single cells wherever possible . We supplemented these studies with conventional analyses of virion production . These studies provide a timeline for key events in the HIV-1 replication cycle and reveal that the interval between the onset of early and late HIV-1 gene expression is only ~3h , but the globular head of MA causes an additional ~6–12h delay in virion assembly . Interestingly , the globular head of MA delays particle assembly to beyond the time at which A3G has largely been removed from the cell . Thus , the need to remove inhibitory molecules , and otherwise prepare the cell to be an efficient producer of virions , may provide an explanation for the apparent requirement that HIV-1 actively inhibits the pace of particle assembly .
To determine the relative timing of the early and late phases of HIV-1 gene expression , we constructed proviral plasmids encoding fluorescent proteins in positions that would be expressed via a completely spliced early transcript or via an unspliced Rev-dependent late transcript ( Fig 1A ) . The early-gene reporter was inserted in place of nef , which is expressed at high levels compared to the other early genes [20] but is not required for viral gene expression or replication . The late gene reporter was inserted near the C-teminus of MA in the Gag polyprotein , at a site known to tolerate such insertions [49] . One reporter construct , termed HIV-1 ( MA-cherry/Nef:GFP ) , had GFP in the early gene position and mCherry in the late gene position , while another , termed HIV-1 ( MA-GFP/Nef:cherry ) carried the reporters in exchanged positions ( Fig 1A ) . By employing both constructs in these assays we could ensure that differences in the measured kinetics of early versus late gene expression were not artifacts of differential fluorophore intensities or maturation rates . The viral reporter genomes contained inactivating mutations in the catalytic sites of the reverse transcriptase and integrase enzymes , and a deletion in Env . Thus , replication was restricted to a single cycle , and reporter genomes were introduced into cells using virions generated by co-transfection of the reporter genomes with HIV-1 GagPol and VSV-G expression plasmids . To track the dynamics of early and late gene expression , we infected MT4 cells as synchronously as possible . This was done by spinoculating cells at 15°C , briefly incubating them at 37°C ( 90 min ) , and then removing unattached virions by washing . Expression of the reporter genes was first detected at 18–24h after infection , and the numbers of cells expressing the early gene reporter alone , or expressing both reporters increased progressively thereafter ( Fig 1B ) . As expected , given that late gene expression is dependent on early gene products , the percentage of cells expressing the early gene reporter was always higher than that expressing the late gene reporter and virtually no cells expressed the late reporter without also expressing the early reporter ( Fig 1B ) . An unanticipated observation was that both GFP and mCherry reporters were expressed at higher and more heterogeneous levels when they were expressed using a late gene transcript than via the early gene transcript ( Fig 1B ) . However , this finding is consistent with previous analyses of viral mRNA levels in infected cell populations [25] . We compared the dynamics of early and late reporter gene expression in MT4 cells with those in primary T-cells ( S1 Fig ) . While the dynamics of reporter gene expression in primary cells varied somewhat with individual donor , and more so with how the cells were activated ( S1A–S1I Fig ) the times after infection at which expression of the early and late reporter genes were detected in cell lines ( MT4 and HOS , S1J–S1L Fig ) was quite similar to that in primary T-cells . Therefore , unless otherwise stated we used MT4 cells for convenience and reproducibility thereafter . To place the timing of the late steps of HIV-1 replication in context , we derived an estimate of the timing of early events in the replication cycle in our MT4 cell experimental system . To accomplish this , we first synchronously infected cells with one of the dual reporter vectors ( HIV-1 ( MA-Cherry/Nef:GFP ) ) . Thereafter , reverse transcription and integration inhibitors were applied at varying time points and the fraction of infection events that were no longer susceptible to the inhibitors at each time of inhibitor addition was determined ( Fig 1C ) . To increase the robustness of this analysis , infections were done at 4 different multiplicities of infection ( MOI ) . We fitted 5-parameter sigmoid functions to these each of these 4 data sets and calculated the times at which each curve reached 50% of its maximum value . This analysis indicated that half of the incoming virions had completed reverse transcription at 14 . 4±0 . 4h and integration at 19 . 3±1 . 1h post-inoculation ( Fig 1C ) . To obtain an estimate of the timing of the onset of early and late gene HIV-1 expression in populations of cells , we again infected cells at the 4 different MOI and used FACS to determine the fraction of cells that expressed detectable levels of early or late reporter genes at various times after infection ( Fig 1C ) . Fitting 5-parameter sigmoid functions to these 4 data sets demonstrated that half of the infected cells expressed detectable levels of the early gene reporter at 30 . 1±0 . 4h and detectable levels of the late gene reporter at 33 . 4±0 . 4h after infection ( Fig 1C ) . Thus , the interval between integration and detection of early gene expression estimated by these methods was 10 . 8h , while the interval between early and late gene expression was 3 . 3h , or ~10% of the duration of the viral replication cycle up to that point . The FACS-based methods described above analyzed gene expression in individual cells , but were based on counting the number of cells in a population whose fluorescence had crossed a threshold detection value . Thus , FACS analyses could not provide information about how the fluorescence of individual cells changes over time , or how the interval between the onset of early and late gene expression might vary among individual cells in a population . For this reason , and to provide a second method for estimating the interval between early and late gene expression , we established a microscopy method to dynamically measure reporter gene expression in individual cells . Following exposure to the reporter virus inoculum , infected MT4 cells were washed and immobilized . Images of cells were collected at 2–5 min intervals from ~12h to ~48h following infection ( Fig 2A and 2B and S1–S4 Movies ) , and fluorescence intensity associated with the early- and late-gene reporters quantified ( Fig 2C ) . While , as expected , there was variability in the timing and level of reporter expression among individual infected cells , their overall behavior was stereotypic ( Fig 2A and 2B and S1–S4 Movies ) . Specifically , the early gene reporter became detectable in individual cells ~14 to 30h after infection , and the late gene reporter became detectable shortly thereafter , 1 . 3 to 4 . 7h later . Both early-gene and late-gene reporters exhibited a rapid increase in intensity for the first several hours after they became detectable . During this phase , the levels of the early and late reporters increased at similar rates , so that the trajectories of the fluorescent intensity measurements were approximately parallel ( Fig 2C ) . A few hours after the time at which early-gene reporter became detectable , there was a clear change in the trajectory of early gene expression , after which the levels of the early gene reporter plateaued , or increased less rapidly . The late gene reporter did not show this behavior . Rather , the levels of late reporter increased for the duration of the experiment , with only marginal tendency for the rate of increase to slow toward the end of a 40–48h imaging experiment ( Fig 2C ) . This finding is concordant with the behavior of populations of cells analyzed by flow cytometry ( Fig 1B ) , in which the fluorescence intensity associated with the late reporter increased over time , ultimately reaching higher levels than the early gene reporter . We used the microscope-based assay to quantitate the interval between early and late gene expression in individual cells . To accomplish this in an unbiased manner , we used a MATLAB script to fit a 5-parameter logistic equation to the relevant regions of each of the fluorescent intensity plots to generate sigmoid best-fit curves , and to identify the points at which fluorescence rose above background ( Fig 2D ) . Background fluorescence was measured in an unoccupied control region close to each cell and subtracted from the cell-associated fluorescence . The resulting fluorescence intensity data had initial levels that were close to zero , corresponding to the lower bound of the best-fit curve . The time at which the intensity curves became detectable above background , was defined as the time at which the fitted curve was at least 7 intensity units above its horizontal lower bound ( Fig 2D ) . Using this approach we found that the mean±s . d . interval between the initiation of HIV-1 early and late gene expression was 2 . 8±0 . 6h with a range of 1 . 6 to 4 . 4h in 59 individual infected cells ( S2 and S3 and 2D Figs ) . Importantly , this result was not significantly affected by the identity of the fluorophore assigned to the early and late gene reporter positions ( S2 and S3 and 2D Figs ) . Although there was substantial variation in the interval between early and late gene expression among individual infected cells , the mean value was in reasonable agreement with the value of 3 . 4h obtained using FACS analysis of populations of cells ( Fig 1C ) . To determine the timing of HIV-1 assembly during a single cycle of HIV-1 replication , we modified a replication-competent viral clone ( NHG ) that carries a single reporter gene ( GFP ) in the Nef position . An NHG derivative lacking the globular head of matrix ( ΔMA ) was used to quantify the effect of the globular head of MA . VSV-G pseudotyped virions were used to infect MT-4 cells , leading to the production of virions that were incapable of initiating a second round of infection in the culture . Although it was not possible to measure virion release by individual cells , the amount of viral protein associated with virions released into the culture supernatant by populations of cells could be measured using western blot assays ( Fig 3A ) . These analyses both showed that the ΔMA virus indeed generated extracellular virions more rapidly and in greater quantities than WT virus ( Fig 3A and 3B and 3C ) . These measurements were quite variable ( Fig 3C ) , and MOI-dependent , presumably because the efficiency of virion assembly is dependent on Gag expression level , particularly in the case of the WT virus . Nevertheless , using the time point at which the yield of extracellular WT virus had reached its maximum level ( at the end of the 48h experiment ) as a reference point , we found that the ΔMA virus achieved this level of extracellular virions 12 . 5±5 . 4h ( n = 7 ) earlier than WT virus ( Fig 3C ) . Moreover , the total deficit in virion production over the course of a 48h experiment that could be ascribed to MA was 4 . 4±3 . 2 fold ( n = 7 ) . Thus , there was clear penalty in replication kinetics and overall particle yield imposed by the globular head of MA . To determine the dynamics of A3G depletion from infected cells relative to HIV-1 gene expression , we constructed two cell lines , based on MT-4 and HOS , stably expressing an mCherry-A3G fusion protein . These cells were infected with HIV-1 ( Nef:GFP ) or HIV-1 ( MA-GFP ) carrying a GFP reporter as an early or late gene . Thus , cells became green fluorescent as HIV-1 early or late gene expression commenced , and they lost their red fluorescence as mCherry-A3G was degraded through the action of Vif . We first determined the kinetics of A3G removal relative to HIV-1 gene expression in populations of cells , using FACS analyses ( Fig 4A and 4B ) . In mCherry-A3G expressing cells infected with HIV-1 carrying a GFP reporter in either early or late gene positions , cells depleted of cherry-A3G became evident shortly after GFP-positive cells became detectable , with GFP-positive/mCherry-low cells predominating at later time points ( Fig 4A and 4B ) . At intermediate time points , a significant number of GFP-expressing infected cells also retained unaltered mCherry-A3G expression ( Fig 4A and 4B ) , consistent with the notion that there is a delay between expression of HIV-1 genes and removal of A3G from infected cells . As expected , and could be seen most clearly in HOS cells ( Fig 4B ) , fewer GFP-positive cells retained mCherry-A3G when the GFP reporter gene was in the late position . In both cell lines , cells that expressed higher levels of the late GFP reporter were more likely to have depleted mCherry-A3G than cells that expressed lower levels of the late GFP reporter . Given that individual cells expressed progressively higher levels of late gene expression over time ( Fig 2 ) , this finding suggests that A3G depletion is more likely to have occurred in cells that have been expressing late genes for a longer period of time . We next used the single-cell microscopy-based assay to measure mCherry-A3G levels in individual MT4 cells infected with HIV-1 ( Nef:GFP ) or HIV-1 ( MA-GFP ) carrying a GFP reporter in either early or late gene positions ( Fig 5 ) . In the presence of Vif , mCherry-A3G expression was visibly and progressively extinguished in infected cells as , or shortly after , GFP expression was detected ( Fig 5A and 5B and S5–S8 Movies ) . Conversely , in the absence of Vif , mCherry-A3G expression levels remained stable over the course of infection ( Fig 5B ) . To quantify the timing of A3G removal , we used the aforementioned MATLAB script to fit curves to the GFP and mCherry-A3G measurements for 28 cells infected with HIV-1 ( Nef:GFP ) and 29 cells infected with HIV-1 ( MA-GFP ) ( Figs 5C and S4 and S5 ) . Thereafter , we identified the time points at which GFP expression rose above background levels and mCherry expression dropped to background levels and calculated the time interval between them ( Fig 5C ) . The mean±s . d . interval between the initiation of early gene expression to the completion of cherry-A3G removal was 4 . 9±1 . 2h , while the mean±s . d . interval between the initiation of late gene expression to the completion of cherry-A3G down-regulation was 2 . 5±0 . 6h ( Fig 5D ) . Notably , the difference between these values , 2 . 4h , is in reasonable agreement with the value of 2 . 8h calculated for the interval between early and late gene expression using single-cell microscopy with dual-reporter viruses ( Fig 2 ) . Fluctuations in the levels of mCherry-A3G fluorescence , and heterogeneity in the trajectories of the best-fit curves made it difficult to assign time points at which A3G removal commenced . However , visual inspection of the fluorescence measurements in many individual cells ( S4 and S5 Figs ) suggested that A3G levels began to decline approximately coincident with the detection of late gene expression , thus A3G removal occurred over a period of ~2 . 5h in a typical cell . Together , these experiments allow the generation of an approximate time-line of events in HIV-1 replication in a typical MT4 cell ( Fig 6 ) . Reverse transcription and integration are completed at 14 . 4h and 19 . 3h after infection in 50% of cells , while early and late gene expression are detected by FACS in 50% of cells at 30 . 1h and 33 . 4h hours post-infection , respectively . The 3 . 4h interval between the detection of early and late gene expression measured by FACS is in reasonable agreement with the interval ( 2 . 8h ) measured using single live cell microscopy . It is somewhat more challenging to relate virion assembly/release measurements to parameters that can be determined for individual cells , because the measured amount of released virions by populations of cells is governed by several parameters including: ( i ) the number of cells expressing Gag ( i ) the level at which they are expressing Gag and ( iii ) the rate at which virions are assembled and released . Both ( i ) and ( ii ) increase with time , given the asynchrony associated with HIV-1 replication , and ( iii ) also increases with time in the case of the WT virus given the cooperative nature of HIV-1 Gag assembly [42] . Nevertheless , the western blot analysis of HIV-1 Gag expression and particle release enabled some conclusions . First , Gag expression in cells was initially detected by western blot analysis at ~18h after infection ( Fig 3 ) . Importantly , this Gag expression was detected at about the same time as a small population of cells ( a few percent ) express detectable levels of the late gene reporter—note that the late gene reporter is embedded in the Gag protein and is , in fact , a surrogate measurement of Gag expression ( Fig 1 ) . Thus , Gag ( or MA-GFP ) detected at 18h represents expression by a small ‘leading’ cohort of cells in the asynchronously infected population that were the first to begin to express Gag . Second , in the case of the ΔMA virus , initial detection of extracellular virions occurred simultaneously with detection of cell-associated Gag and late gene reporter expression ( Figs 1 and 3 ) . Third , graphs describing extracellular virion accumulation for the ΔMA virus with time and the fraction of cells expressing the late gene ( Gag ) reporter exhibit similar trajectories ( Fig 6A ) . Together , these finding suggest that , in the case of the ΔMA virus , there is no measurable delay between late gene ( Gag ) expression and virion assembly/release . Thus , if we make the assumption that a typical cell in the population is able to support virion assembly with similar kinetics to the ‘leading’ cohort of cells , then a typical ΔMA virus-infected cell would begin to release virions at 33 . 4h after infection ( the same time as it begins to express late genes , including Gag ) . Conversely , WT virus infected cells generate extracellular virions only about 6–12h after detection of late reporter or Gag expression ( Fig 3 ) , leading to the conclusion that a typical WT virus infected cell would begin to generate extracellular virions at about 40 to 46h after infection . Notably , the interval of about 2 . 8h between the onset of late gene expression and the completion of cherry-A3G removal ( Fig 5 ) would place cherry-A3G removal between the times at which ΔMA and WT virions would be released . Thus , the delay in virion assembly imposed by MA results in the generation of virions only after A3G has been removed from the cell .
The relative paucity of information on the timing of the individual steps during the post-integration stages of the HIV-1 life cycle prompted us to develop approaches for measuring the progression of these processes . Specifically , we used viruses encoding fluorescent proteins that report levels of early and late ( Gag ) gene expression , coupled with flow cytometry , quantitative live cell microscopy , and quantitative western blotting to measure gene expression , virion production and removal of A3G from infected cells . Even though we synchronized infection by employing a spinoculation and washing protocol , there was a significant degree of asynchrony in infected MT4 cell cultures as the HIV-1 replication cycle proceeded . This asynchrony primarily manifested itself prior to or during reverse-transcription , as evidenced by the significant variation ( >18h ) in the time required for infection events to escape the effect of RT inhibitors . Additional variation in the overall length of the viral life cycle , introduced between reverse transcription and gene expression , appeared comparatively modest , as evidenced by the rather parallel nature of the curves describing RT inhibitor escape and detection of early and late gene expression . Nevertheless , inherent asynchrony introduced early in the HIV-1 replication cycle complicates the measurement of the dynamics of the late stage of the viral life cycle . Therefore , a key feature of our analysis was the measurement of parameters in individual cells wherever possible , and enumeration of the numbers of individual cells in a population in which the viral replication cycle had progressed beyond a point that could be measured . Using these approaches , estimates of the timing of events in a typical infected cell were generated , as well as an appreciation of how much these values vary from cell to cell . Our data revealed a significant interval between integration and the detection of early gene expression ( ~11h ) , a smaller interval ( ~3h ) between early and late gene expression , and a longer interval ( ~6–12h ) between late gene expression and the release of virion particles . The overall replication cycle in a typical cell required ~42h but was subject to a large degree of variation ( as much as 24h ) among individual cells , even in this relatively homogeneous cell population . A few caveats accompany this analysis . First , we used VSV-G to mediate viral entry , and so the precise timing of viral entry may differ from that mediated by HIV-1 Env . However , the timing of entry and other steps in the HIV-1 replication cycle is likely to be intrinsically variable according to the cell type in which it is measured [1–5 , 7 , 8 , 10 , 50] , and determined in part by the levels of receptors and dNTPs . The kinetics of other processes in the viral replication cycle measured here may also be cell-type dependent , depending on the availability of cofactors required for their execution . We performed our experiments using a highly permissive T-cell line , MT4 , in which some steps may be more rapid than in primary cells . Nevertheless , our analysis of early and late gene expression in primary cells stimulated in various ways indicated that MT4 cells reasonably , albeit imperfectly , represented the properties of authentic HIV-1 target cells . Thus , the values given herein should be regarded as estimates of the timing of each occurrence in a typical MT4 cell and subject to variation within and between cell populations . Additionally , the timing of some events is obviously influenced by the techniques used to detect them . For example our measurements of gene expression involved detection of the protein products of early and late transcripts . Our measurements did not determine whether HIV-1 transcription is initiated immediately following integration ( and 11h is required for HIV-1 mRNAs to be spliced and exported , and their translation product to reach a detection threshold ) or whether there is a biologically determined delay between integration and transcription ( perhaps , for example , governed by the cell cycle ) . Finally , our measurements of the removal of APOBEC3G make the assumption that the mCherry-fused protein is degraded with similar kinetics to that of the endogenous unfused protein . Nevertheless , the relative timing of late events in the HIV-1 replication described herein is likely to be broadly correct . For example , the interval between early and late gene expression of about 3h was quite similar when measured in different ways: ( i ) using flow cytometric analysis of populations of cells expressing two different fluorophores as early and late genes , ( ii ) using microscopic analysis of individual cells expressing two different fluorophores as early and late genes ( iii ) using microscopic analysis of individual cells expressing a single fluorophore as an early or late gene and measuring the timing of its expression relative to the removal of cherry-A3G . An unexpected finding was that early gene expression increased rapidly for several hours and then plateaued or increased at a slower rate thereafter , while late gene expression ( Gag with an embedded fluorophore ) continued to increase throughout the course of an ~48h experiment . This finding may reflect a degree of autoregulation of early versus late HIV-1 gene expression . Perhaps accumulated of Rev protein promotes diversion of incompletely spliced HIV-1 transcripts to the cytoplasm , limiting the number of nascent of transcripts that are available to be spliced for expression of early genes . A possible caveat is that the early reporter is expressed in an authentic unfused fluorescent protein , while the late reporter is a fluorescent protein embedded in Gag . While differential protein stability may affect accumulation of the two reporters , Gag is significantly less stable than unfused fluorescent proteins [51–53] , and therefore differential protein stability cannot account for the elevated expression of late over early reporter genes at later time points . Moreover , one previous report has also suggested that the ratio of late:early HIV-1 mRNAs increases with time of infection [25] . A key conclusion of our studies is that , in the absence of the MA globular head , HIV-1 virion assembly proceeds promptly after late gene-expression . However , in the context of an intact Gag protein , virion egress is delayed by ~6–12h . Our own and others’ previous findings indicate that the MA globular head inhibits membrane binding , particularly at low Gag expression levels [39–42 , 54] . Once the formation of an individual virion has been initiated at the plasma membrane , the assembly process itself occurs in minutes [55] . Thus , binding to the plasma membrane is likely a rate-limiting step in HIV-1 assembly and the MA globular head therefore causes a significant prolongation of the HIV-1 replication cycle . Two properties of MA appear to underlie its ability to retard Gag-membrane binding . First , when Gag is first expressed in the cytoplasm , it appears to be largely monomeric [56] , a state in which the N-terminal myristate in sequestered in a hydrophobic pocket in the MA globular head [36] . Second , lysine residues in the N-terminal ~30 amino acids of the MA globular head that contribute to membrane binding also bind to particular tRNAs , specifically in the cytoplasm [33] . Thus , the two major signals in MA that drive Gag membrane binding are at least partly occluded when Gag is present in the cytoplasm . This autoinhibition of assembly caused by the MA globular head appears to be very significant in the context of a single cycle of HIV-1 replication , causing both a delay in assembly ( about 6–12h ) and an overall decrease in virion yield . Previous pulse-chase analyses of HIV-1 assembly have indicated that virions are released from the cell over a time period of 2–8h following the synthesis of WT Gag [52 , 53 , 57] , thus demonstrating a significant time interval between WT Gag synthesis and its appearance in virions . While the values obtained using pulse chase analyses might appear to indicate that WT Gag assembles into virions more rapidly than do our experiments , it is important to note that these earlier studies were done under conditions where Gag proteins had already accumulated in transfected cells . Our analysis was done under conditions of a single infection cycle , in which the level of Gag expression increases with time . Because the rate of virion assembly increases with Gag expression levels , the descriptions of the kinetics of particle generation from the two different approaches are entirely compatible with each other . The existence of antiviral proteins in infected cells may provide one impetus for this apparently programmed delay in HIV-1 egress . Molecules that inhibit the release of HIV-1 particles ( CD4 and tetherin ) or effectively poison progeny virions ( A3G and other APOBEC proteins ) are often present constitutively at significant levels in HIV-1-infected cells [45 , 58 , 59] . To accomplish the removal of these inhibitory proteins , HIV-1 employs accessory proteins ( Nef , Vif and Vpu ) that deplete or remove them from their sites of action . Our analysis shows that one consequence of the MA-imposed inhibition of HIV-1 assembly is to delay particle genesis to a time where A3G has been removed from the cell . The removal of other inhibitory molecules is likely to require similar delays in the replication cycle in order to be completed prior to virion production . Other processes in HIV-1 replication might further impose a requirement for a delay in viral assembly . In order to generate infectious virions , Gag , Gag-Pol , Env and the viral genome must co-assemble at the plasma membrane . The dynamics of the accumulation of Gag-Pol and the viral genome might be reasonably assumed to be similar , given that the same RNA species serves as both the viral genome and the mRNA for Gag and Gag-Pol . However , given the complex modifications and trafficking that govern the localization of the HIV-1 Env protein , it is possible that virion assembly is delayed to enable the accumulation of functional Env spikes at the cell surface . Two features of HIV-1 replication are emphasized by our findings: ( i ) there is significant heterogeneity in the length of the HIV-1 replication cycle , even in relatively homogenous MT4 cells , ( ii ) a typical infected cell generates new virions for only a few hours at the end of a 48h observation period . Notably , a typical infected T-cell in vivo lives for only ~48h [13] . If we assume that the length of HIV-1 replication cycle in T-cells in vivo is not significantly shorter than in highly permissive MT4 cells , then many infected cells generate virions for only a few hours at the end of their life . Therefore , interventions or cytotoxic immune responses that have only a small effect on the lifespan of infected cells might have a large effect on overall viral yield in a single cycle and the overall clinical course in an infected individual .
293T ( ATCC #: CRL-11268 ) and HOS ( ATCC #: CRL-1543 ) cells were obtained from ATCC and grown in DMEM supplemented with 10% fetal bovine serum . MT-4 cells , obtained from the NIH AIDS Research and Reference Reagent Program , were cultured in RPMI supplemented with 10% fetal bovine serum . Derivatives of HOS and MT-4 cells stably expressing cherry-A3G ( cherry-A3G ) were generated by transduction with a retroviral vector ( pBMN-IRES-Blasti ) , that allowed expression of the gene of interest and Blasticidin resistance gene from the same transcript separated by an IRES , and were grown in medium supplemented with 10μg/ml Blasticidin . Stocks of the pBMN-cherry-A3G-IRES-Blasti retroviral vector were generated by co-transfection of the vector plasmid with plasmids expressing MLV Gag-Pol , VSV-G and HIV-1 Vif to block A3G-mediated effects on retroviral transduction . Peripheral blood mononuclear cells were isolated from blood using Ficoll-paque gradient centrifugation and CD4+ T-cells were isolated by negative selection ( RosetteSep Human CD4+ T Cells Enrichment Cocktail , StemCell Technologies ) . Cells were activated with phytohemagglutinin ( PHA-P , Sigma , 5μg/ml ) or anti-CD3/CD28 beads ( Dynabeads Human T-Activator CD3/CD28 , Gibco ) for 48h , and then grown in RPMI with 10% fetal bovine serum in the presence of IL-2 ( 20 or 30 U/ml ) for a further 2 to 8 days before use . For some experiments ( S1H and S1I Fig ) , CD4+ T-cells were stimulated with CD3/CD28 Dynabeads 6 days after initial stimulation with phytohemagglutinin , 48h h before infection . The HIV-1 reporter viruses HIV-1 ( Nef:GFP ) , HIV-1 ( MA-GFP ) , HIV-1 ( MA-GFP/Nef:cherry ) , HIV-1 ( MA-cherry/Nef:GFP ) were constructed using overlap PCR to insert GFP or cherryFP in either the Nef or Gag positions within the HIV-1 proviral clone HIV-1NL4-3 ( dINdRTdEnv ) . The HIV-1NL4-3 ( dINdRTdEnv ) carries inactivating mutations in both the IN and RT open reading frames and a deletion in Env , rendering it replication-incompetent [55] . Reporters in the Nef position were inserted with the start codon of the fluorescent protein positioned identically to the native start codon of Nef . Reporters embedded in Gag were inserted in the stalk region of MA , immediately N-terminal to the protease cleavage site at the MA-CA junction , as previously described [55] . The protease site was duplicated immediately N-terminal to the fluorescent protein . For experiments in which virus particle yield was measured , an HIV-1 proviral clone NHG carrying GFP in the Nef position [60] was used . NHG was modified by the addition of a stop codon at nucleotide 2133 of the env gene removing the 142 C-terminal residues of the cytoplasmic tail of the Env protein , and by replacement of the V3 encoding region of env ( nucleotides 915–975 ) with the V3 region from the R5-tropic proviral clone YU2 . This proviral plasmid served as the backbone for the construction of ( referred to as HIV-1 ( ΔMA ) ) by the deletion of sequences encoding amino acids 7–110 of the MA protein . The presence of the cytoplasmic tail-deleted R5 tropic Env protein allowed the demonstration that the manipulations allowed the demonstration that the HIV-1 ( ΔMA ) virus was infectious , but was limited to a single cycle of infection in MT4 cells , which lack CCR5 . Because the infectiousness of the HIV-1 ( ΔMA ) virus was significantly reduced compared to the WT virus we limited our analyses to production of physical particles . All DNA manipulations were performed using overlap-extension PCR and standard molecular biology techniques . All viral stocks used in these studies were pseudotyped with VSV-G and produced by transfection of semiconfluent 10cm plates of 293T cells with 10ug of proviral plasmid and 1ug of VSV-G expression vector , using polyethyleneimine ( PEI; PolySciences ) at a DNA:PEI ratio of 1:4 . Single-cycle reporter viruses that carried inactivating mutations were produced by co-transfection of the proviral plasmid , an HIV-1NL4-3 GagPol-expression plasmid and a VSV-G expression plasmid , at a ratio of 5:5:1 . Viral supernatants were filtered through a 0 . 22μm filter and stored in aliquots at -80°C . All viruses carried at least one fluorescent reporter gene , and viral titers were determined by infection of MT-4 cells with serially diluted viral stocks for 48 hours , and flow cytometry to measure the number of infected cells . All infections employed a synchronized infection protocol . Single-cycle virus containing supernatant was added to cells in 6-well or 12-well dishes in the presence of 5μg/ml polybrene . Cells were centrifuged at 350g , 15°C for 40 min , incubated at 37°C for 90 min ( or 120 min for experiments where virion yield was determined ) and then washed three times and fed with fresh medium . At various time points thereafter , cells were collected ( following trypsinization for adherent cells ) and fixed in a 2% final concentration of paraformaldehyde . Flow cytometry was performed on a CyFlow Space flow cytometer ( PARTEC ) using a blue 488nm laser for the excitation of GFP and a yellow 561nm laser for excitation of mCherry . Samples were loaded using a Hypercyte Autosampler 96-well plate-sampling machine ( Intellicyt ) . Results were analyzed using FlowJo flow cytometry analysis software ( Treestar Inc . ) . Anti-retroviral drugs were used in the inhibitor time course experiments and as controls in the assembly assays . Elvitegravir , an integrase inhibitor was used at 400nM . Efavirenz , a reverse transcriptase inhibitor was used at 200nM . Cell-Tak adhesive ( BD Biosciences ) coated glass-bottom dishes were prepared on the day of use by adsorption with sodium bicarbonate and NaOH . MT4 cells , infected at a sufficiently low MOI so that each infected cell contained a single provirus were dispensed into these plates after washing , centrifuged at 350g for 8 minutes and immediately transferred to the microscope incubator . Time-lapse microscopy was performed using a VivaView FL incubator microscope ( Olympus ) . Images of infected MT-4 cells were captured every 2–5 minutes using GFP , RFP , and DIC filter sets . Preparation of movies and quantitation of microscope data was done using Metamorph software ( Molecular Devices ) . For quantitation , regions were drawn closely around immobilized cells and the maximum fluorescence intensity within each region was logged in both the GFP and mCherry channels . Fluorescence in unoccupied regions proximal to each cell was also quantified to provide background subtraction values for each cell . The background-corrected fluorescence intensities were analyzed using a custom MATLAB script that fit each set of fluorescent intensity data to a 5-parameter sigmoid logistic function . The function was used to identify time points where the signal was 7 intensity units above the baseline level . ( Images had a bit-depth of 12 , i . e . , an intensity range of 0–4095 ) . For experiments where virion yield was determined , a portion of the cells collected at each time point was resuspended in SDS-PAGE loading buffer . Simultaneously collected culture supernatants were clarified by centrifugation at 1000 rpm for 5 min filtered ( 0 . 22 μm ) , layered over 20% sucrose in 1X PBS and centrifuged at 14 , 000 rpm in an Eppendorf 5417R microfuge for 2 hours at 4°C . Virion pellets were resuspended SDS-PAGE loading buffer . Cell and virion lysates were separated on 4–12% acrylamide gels ( Novex ) and proteins transferred to nitrocellulose membranes which were then probed with antibodies against HIV-1 Gag ( 183-H12-5C , NIH AIDS Research and Reference Reagent Program ) . The blots were then probed with anti-mouse IgG conjugated to IR Dye800 CW ( LiCOR ) , scanned with a LiCOR Odyssey IR imager and quantified using Odyssey quantification software .
|
The HIV-1 replication cycle is composed of several sequential steps . While the timing of the early steps of HIV-1 replication is quite well understood , measuring the duration of later steps is complicated by the fact that asynchrony is introduced into populations of infected cells during early steps . We devised imaging methods for measuring the duration of late steps in HIV-1 replication in single infected cells , circumventing the problems associated with measurements in populations of asynchronously infected cells . By combining these measurements with conventional analyses of HIV-1 replication in populations of cells , we derived a time-line of key events during the late steps of the HIV-1 life cycle . We find that the delay between early and late gene expression is small but that a subsequent programmed delay in virus assembly enables HIV-1 to remove a host antiviral protein from infected cells before new virions are generated . In so doing , HIV-1 may prevent futile virion production .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
|
Single-Cell and Single-Cycle Analysis of HIV-1 Replication
|
Visceral leishmaniasis is a parasitic disease associated with high mortality . The most important foci of visceral leishmaniasis in Ethiopia are in the Northwest and are predominantly associated with high rates of HIV co-infection . Co-infection of visceral leishmaniasis patients with HIV results in higher mortality , treatment failure and relapse . We have previously shown that arginase , an enzyme associated with immunosuppression , was increased in patients with visceral leishmaniasis and in HIV seropositive patients; further our results showed that high arginase activity is a marker of disease severity . Here , we tested the hypothesis that increased arginase activities associated with visceral leishmaniasis and HIV infections synergize in patients co-infected with both pathogens . We recruited a cohort of patients with visceral leishmaniasis and a cohort of patients with visceral leishmaniasis and HIV infection from Gondar , Northwest Ethiopia , and recorded and compared their clinical data . Further , we measured the levels of arginase activity in the blood of these patients and identified the phenotype of arginase-expressing cells . Our results show that CD4+ T cell counts were significantly lower and the parasite load in the spleen was significantly higher in co-infected patients . Moreover , our results demonstrate that arginase activity was significantly higher in peripheral blood mononuclear cells and plasma of co-infected patients . Finally , we identified the cells-expressing arginase in the PBMCs as low-density granulocytes . Our results suggest that increased arginase might contribute to the poor disease outcome characteristic of patients with visceral leishmaniasis and HIV co-infection .
Visceral leishmaniasis ( VL ) , a neglected tropical disease caused by the parasite Leishmania ( L . ) donovani , is one of the most significant vector-borne diseases in Ethiopia , with an estimated 4500 to 5000 new cases every year [2] . VL is associated with high mortality and morbidity and hinders economic development . VL is worsened by malnutrition and co-infections with HIV . In the region with the highest endemicity for VL in Northwest Ethiopia , the prevalence of HIV seropositivity among patients with VL has been shown to be as high as 38% [1] . Patients with visceral leishmaniasis and HIV infection ( VL/HIV patients ) display an increased susceptibility to drug toxicity , increased leishmaniasis relapses and increased mortality [1] , [3] , [4]; and accelerated progression to AIDS [5] . Both Leishmania and HIV are able to infect and replicate in monocytes and macrophages and both pathogens mutually promote their replication in these host cells . Several studies have shown that infection of myeloid cells with Leishmania parasites promotes HIV replication [6] , [7] , [8] . Equally , HIV not only promotes Leishmania uptake by macrophages [9] , but also increases parasite replication in monocytes [10]; this is in agreement with the observation of increased parasitemia in VL/HIV patients [11] . One of the immunological hallmarks of VL and HIV infections is a diminished ability of PBMCs from these patients to respond to recall antigens [12] , [13] , [14] . We have recently shown that the activity of the enzyme arginase is increased in patients with visceral leishmaniasis ( VL patients ) and in HIV seropositive patients ( HIV patients ) with low CD4+ T cell counts [15] , [16] . Arginase hydrolyzes L-arginine to urea and ornithine , which is further metabolized into polyamines . Arginase can also be upregulated in myeloid cells and has been shown to impair T cell responses by reducing the bioavailability of L-arginine in the microenvironment . Since L-arginine is essential for efficient T cell activation , this decrease results in impaired T cell responses [17] , [18] , [19] . In both our HIV [20] and VL [16] studies , increased arginase activity in PBMCs was a marker of disease severity , and coincided with lower L-arginine levels and impaired T cell responses . Here , we tested the hypothesis that a synergistic increase in arginase activity occurs in VL patients co-infected with HIV as compared to VL patients , and therefore contributes to exacerbated disease outcomes .
The study was approved by the IRB of College of Medicine and Health Science , University of Gondar , reference number 09/07/2003 . For this cross-sectional study , a cohort of 26 patients with visceral leishmaniasis , but HIV seronegative uninfected ( VL patients ) and 14 VL/HIV co-infected patients was recruited from the Leishmaniasis Treatment and Research Center of Gondar University Hospital; informed written consent was obtained from each patient . All patients recruited in our study were migrant workers and male . The diagnosis of VL was based on positive serology ( rK39 , DiaMed IT Leish , DiaMed AG , Cressiers/Morat , Switzerland ) and presence of amastigotes in spleen or bone marrow aspirates . HIV seropositivity was based on the following tests: KHB Shanghai Kehua Bio-engineering Co . Ltd and Chembio HIV 1/2 STAT-PAK; Uni-Gold ( Trinity Biotech PLC ) was used to resolve ambiguous results . Out of the 14 co-infected patients , 10 were primary VL patients and 4 had a relapse of VL . Relapse is defined as follows: individuals diagnosed with visceral leishmaniasis ( clinical features and positive parasitology ) , after having been successfully treated for primary VL and been discharged with an improved medical condition or with a negative test of cure . Six patients were already on anti-retroviral therapy ( ART ) , the 8 remaining patients were initiated on ART after the treatment against VL . The treatment was given according to the recommendation of the National Guideline for Diagnosis , Treatment & Prevention of Leishmaniasis in Ethiopia: VL patients HIV+ already on ART were treated with AmBisome ( Gilaed Sciences Ltd . ( Ireland ) ) at a dose of 3–5 mg/kg daily or intermittently for 10 doses , up to a total dose of 40 mg/kg . VL patients HIV+ not on ART were treated with SSG ( 20 mg/kg for 30 days ) first and started ART upon completion of the treatment against VL . All the VL patients were treated with SSG for 30 days at a dose of 20 mg/kg . Informed written consent was obtained from each patient . The median age of the VL patients was 22 . 0±1 . 0 years ( range: 16–32 years ) and that of VL/HIV co-infected patients was 33 . 0±1 . 9 years ( range: 28–38 years ) . Saliva was collected by spitting in a sterile 50 ml tube , after the patients rinsed their mouth with clean water . The saliva sample was immediately frozen at −20°C . Ten to twenty ml of venous blood was collected into EDTA tubes before the antileishmanial treatment started . Plasma was isolated by centrifuging 1 ml of blood at 1800 rpm for 10 minutes and was frozen at −20°C until further use . Peripheral blood mononuclear cells ( PBMCs ) were isolated by density gradient centrifugation on Histopaque-1077 ( Sigma ) . Cells were washed in phosphate buffered saline ( PBS ) and used straight away for flow cytometry; PBMCs used for arginase and protein determination were immediately resuspended in lysis buffer ( 0 . 1% Triton X-100 , 25 mM Tris-HCl and 10 mM MnCl2 , Sigma ) and frozen at −20°C until further use . CD4+ and CD8+ T cell counts were determined using a BD Multi TEST kit ( BD Biosciences ) and acquisition was performed using a FACSCalibur ( BD Biosciences ) . Liver and renal function tests were performed as part of the routine management practice . The following reagents ( HUMAN Diagnostics ) were used according to the manufacturer's protocol: GOT ( ASAT ) ; Aspartate aminotransferase liquiUV test; GPT ( ALAT ) , Alanine aminotransferase liquiUV test; ALP , Alkaline Phosphatase liquicolor test; BUN ( Blood Urea Nitrogen ) , Urea liquicolor test; Creatinine , creatinine liquicolor test . Determination of parasite burden: grading was assessed as previously described in the splenic aspirates [21] and the same grading system was applied for the bone marrow aspirates . The enzymatic activity of arginase was measured as previously described [15] . To activate arginase , 50 µl of PBMCs ( resuspended in lysis buffer = 0 . 1% Triton X-100/10 mM MnCl2/25 mM Tris-HCl ) were incubated for 10 min at 56°C . Arginine hydrolysis was achieved by incubating the lysate with 50 µl of 0 . 5 M L-arginine ( pH 9 . 7 ) at 37°C for 15–120 min . The reaction was stopped with 400 µl of H2SO4 ( 96% ) /H3PO4 ( 85% ) /H2O ( 1/3/7 , v/v/v ) . Urea concentration was measured at 550 nm after addition of 20 µl α-isonitrosopropiophenone ( dissolved in 100% ethanol ) , followed by heating at 100°C for 45 min . To determine arginase activity in the plasma , urea concentrations were first determined in the sera , without the activation and hydrolysis steps; these values were subtracted from those obtained by measuring the urea levels as described above . One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of urea per min . To determine the protein concentration of lysed PBMC samples , serial dilutions of each PBMC lysate were made in PBS ( Sigma ) . BCA Protein Assay Reagent ( Pierce ) was added to each PBMC dilution following supplier's recommendations . A bovine serum albumin ( BSA ) standard ( Pierce ) was serially diluted using PBS . Following 30 minutes incubation at 37°C , the optical density ( OD ) was measured at 570 nm . Antibodies used were as follows: anti-CD14 ( BD Pharmingen: cloneM5E2 ) , and anti-CD15 ( Clone H198 , BD Pharmingen ) . Anti-arginase I ( HyCult Biotechnology: clone 6G3 ) and the isotype control ( BD Pharmingen: clone MOPC21 ) were coupled with Alexa FluorR 488 ( Molecular Probes ) . Cells were washed with PBS , the fixation step was performed with 2% formaldehyde in PBS and the permeabilization step with 0 . 5% saponin in PBS . The determination of intracellular arginase was performed as described in [15] . The percentages for the isotype controls were <1 . 23% . Acquisition was performed using a FACSCalibur ( BD Biosciences ) and data were analyzed using Summit v4 . 3 software . Data were evaluated for statistical differences using a two-tailed Mann-Whitney test ( GraphPad Prism 5 ) and differences were considered statistically significant at p<0 . 05 . Unless otherwise specified , results are expressed as median±SEM .
For this study , a cohort of 26 VL patients and 14 VL/HIV co-infected patients was recruited . All patients were male . The duration of illness was defined as the number of weeks since the patients noticed symptoms associated with visceral leishmaniasis: the most common symptom that they refer to was fever , but could also be weight loss , epistaxis , fatigue and abdominal swelling ( as a sign of enlarged spleen or liver ) . For those patients with relapsing VL , the duration of illness only applies to the onset of the relapse . Duration of illness ranged from 2–24 weeks among the VL patients ( 4±1 . 2 weeks ) and from 4–96 weeks among the VL/HIV patients ( 24±6 . 4 weeks ) ( data not illustrated ) . All patients had an enlarged spleen and liver , but the differences between the two groups were not significant ( Table 1 , p>0 . 05 ) . Their liver and kidney functions were assessed , and as shown in Table 2 , all the values were similar between VL and VL/HIV patients ( p>0 . 05 ) . The parasite grade was significantly higher in splenic aspirates from co-infected patients as compared to VL patients ( 6 . 0±0 . 3 vs 4 . 0±0 . 3 , p<0 . 0001 , Figure 1A ) . Parasite grade in the bone marrow aspirates appeared similar in both groups , however , it is difficult to draw a definite conclusion from these data , as there were only 3 values for the VL/HIV co-infected patients ( Figure 1B ) . The nutritional status of the two cohorts was assessed by calculating their body mass index ( BMI ) and measuring their upper arm circumference . As shown in Figure 2A , the medians BMI of both VL and VL/HIV groups were remarkably low and the median BMI of VL/HIV patients was similar to the BMI of VL patients ( VL: 16 . 5±0 . 3 and VL/HIV: 15 . 8±0 . 6 , p = 0 . 06 ) . Twenty-three out of 25 VL patients and 13 out of 14 VL/HIV patients had a BMI<18 . 5; 12 out of 25 VL patients ( 48 . 0% ) and 11 out of 14 VL/HIV patients ( 78 . 6% ) had a BMI <16 . 5; indicative that the VL/HIV cohort was more severely malnourished . Although the median upper arm circumference was lower in the VL/HIV cohort than the VL cohort , this difference was not statistically significant ( Figure 2B , 21 . 0±0 . 5 cm vs 19 . 0±0 . 7 cm , p = 0 . 1210 ) . Both CD4+ and CD8+ T cell counts were below the normal range in both groups of patients ( Figures 3 ) . Moreover , CD4+ T cell counts were significantly lower in VL/HIV patients ( VL: 173±24 . 1 , VL/HIV: 34 . 5±13 . 5 , p<0 . 0001 , Figure 3A ) . Although CD8+ T cell counts were below the normal range in both VL and co-infected patients ( 164±22 . 3 and 195±41 . 9 respectively , Figure 3B ) , the difference between the two groups was not statistically significant ( p = 0 . 5799 ) . Our previous study had shown that arginase activity was higher than normal in the blood of HIV+ patients [15] and in the blood of VL patients [16] . In the present study , we tested the hypothesis that arginase activity was even further increased in co-infected patients . In order to reduce the number of venipunctures , we tested whether arginase is detectable in saliva; our results show that it is clearly measurable in saliva ( Figure 4A ) , however , the values obtained in each group were similar: VL: 70±28 . 2 mU/ml of saliva and VL/HIV: 80±25 . 1 mU/ml ( p = 0 . 2925 ) . Next we measured the levels of arginase activity in PBMCs and in plasma . The results in Figure 4B show that arginase activity was statistically significantly higher in PBMCs from co-infected patients ( 203±148 . 1 mU/mg protein ) than those from VL patients ( 94 . 2±16 . 3 , p = 0 . 0308 ) . Similar results were obtained with the plasma: VL: 6 . 9±1 . 3 mU/ml vs VL/HIV: 13 . 1±1 . 6 mU/ml ( p = 0 . 0162 ) . These results show that arginase activity is even higher in PBMCs and plasma of co-infected patients as compared to VL patients . Finally , we determined the phenotype of arginase-expressing cells among the PBMCs . Our results are in agreement with our previous results [15] , [16] , [22] and show that neutrophils are the main type of cells in the PBMCs that express arginase . Indeed , in all 8 VL patients tested >98 . 9% of CD15+ and <0 . 24% were CD15− arginase+ ( Figure 5A ) . Similarly , in all 5 VL/HIV patients tested , >98 . 2% of CD15+ cells expressed arginase and <0 . 31% are CD15− arginase+ ( Figure 5B ) . These results demonstrate that neutrophils are the main cells expressing arginase in the PBMCs of VL and VL/HIV patients .
The results of our study show that arginase levels are significantly higher in VL/HIV coinfected patients than in VL patients . We propose that increased arginase-mediated L-arginine metabolism contributes to T cell dysfunction and as a consequence , to the poor outcome associated with VL/HIV co-infection . We and others have shown that higher arginase activity coincides with lower levels of L-arginine and lower expression levels of CD3ζ in T cells [15] , [16] , [23] , suggesting that T cell functions are compromised when activation occurs in the presence of suboptimal levels of L-arginine . One of the main immunological characteristics of both HIV and VL patients is the failure of their PBMCs to respond efficiently to antigen-specific restimulation [12] , [13] , [14] , [24] , [25] . This is even further impaired in VL/HIV co-infected patients: PBMCs from these patients proliferate less and produce considerably lower levels of IL-2 and IFN-γ in response to Leishmania and HIV antigenic restimulation [26] . And indeed , the CD4+ T cell counts , which are closely associated with disease severity and immune suppression [12] , [27] , [28] , [29] were significantly lower in VL/HIV patients as compared to VL patients . The results presented in our study could explain this weakened immune response as we show that arginase , an enzyme with potent immunosuppressive properties , is even further increased in the PBMCs from VL/HIV patients . These higher levels of arginase activity may also explain why VL/HIV patients develop more frequent opportunistic infections , such as tuberculosis and pneumonia [5] , [30] . In addition to impairing T cell functions , arginase activity has also been shown to favour parasite growth in macrophages: arginase catabolises L-arginine into polyamines , which are the main intracellular source of the polyamines necessary for the growth of the parasites [31] . Similarly , polyamines also appear be important for HIV replication , since blocking of a key enzyme in polyamine synthesis efficiently suppresses HIV-1 replication [32] . Therefore it is possible that polyamines resulting from the catabolism of L-arginine by arginase benefit both Leishmania and HIV replication . This might also account for the higher parasite grade in VL/HIV co-infected patients . Arginase-expressing macrophages have been shown to promote parasite growth in vitro in experimental models of leishmaniasis [31] , [33] , and increased arginase activity at the site of pathology is a hallmark of nonhealing infections [31] , [34] . However , the phenotype of arginase-expressing cells in the lesions has not yet been established . In human blood , the main arginase-expressing cell types are neutrophils [35] , myeloid suppressor cells [36] and alternatively activated macrophages [37] . Here , we show that over 98% of CD15+ cells express arginase and only a minority of CD14+ monocytes express arginase . We and others have previously shown that a population of neutrophils co-purify with PBMCs following density gradient centrifugation [15] , [37] , [38] , [39] . Because of their difference in density , these cells are referred to as low-density granulocytes ( LDGs ) . Our data from HIV patients [20] and VL patients [16] suggest that these neutrophils have degranulated and that an increased frequency of LDGs coincides with disease severity: in HIV+ patients , we found a strong inverse correlation between the percentages of LDGs and CD4+ T cells counts [20] , similar to that we found between arginase activity and CD4+ T cell counts [15]; in our VL study , we showed that the frequency of LDGs was significantly increased in PBMCs of patients before treatment . Further characterization of these cells in VL/HIV patients will be essential to define how they contribute to exacerbated disease outcomes . Severe malnutrition was remarkably common in VL/HIV patients: 78 . 6% of these patients had a BMI below 16 . 5 . Malnutrition is associated with lower levels of L-arginine in the plasma [40] , [41] , [42] and we cannot exclude the possibility that malnutrition in these patients also contributed to the diminished levels of L-arginine in the plasma . The spleen and liver sizes and the liver and kidney functions were similar in the VL and the VL/HIV patients , this is in agreement with the study by Hurisssa et al [1] , that shows that the clinical presentation were similar in both groups of patients . In agreement too with the publication by Hurissa et al [1] was the intriguing finding that the duration of illness was longer in VL/HIV patients . The duration of illness was defined by the patient , from the onset of his symptoms , and it is therefore difficult to determine whether this information was accurate . However , it is possible that since T cells from co-infected patients display significantly more impaired immune functions [26] , VL/HIV patients might therefore experienced symptoms earlier than the VL patients . It was also noticed that in contrast to VL patients , co-infected patients may not mount fever or only low grade fever , this would delay the process of seeking medical attention . The development of new treatments that address the specific requirements associated with co-infection is of utmost importance . Our results have important implications for understanding the pathogenesis of VL/HIV co-infections . This may open up new therapeutic avenues to target dysregulated T cells responses . Further , the results of our study are not only applicable to understanding VL/HIV co-infections , but might also be relevant in understanding the exacerbated disease outcome associated with other co-infections .
|
Visceral leishmaniasis ( VL ) in Ethiopia is caused by the parasite Leishmania donovani . This disease has one of the highest mortality rates: if left untreated , it is almost always fatal . VL belongs to the most neglected tropical diseases , affecting the poorest populations , for whom access to diagnosis and effective treatment are often not available . VL is worsened when the patients are co-infected with HIV . We have recently shown that patients with visceral leishmaniasis ( VL patients ) and HIV seropositive patients ( HIV+ patients ) have increased levels of an enzyme , arginase , in their blood . This enzyme has been shown to prevent cells of the immune system from mounting an efficient response and controlling the replication of the virus in HIV+ patients or the parasites in VL patients . In this study , we show that arginase is considerably higher in the blood of VL/HIV co-infected patients as compared to VL patients . Our results suggest that this abnormally high arginase might contribute to the poor prognosis associated to VL/HIV patients .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"biochemistry",
"infectious",
"diseases",
"enzyme",
"metabolism",
"enzymes",
"neglected",
"tropical",
"diseases",
"biology",
"viral",
"diseases",
"parasitic",
"diseases"
] |
2013
|
Arginase Activity in the Blood of Patients with Visceral Leishmaniasis and HIV Infection
|
It has long been thought that signal joints , the byproducts of V ( D ) J recombination , are not involved in the dynamics of the rearrangement process . Evidence has now started to accumulate that this is not the case , and that signal joints play unsuspected roles in events that might compromise genomic integrity . Here we show both ex vivo and in vivo that the episomal circles excised during the normal process of receptor gene rearrangement may be reintegrated into the genome through trans-V ( D ) J recombination occurring between the episomal signal joint and an immunoglobulin/T-cell receptor target . We further demonstrate that cryptic recombination sites involved in T-cell acute lymphoblastic leukemia–associated chromosomal translocations constitute hotspots of insertion . Eventually , the identification of two in vivo cases associating episomal reintegration and chromosomal translocation suggests that reintegration events are linked to genomic instability . Altogether , our data suggest that V ( D ) J-mediated reintegration of episomal circles , an event likely eluding classical cytogenetic screenings , might represent an additional potent source of genomic instability and lymphoid cancer .
V ( D ) J recombination is a unique mechanism of somatic recombination aimed to provide a large antigen receptor repertoire in T and B cells ( for review , see [1] and references therein ) . During this process , the variable ( V ) diversity ( D ) and joining ( J ) gene segments present within the immunoglobulin ( IG ) and T-cell receptor ( TCR ) loci , are assembled to form a complete VDJ exon encoding the variable region of the IG/TCR ( Figure 1A ) . The recombination requires the presence of specific motifs flanking all the V , D , and J gene segments ( 12– and 23–recombination signal sequences [RSSs] ) , and allowing the recruitment , binding , and proper positioning of the products of the recombination activating genes 1 and 2 ( RAG-1/2 ) . Recent data suggest that in vivo , RAG-1/2 proteins initiate the rearrangement by performing a first single-strand nick at the exact border between a 12-RSS and its adjacent coding gene segment [2] . This leads to the capture of a 23-RSS , the formation of a 12/23 synaptic complex in which the two DNA/protein structures are held in close juxtaposition , and the generation of another nick at the captured 23-RSS . Within this complex , RAG-1/2 catalyzes a trans-esterification reaction in which each liberated hydroxyl group attacks the opposite DNA strand [3]; this generates four broken ends held in a postcleavage synaptic complex: two blunt RSSs or signal ends ( SEs ) , and two covalently sealed hairpin coding ends ( CEs ) . The broken ends are then efficiently repaired by the nonhomologous end-joining ( NHEJ ) pathway [4–6]; on the one hand , hairpins present at the CEs are resolved through the Artemis endonuclease activity; when opened at bases off the apex , hairpin opening generates overhanging flaps , which , if filled in by a DNA polymerase activity , form palindromic ( P ) stretches . Nontemplated ( N ) nucleotides may be added de novo by the terminal deoxynucleotidyl transferase ( TdT ) , and/or nucleotides may be deleted from the CEs . Ligation of the processed CEs forms a highly diversified coding joint ( CJ ) . By contrast , the two SEs present in the synaptic complex undergo only limited processing ( some N addition and rare nucleotide deletion ) before joining into signal joints ( SJs ) . While CJs give rise to the functional recombination products , the SJs are merely the byproduct of V ( D ) J recombination . SJs have until recently been assumed to be “harmless” and irrelevant in the dynamics of the V ( D ) J recombination process , but evidence now starts to accumulate that this is not the case , and that they play unsuspected roles in events which might compromise genomic integrity [7 , 8] . SJs are indeed constituted of two functional RSSs fused back to back , each of which therefore potentially capable of further V ( D ) J rearrangement in presence of RAG-1/2 . The issue of SJ reactivity was initially addressed ex vivo by the use of integrated minilocus and transient extrachromosomal recombination substrates containing germline gene segments flanked by their RSSs , and undergoing rearrangement in culture [9–11] . Both integrative and extrachromosomal experiments indicated that , following a first rearrangement by inversion , the SJ produced was indeed reactive , and could engage into further cycles of rearrangement with RSS partners in cis ( similar to Figure 1C and 1D ) . In vivo and ex vivo observations have revealed that the products resulting from such secondary SJ rearrangements consist of one new SJ and one hybrid RSS/coding-segment junction ( hybrid joint [HJ] ) , albeit with the molecular features of a CJ ( i . e . , with N nucleotide insertion , and extensive nucleotide deletion and P nucleotide addition at both the RSS and coding segment sides; Figure 1D ) [8–10 , 12] . This junction , which we refer to as a “pseudo-hybrid” joint ( ΨHJ ) , is thereby morphologically distinguishable from CJs , SJs , and to a large extent from “genuine” HJs [13–18] . ΨHJs constitute therefore specific signatures of such ongoing SJ rearrangement events . Interestingly , recent in vivo data suggest that IGK/IGL rearrangement hierarchy and isotypic exclusion might in part be achieved by ongoing SJ recombination [12] . Thus , SJ reactivity might have also evolved as part of the dynamics of the V ( D ) J rearrangement process . Eventually , the pathological counterpart of this possible physiological extension of the V ( D ) J recombination capability has also been shown to occur in cases of oncogenic chromosomal translocation , in which ongoing rearrangement of the resulting chromosomal SJ ( CSJ ) constitutes the source of oncogene activation [8] . In the normal process of V ( D ) J recombination , the large majority of SJs produced is however not retained on the chromosome , but excised on episomal circles ( ECs; Figure 1A ) . Because ex vivo RAG binding ( or rebinding ) also efficiently takes place on episomal SJs ( ESJs ) , leading to SJ recleavage and , at least in vitro , to RAG transposition [7] , we reasoned that ongoing trans-V ( D ) J recombination might also and concurrently occur ( Figure 1B ) . This might result in the same type of insertion of the whole circle into the genome as previously observed in vivo for RAG-mediated transposition [19] , with the important difference that it would in this case employ trans-V ( D ) J recombination [20–23] , a process potentially more efficient than RAG transposition [15 , 24–28] . Both mechanisms might obviously lead to similar genomic instability events , including oncogenic activation/deregulation . In this report , we investigated V ( D ) J-mediated ESJ insertion as an additional potent source of genomic instability and oncogenic deregulation in lymphoid cells .
To investigate the possibility that excised episomes can reintegrate the genome through ongoing recombination of the SJ , we first assessed the ability of an ESJ to undergo trans-V ( D ) J recombination in an ex vivo trans-recombination substrate assay [20] ( Figure 2A ) . Three different human ESJs and two standard RSSs were cloned in separate extrachromosomal recombination substrates; two genuine SJs were used as “donor” ESJ plasmids: Jδ1/Dδ3 and Ki/Jκ3 [12]; furthermore , an artificial Dβ1Δ ESJ was generated by mutagenesis deletion of the 12-bp Dβ1 coding sequence located between the Dβ1 5′ and 3′ RSSs; the human Jβ2 . 7 and VκA27 gene segments were used as 12-RSS “target” substrates . NIH3T3 fibroblasts were cotransfected with donor and target plasmids either with or without RAG-1/2 and TdT expression vectors . Bulk plasmid DNA was recovered after 48 h of culture , and junctions resulting from trans-recombination between the ESJ and the target RSS were amplified in a single round PCR . Primer combinations were designed to detect the 2 expected integration breakpoints complying with a 12/23 synapsis: combination ( 3 + 2 ) was used to detect putative SJs , and combination ( 1 + 4 ) was used to detect putative ΨHJs ( Figure 2A ) . PCR products were revealed by an IRD800-labeled primer extension ( PE ) assay allowing a precise to-the-base resolution of the amplified species ( see Materials and Methods ) . In absence of the RAGs , faint and nonrecurrent PCR products scattered at various sizes were obtained for both primer combinations ( e . g . , Figure 2B , ΨHJ T2 , ~160 bp; additional bands are also present outside the visualized parts of the gels ) . To assess the identity of such junctions , double-nested secondary PCR were performed , and the amplification products were cloned and sequenced . Sequence analysis confirmed the occurrence of junctions that were not generated by RAG-mediated recombination , but rather due to random breaks joined through NHEJs and/or homologous recombination pathways ( not shown ) . Such junctions , collectively referred to as “break/repair , ” have been previously described , and represent the RAG-independent recombination background of the trans-V ( D ) J assay [29] . In presence of RAGs , however , more intense and recurrent PCR products of the expected sizes were obtained for both PCR combinations ( typical examples are illustrated for the Dβ1Δ × Jβ2 . 7 combination in Figure 2B ) . SJ primer combination ( 3 + 2 ) usually displayed one major band and few minor species around that position , as expected from a standard SJ , which generally presents limited nucleotide processing . ΨHJ primer combination ( 1 + 4 ) , on the other hand , displayed a pattern of intense bands around the expected size , representing the typical spectra of largely processed junctions . Sequence analysis of cloned products from double-nested secondary PCR confirmed the presence of the two specific breakpoints expected from V ( D ) J-mediated ongoing ESJ recombination in most cases ( Figures 3 and 4; also , see below ) . Thus , and as anticipated from previous studies describing ongoing recombination of SJ with targets in cis , our results demonstrate that ESJs are also capable of ongoing efficient RAG-mediated recombination with RSS targets in trans in the context of a 12/23 synapsis . However , as the ESJ is formed by a functional 12-RSS and a functional 23-RSS , both potentially able to bind the RAGs , we next wondered if this particular structure might allow to bypass the 12/23 rule for synapsis and give rise to additional recombination products that we would fail to detect with the two primer combinations used above . Double-nested PCR with the two complementary primer combinations ( 1 + 3 ) and ( 2 + 4 ) ( Figure 2A ) corresponding to a 12/12 synapsis were thus performed on the same bulk DNA . Such combinations , however , gave rise to only weak amplification products . Cloning and sequencing confirmed in most cases the occurrence of the symmetrical 12/12 SJ ( 1 + 3 ) and ΨHJ ( 2 + 4 ) ( Figure S1A ) . This suggests that although a fraction of the trans-recombination can occur in violation of the 12/23 rule ( as in normal V ( D ) J recombination ) , this represents a minor population , and the large majority of the RAG-mediated recombinants are detected with primer combinations ( 3 + 2 ) and ( 1 + 4 ) , in accordance with a 12/23 synapsis between the ESJ and its RSS target . Accordingly , the use of ESJ donors made of two 12-RSS in the context of a 12-RSS target did not give rise to any specific amplified signal , while each of the two RSS from an ESJ donor made of two 23-RSS gave rise to efficient recombination in the context of the same 12-RSS target ( Figure S1B ) . In addition , the use of ESJ donor constructs made of one functional and one nonamerless RSS through deletional mutagenesis showed that while deletion of the nonamer from the reactive 23-RSS completely abolished recombination , deletion of the nonamer from the bystander 12-RSS did not modify the overall efficiency ( Figure S1C ) . Altogether , our data clearly indicate that trans-V ( D ) J recombination of ESJ obeys the 12/23 rule and is not dependent on alternative mechanisms such as ESJ opening by nick–nick ( see below for a potential role of nick–nick in the generation of some junctions ) . We next analyzed in details the sequences issued from recombination of the ESJ ( Figure 3 and 4 ) . SJs obtained were morphologically undistinguishable from standard SJs , with the presence of some N insertion and limited nucleotide deletion ( Figure 3; 3T3 ) . Likewise , detailed analysis of the ΨHJ revealed that in most sequences , end processing similar to that of a standard CJ ( nucleotide deletion , N and P nucleotide addition ) occurred at both the SE ( the bystander 12-RSS of the ESJ ) and the CE sides of the joint ( Figure 4; 3T3 ) . This strongly suggests that once engaged in synapsis , and despite its ability to bind the RAGs , the bystander RSS of the ESJ mostly behaves as a coding segment , and undergoes therefore the hairpin formation step , followed by hairpin resolution and further processing ( Figure S2 , left panel ) . Nevertheless , the presence of sequences containing a full-size ESJ bystander RSS ( e . g . , Jδ1; Figure 4 ) together with the ambiguity in the assignment of P nucleotides in presence of TdT , suggested possible RAG binding on both ESJ RSSs , and involvement of alternative pathways of RAG-mediated recombination . To confirm the involvement of trans-V ( D ) J recombination and to test the potential contribution of alternative pathways , the ESJ assay was repeated in the GUETEL Artemis-deficient cell line [30] . During standard V ( D ) J recombination , failure to resolve hairpin CEs in absence of the Artemis endonuclease results in the absence of CJ formation without impeding SJ formation [31–33] . Similarly , the absence of Artemis in the ESJ assay should prevent ΨHJ formation without hindering the generation of SJs . Following cotransfection of the GUETEL cell line with the ( Ki/Jκ3 ) ESJ/VκA27 couple , PCR was performed as above and the amplification products cloned and sequenced . As expected , SJs were obtained in absence of Artemis and were undistinguishable from SJs obtained both in the Artemis-proficient 3T3 cells and in the GUETEL cell line complemented with Artemis ( GUETEL-A; [30] and Figure 3 ) . Despite the absence of Artemis , weak amplification products were also obtained for the ΨHJ PCR combination ( 1 + 4 ) . However , the sequenced junctions displayed virtual absence of N insertion and nucleotide processing ( Figure 4 ) , in sharp contrast to the morphology of ΨHJs obtained in Artemis-proficient cells ( 3T3 and GUETEL-A ) . The features of the Artemis−/− junctions are however strongly reminiscent of HJs generated by the “RAG-mediated joining” pathway [15–18] . RAG-mediated joining is an NHEJ-independent pathway related to RAG transposition in which a direct attack of a free 3′ hydroxyl group from the SE into the hairpinned CE bypasses the hairpin resolution step ( illustrated in Figure S2 , right panel ) ; this usually results in the generation of a class of HJs displaying a full-size RSS joined to a coding sequence with limited processing ( depending on the position of the attack in the hairpin ) . The presence of such junctions in the absence of Artemis suggests that both trans-V ( D ) J recombination and RAG-mediated joining concurrently occur to generate ΨHJs and HJs , respectively . However , the virtual absence of such junctions in Artemis-proficient cells ( 3T3 , Artemis-complemented ) strongly suggests that RAG-mediated joining constitutes a minor recombination pathway compared to trans-V ( D ) J recombination . Thus , the junctions with a full-size ESJ bystander RSS initially observed in Artemis-proficient cells are Artemis dependent , and are either ΨHJs in which processing is limited due to RAG-binding on the bystander RSS , or HJs generated through “RSS swapping” ( Figure S2 , middle panel ) . Altogether , our results indicate therefore that standard trans-V ( D ) J recombination is the major pathway of RAG-mediated ESJs ongoing rearrangement . How efficient is trans-V ( D ) J recombination of ESJs ? In the case of recombination between two standard coding-segment RSSs , the frequency of trans-V ( D ) J recombination has been previously shown to be reduced compared to cis-V ( D ) J recombination events [20 , 21] . In the present case of recombination between an ESJ and its RSS target , the presence of the second RSS in the SJ could structurally impede—or on the contrary stimulate—RAG fixation and/or activity on the reactive one , and modify the overall recombination efficiency . We therefore compared in the ex vivo assay the efficiency of trans-V ( D ) J recombination of an ESJ and a RSS target with the efficiency of trans-V ( D ) J recombination between two standard coding-segment RSSs ( Figure 5A ) . To do so , we tested two pairs of plasmids: ( Jδ1/Dδ3 ) ESJ × Jβ2 . 7 versus Dδ3 × Jβ2 . 7 , and ( Dβ1ΔESJ × Jβ2 . 7 versus Dβ1 × Jβ2 . 7 . Following transfection and harvesting carried out as above , semiquantitative primary PCR amplification of the breakpoints was performed , and serial dilutions were revealed by PE . As illustrated in Figure 5B for the ( Jδ1/Dδ3 ) ESJ × Jβ2 . 7 versus Dδ3 × Jβ2 . 7 couples , no significant difference could be seen in the formation rate of SJs in the presence of an ESJ . Similarly , results showed comparable rates in the formation of a ΨHJ relative to a CJ . We conclude that ESJs are at least as efficient as standard RSSs to undergo trans-V ( D ) J recombination . Altogether , this suggests that the presence of the bystander RSS does not impede the recombination process , whether structurally ( through steric constraints ) or functionally ( through nick–nick activity ) . From the mechanistic point of view , this data predicts therefore that in vivo , V ( D ) J-mediated reintegration of ESJs ( Figure 1B ) should not be different from V ( D ) J-mediated translocation ( Figure 1C ) ; most important , both processes should use the same RSS targets with the same efficiency . V ( D ) J-mediated translocations have been shown to occur not only between authentic RSSs from distinct IG/TCR loci , but also between authentic RSSs and fortuitous sequences in the genome resembling a RSS ( cryptic RSSs ) [34] . The mistargeting of the RAGs towards cryptic RSSs located in the vicinity of a silent proto-oncogene is a recurrent source of genomic instability and oncogenesis . Erroneous targeting of cryptic sites located near the LMO2 and TAL2 proto-oncogenes in t ( 11;14 ) ( p13;q11 ) and t ( 7;9 ) ( q34;q32 ) translocations , respectively , represent prototypical examples of such oncogenic translocations in T-cell acute lymphoblastic leukemia ( T-ALL ) [8 , 29 , 35–37] . Our data above suggest that in vivo , such cryptic sites might provide efficient targets for ESJ reintegration . To further define the potential oncogenic properties of episomal reintegration , we next investigated in our ex vivo assay the capacity of ESJs to target oncogenic cryptic RSS . The human LMO2 and TAL2 cryptic RSSs and flanking sequences were cloned in a recombination substrate plasmid ( Figure 6A ) and assayed in parallel to the Jβ2 . 7 segment as a target for the ( Jδ1/Dδ3 ) ESJ , using the PCR/PE assay described above . Our results show a similar considerable high rate of V ( D ) J-mediated recombination of the ESJ with the LMO2 and TAL2 cryptic RSSs than with the Jβ2 . 7 authentic RSS ( Figure 6A ) . To further estimate the likelihood in vivo of SJ insertion in such cryptic RSS , the LMO2 versus the HPRT intron 1 region were assayed as competitive targets for the ( Jδ1/Dδ3 ESJ ( Figure 6B ) . The HPRT intron 1 region was chosen as a competitor because it contains a well-described cryptic RSS classically involved in illegitimate V ( D ) J-mediated deletion of exons 2–3 in vivo [38] , and has also been identified as a site of ESJ reintegration in vivo ( [39]; see Discussion ) as well as a region of RAG transposition in vivo [19] . While recombination with the LMO2 cryptic RSS constituted a hotspot of integration , no recombination could be observed in the HPRT region , neither specifically at the cryptic RSS , nor in the surrounding sequences ( Figure 6B ) . Similar results were obtained when using a target plasmid containing a second copy of the HPRT fragment in a head-to-tail orientation , and forming a cruciform structure ( not shown ) . Altogether , these data strongly suggest that cryptic sites such as LMO2 with much higher recombinogenic potential than the HPRT intron 1 cryptic RSS might also be targeted in vivo by ESJ insertion and could potentially lead to oncogenic activation . We next investigated the physiological relevance of the reintegration of excised episomes through ongoing SJ recombination . As a first approach , we sought to assess if trans-chromosomal ΨHJ breakpoints were present and detectable in vivo , and if so , to estimate their formation rate . To do so , we designed primer combinations allowing the amplification of trans-TCR ΨHJ in mouse thymocyte DNA ( Table 1 ) . Additional combinations designed to amplify trans-TCR CJs and trans-TCR SJs were also performed as reference . To detect such relatively rare trans-TCR recombination events , we used a sensitive fluctuation PCR assay allowing the detection of less than one recombination event in a million cells ( see Materials and Methods ) . Positive PCR-amplification replicates were obtained for all trans-TCR combinations in the broad range of ~1 in 1 , 000 , 000 cells to 1 in 10 , 000 cells . Sequence analysis of the PCR-amplification products was carried out to assess the identity of the junctions , and revealed the presence of ΨHJs ( Figure S3 ) , CJs , and SJs ( not shown ) . Importantly , the ΨHJs obtained in vivo displayed the same molecular features as observed in the ex vivo assay , with extensive nucleotide processing on both sides of the junctions , and N/P addition . These results clearly demonstrate that ΨHJ breakpoints are indeed generated in vivo . Furthermore , such junctions are readily detectable in mouse thymocytes at a range similar to that of equivalent trans-CSJs and CJs . In line with our ex vivo results , this indicates that in vivo , SJs undergo efficient ongoing cis- and/or trans-rearrangement with RSS targets in the genome with surprisingly high frequency . This suggests further that in presence of RAGs , CSJs and/or ESJs are indeed very recombinogenic structures . Although we demonstrated above that trans-chromosomal ΨHJs are readily detectable in vivo , such breakpoints do not represent exclusive signatures of ESJ reintegration , as they may also derive from ongoing recombination of trans-CSJs with neighboring gene segments in cis ( compare Figure 1B and 1D ) . To ensure that the ΨHJs observed above did not exclusively derive from CSJs issued from trans-TCR translocations , and to estimate the rate of episomal reintegration in vivo , we generated double mutant mice in which the generation of trans-CSJs is abolished ( Figure 7 ) . Eβ−/− knockout mice , in which deletion of the 560-bp Eβ core generates an >100-fold reduction in TCRβ rearrangements [40] and TCRδ/β translocations ( Table 1 ) , were crossed with a Dβ1GFP knockin mouse , in which the introduction of the GFP in the Dβ1 gene segment and flanking 23-RSS abolishes Dβ1-Jβ1/2 rearrangements ( SS , OC , PF , unpublished data ) . In the ( Dβ1GFP × Eβ− ) double-mutant ( DE ) mice , all TCRβ chains are consequently produced via a Vβ-Dβ2-Jβ2 rearrangement from the Dβ1GFP allele ( unpublished data ) , and all TCR excision circles issued from Dβ-Jβ rearrangements carry a ( Dβ2Jβ2 ) ESJ ( Figure 7A ) . Because one allele is knocked out for Eβ , and the other produces a TCRβ chain , translocations to TCRβ cannot be present in TCRαβ+ cells from DE mice . In absence of translocation to TCRβ , no trans-TCRβδ CSJ is produced , and all Jβ2 . 7/Jδ1 ΨHJs are consequently issued from episomal reintegration . As shown in Table 1 , while the rate of Jβ2 . 7/Jδ1 ΨHJs was decreased over ~40-fold in total thymocytes from the Eβ−/− mice compared to WT , the rate of Jβ2 . 7/Jδ1 ΨHJs was only decreased 1 . 2-fold in sorted TCRαβ+ cells from DE mice compared to WT . Altogether , and in agreement with the ex vivo data , these results clearly demonstrate that V ( D ) J-mediated episomal reintegration is indeed occurring in vivo , and at a rate comparable to that of V ( D ) J-mediated chromosomal translocation . During our screen of trans-TCR SJs described above , we noticed the presence of amplification products larger than expected . Out of a total of 310 PCR replicates of seven distinct trans-TCRδ/β SJ combinations , 120 were PCR positive and four were of unexpected larger size ( unpublished data ) . Sequencing of the cloned products revealed that large amplicons resulted from cis-V ( D ) J recombination to a cryptic site in one case , and to a downstream gene segment in another case; eventually , two of the four cases showed the insertion of a sequence from the TCRδ locus with features compatible with RAG-mediated ESJ reintegration . In one of these two cases , a ~900-bp Jδ1-Dδ2 fragment was inserted into a Jβ2 . 7/Vδ2 CSJ target ( Figure 8A ) . The breakpoints consisted of a perfect SJ on the right arm of the insertion; on the left arm , a junction compatible with a ΨHJ was found , displaying a deletion of three nucleotides on one side of the joint , a deletion of one nucleotide on the other side , and one N nucleotide addition; in the second case , the same ~900-bp Jδ1-Dδ2 fragment was observed inserted into a Dβ1/Vδ2 CSJ target ( Figure 8B ) . Similarly , one of the breakpoints displayed a perfect SJ , and the other breakpoint consisted of an SJ with 2 N insertions . Considering the structure of the target , this last junction was ambiguous to assign as an SJ or a ΨHJ , and could have occurred through nick–nick , V ( D ) J , and/or RAG-mediated joining . Nevertheless , all junctions complied with the 12/23 rule and with features of RAG-mediated breaks , and it is therefore very likely that both cases represent in vivo examples of RAG-mediated reintegration of a Jδ1-Dδ2 ESJ . In the two cases , both ESJ reintegration and translocation events occurred , and two mechanisms could therefore account for their formation: either the trans-TCR translocations occurred first , and provided a trans-TCR SJ target for ESJ reintegration; or , alternatively , ESJ reintegration in a standard RSS target ( e . g . , Vδ2 ) could have occurred first , and provided a trans-TCR structure and/or breakpoints prone to V ( D ) J-mediated translocation . Intriguingly , the actual frequency of this double translocation/insertion event ( 2/120 = ~10−2 trans-recombination events ) was not compatible with the expected combined frequency of each independent event: ( trans-TCR translocation ~10−4–10−6 × reintegration ~10−4–10−6 = ~10−8–10−12 ) . Furthermore , as the assay is limited to PCR-amplifiable sizes of the inserted fragment ( such as the relatively short Jδ1-Dδ2 episome ) , the apparent rate of reintegration is probably vastly underestimated . This strongly suggests that the two events are linked , either because CSJs constitute preferential targets for ESJ reintegration , or because ESJ reintegration generates genomic instability leading to further chromosomal abnormalities; such abnormalities could consist of chromosomal translocations as detected here , or potentially more complex events which could not be detected in the present in vivo assay . Altogether , these data demonstrate that in vivo , SJs located on excised ECs may be reintegrated into the genome through trans-V ( D ) J recombination using standard and cryptic RSS targets , and further suggest that this event is associated with additional genomic instability .
We have demonstrated in ex vivo assays that the efficiency of trans-V ( D ) J recombination of an ESJ with a RSS target is not quantitatively different from the trans-V ( D ) J recombination occurring between two RSSs . This is somehow unexpected , because its particular structure confers additional properties to the ESJ . ESJs are efficiently cleaved ex vivo and in vitro by the nick–nick mechanism , a symmetrical nick occurring 5′ of each RSS ( simultaneously or sequentially ) and generating two flush SEs ending with 3′ hydroxyl groups [7] . Remarkably , this process bypasses both the formation of a hairpin intermediate and the need of synapsis with another RSS . In the context of ongoing SJ recombination , one could think that efficient nick–nick opening of lone ESJs upon RAG binding might prevent the occurrence of synapsis with a RSS target , and thus considerably reduce the overall frequency of trans-V ( D ) J rearrangement . Our ex vivo data clearly argue against this assumption , and suggest that trans-V ( D ) J rearrangement is largely independent of nick–nick . This however does not preclude that ESJ opening by nick–nick might occasionally participate in the synapsis , and the recent in vivo demonstration of synapsis by capture [2] provides a plausible two-step scenario of the occurrence of nick–nick within a 12/23 synapse ( Figure S4 ) . Incidentally , our data provides additional evidence that HJs can be formed ex vivo through both NHEJ-dependent and NHEJ-independent pathways ( Figure S2 ) . RAG-mediated joining has been initially proposed as an efficient alternative pathway of NHEJ-independent HJ formation [16–18] . However , conflicting data have been reported on the relevance of this pathway ex vivo and in vivo [15 , 25–27 , 45 , 46] . In particular , recent data indicated that at least ex vivo , RAG-mediated joining is seldom observed in presence of full-length RAGs , even in the absence of the concurrent NHEJ pathway [15 , 46] . We find here the presence of HJ with features of RAG-mediated joining in the absence , but not in the presence of Artemis , suggesting that at least ex vivo , HJ formation is a mixed process consisting of efficient RSS “swapping” and inefficient RAG-mediated joining . Interestingly , it is possible that the frequency of RAG-mediated joining would be favored in our assay , due to the participation of an ESJ . As mentioned above , nick–nick opening of the ESJ in the context of a 12/23 synaptic complex would generate two flush SEs ending with 3′ hydroxyl groups . While the two reactive RSSs might be sequestered by tight binding in the SE postcleavage complex ( Figure S4 ) , this additional free 3′ OH would provide an available substrate for hairpin attack , at least in absence of concurrent hairpin resolution by Artemis . Thus , nick–nick opening might participate in both trans-V ( D ) J recombination and RAG-mediated joining . Using WT and DE double-mutant mice , we could estimate that ESJ reintegration in authentic TCR RSS targets occurs in vivo in the broad range of 1 in 1 , 000 , 000 to 1 in 10 , 000 mouse thymocytes , depending on the integration site . In agreement with our results , recent data provide further evidence for the existence of V ( D ) J-mediated ESJ reintegration in vivo , and concur with the idea that it constitutes a significant source of genomic instability . Using a screen for HPRT mutants , Finette and colleagues recently identified a case in which a Vα/Jα episome was found inserted in the cryptic 23-RSS from HPRT intron 1 [39] . This finding is all the more remarkable given that this HPRT cryptic site has been shown to exhibit a very low recombinogenic potential in functional assays ( [47] , our unpublished data ) , and provides direct evidence that in vivo , human cryptic RSSs constitute efficient targets for ESJ reintegration . In the same line , using a double selection assay allowing for the recovery and quantification of excision/reinsertion events in a pre–B-cell line , Reddy et al . recently identified three cases of V ( D ) J-mediated reintegration [48] . In full agreement with our in vivo estimation , they evaluated a rate of one reintegration out of every 100 , 000 V ( D ) J recombinations . Considering the average number of V ( D ) J recombination per B or T cell , these data suggest that the daily lymphocyte output in human could be accompanied by as many as 5 , 000 ESJ reintegration events . Authentic RSSs from IG/TCR must without doubt constitute preferential targets for such reintegration . However , there are an estimated 10 million functional cryptic sites dispersed throughout the human genome [34 , 47] , some of them displaying much higher recombinogenic potential than the HPRT intron 1 cryptic RSS in which ESJ reintegration was found [29 , 37] . Most important , some of them are already known to provide recurrent targets for oncogenic ( type 1 ) V ( D ) J-mediated translocations . In T-ALL , for example , the mistargeting of the RAGs towards such cryptic RSSs located in the vicinity of a silent proto-oncogene is a recurrent source of genomic instability and oncogenesis [34] . We demonstrate in our ex vivo assay that cryptic sites involved in oncogenic V ( D ) J-mediated translocations are also hotspots for ESJ recombination; it seems thus reasonable to think that in vivo , such cryptic RSSs might provide as efficient targets for ESJ reintegration than for chromosomal translocations . ESJ reintegration could lead to similar oncogenic activation/deregulation , either through the insertion of active immune regulatory elements ( e . g . , the TCRδ enhancer excised during ΔRec/ΨJα or Vα/Jα rearrangements [42 , 49] , or the IGK enhancer excised during KDE/Ki rearrangements [12 , 50] ) or through the disruption of locus silencing ( e . g . , the negative regulatory element upstream of LMO2 [51 , 52] , WA Dik; BN et al . , unpublished data ) . If ESJs are at least as efficient as any standard coding-segment RSS to undergo V ( D ) J recombination with a RSS target in trans , the two processes are nearly identical mechanistically , and the RSS/cryptic RSS targets are the same , why then has V ( D ) J-mediated oncogenic translocation been largely documented in the literature , but V ( D ) J-mediated reintegration never been reported up to now in lymphoid malignancies ? Higher-order spatial genome organization is a contributing factor in the formation of recurrent translocations [53] . In contrast , increased mobility of the EC might favor interactions and recombination of ESJs , with targets located in parts of the genome generally segregated . Oncogenic targets might thus be distinct for reintegration and translocation . However , due to the multiplicity and size range ( from <1 Kb to several Mb ) of the excised episomes , as well as the diversity of insertion targets , reintegration events are very unlikely to be detected by routine analysis . Particularly , and in contrast to chromosomal translocations , episomal reintegration is invisible by standard karyotype analysis [54 , 55] . Few examples of identified class-switch recombination– and RAG-mediated episomal reintegration in lymphoid neoplasia provide proof of principle that episomal reintegration can indeed lead to cancer , and illustrate the complexity and unlikelihood of identifying such events without specifically designed screens [55–57] . For example , using complex DNA fiber-FISH and three-color interphase FISH techniques on samples from follicular lymphoma patients devoid of the hallmark t ( 14;18 ) ( q32;q21 ) translocation , Vaandrager et al . identified two cases in which the BCL2 gene was excised from 18q21 and inserted into the IGH locus at 14q32 [58] . The relatively high frequency ( 5% ) of such events , discovered relatively recently by Vaandrager et al . out of an abundantly studied and characterized pathology , illustrate well the extent to which insertional events might be missed by routine cytogenetic and molecular analysis . Another possibility which could account for the rarity of cases of episomal reintegration observed in lymphoid neoplasia is that episomal reinsertions might generate unstable transitory structures in the genome , leading to additional aberration events and more complex genomic configuration . This possibility is supported by the unexpected high frequency of two in vivo cases observed in this study , combining ESJ reintegration and translocation events . Similar examples of genomic instability following reintegration of episomal structures have been previously documented . In mouse plasmacytoma , Kovalchuk and colleagues have shown that class-switch–mediated Eμ/Sμ episomal reintegration into c-MYC favors t ( 12;15 ) translocation [56] . Although the ground of such instability is not yet clear , an obvious possibility is that a fraction of the recombination events might lead to incomplete insertion in vivo . Alternatively , the reinserted episomes might in vivo retain their initial “open” chromatinized structures , and provide preferential accessible targets for the recombination machinery . The V ( D ) J-mediated instability of reinserted ESJs would be especially relevant in neoplastic cells such as T-ALLs , in which arrest of differentiation at early stages of T-cell development leads to sustained RAG levels . Gene profiling studies have shown that a substantial fraction of the T-ALL cases display oncogenic activation in absence of detectable chromosomal alterations [59] , suggesting the presence of alternative pathways of oncogenesis . Some of them have indeed been recently discovered , and involve episomal structures [60] . Considering the large number of ESJs produced daily and the mechanistic similarities between ESJ reintegration and oncogenic translocations , our data suggest that reintegration of excised ECs by the V ( D ) J recombinase might also account for some of these cases , and constitute an additional potent source of genomic instability . Definitive answer to this open question will however await large-scale screens of human lymphoid cancer samples with specifically adapted strategies .
Recombination substrates were derived from a series previously described [29] , and the constructs are summarized in Figure S5 . The various gene segments containing the regions to recombine were amplified from human DNA with appropriate tailed primers and cloned in the recombination substrate using unique restriction sites ( Mlu1 , Sac2 , Not1 ) . The ( Ki/Jκ3 ) ESJ was cloned ( Not1/BamH1 ) in the pPCR-scriptAmp vector ( Stratagene , http://www . stratagene . com ) . Some constructs were flanked by “tag” sequences , which were previously tested to be devoid of functional cryptic RSSs , and in which specific PCR primers were designed . NIH 3T3 Swiss mouse fibroblasts , and human Artemis-deficient GUETEL or Artemis-complemented GUETEL-A cell lines [30] ( generously supplied by J . -P . de Villartay ) were cultured in standard conditions ( DMEM/10% FCS ) . Cells ( 2 × 106 ) were transfected with 3 μg of each recombination substrate , 2 μg pEBB-RAG1 , 2 μg pEBB-RAG2 expression vectors ( generously supplied by C . Roman and S . Cherry ) , and 2 μg pCDNA3TdT expression vector ( TdT cDNA from the pTDT expression vector [a generous gift from N . Doyen] recloned into pcDNA3 [Invitrogen , http://www . invitrogen . com] ) using Superfect ( Qiagen , http://www . qiagen . com ) according to the instructions recommended by the manufacturer . Transfected cells were transferred to 10 ml DMEM supplemented with 10% FCS and cultured for 48 h . Cells were subsequently trypsinized , and plasmids recovered by alkaline lysis and phenol-chloroform extraction as previously described [29] . To test the possibility that trans-rearrangements could occur through a first step of homologous recombination between the identical core regions of the donor/acceptor plasmids , the trans-recombination assay was also performed with “coreless” excised linear fragments carrying the RSS target , and gave rise to similar results ( Figure S6 ) . Although we cannot formally exclude the possibility that when present , stretches of homology can facilitate the recombination process both ex vivo and in vivo , our data suggests that recombination efficiently occurs through direct trans-V ( D ) J synapsis as previously assumed for the assay [20] . Breakpoints were PCR amplified from 1 μl ( 1/20 ) harvested bulk DNA with appropriate primers ( summarized in Table S1 ) in the following conditions: 4 min at 94°C for 25 cycles ( 30 s at 94°C , 30 s at 64°C , and 30 s at 72°C ) , and 7 min at 72°C . Secondary double-nested PCRs were performed in the same conditions , and the amplification products cloned and sequenced as previously described [29] . Breakpoints were amplified in a single round PCR from 1 μl ( 1/20 ) harvested bulk DNA with appropriate primers ( summarized in Table S2 ) in the same conditions as above , and otherwise undetectable amplification products were revealed by PE . PE is a sensitive alternative to Southern blot consisting of several cycles of DNA polymerization extending from a labeled primer until the end of a matrix DNA fragment ( here a PCR product ) . This generates linear ( nonexponential ) accumulation of labeled fragments of specific size . PE assays were performed on 1 . 5 μl , 1 . 0 μl , and 0 . 75 μl of primary PCR using a nested IR800-labeled primer in the following conditions: 5 min at 95°C for 20 cycles ( 30 s at 95°C , 15 s at 60°C and 1 min at 70°C ) , and 1 min at 70°C with the EXCEL II kit ( Epicentre Biotechnologies , http://epicentre . com ) , using an equal amount of all dNTPs and omitting ddNTPs . A portion ( 1 . 2 μl ) of the reaction was used for running on a Li-COR 4200 DNA sequencer ( http://www . licor . com ) . A sequencing reaction was performed in parallel on the appropriate unrecombined purified plasmid , using the same IR800-labeled primer and in the same reaction conditions ( at the exception of the dNTP/ddNTP mix ) , and was run on the same gel , providing both a precise to-the-base size marker and a positive control for the reaction . Semiquantitative conditions were calibrated on serial dilutions of bulk DNA and of PCR amplifications ( Figure S7A–S7C ) . Furthermore , to exclude possible bias due to difference in the efficiency of the PCR primer combinations used in the semiquantitative experiments , each comparison using different sets of primers was calibrated ( Figure S7D–S7E ) . All PCR/PE assays were performed on at least four independent transfections with similar results . Thymocyte preparation and cell sorting were performed as described previously [61] . Phycoerythrin-conjugated mAb against TCRβ ( H57–597 ) , purchased from BD PharMingen ( http://www . bdbiosciences . com ) , was used for cell sorting of TCRβ+ thymocytes . The sorting windows were defined in such a way that only cells expressing high levels of TCRβ were purified . The principle of the assay has been previously described [29] . Detection of rare events by sensitive double-nested PCR gives rise to fluctuation in target amplification , depending on the presence or not of the event in the aliquot taken from the sample . Junctions were PCR amplified from multiple replicates of 2 μg DNA ( or 50 ng for positive replicates out of the fluctuation range ) isolated either from total thymocytes of WT or Eβ−/− mice [40] , or from TCRαβ+ sorted thymocytes from WT or DE double-mutant mice , using appropriate primers ( see list in Protocol S1 ) , and in the following conditions: 4 min at 94°C for 30 cycles ( 30 s at 94°C , 30 s at 64°C , and 30 s at 72°C ) , and 7 min at 72°C . Secondary double-nested PCRs were performed on 1 μl of each primary PCR replicate in the same conditions , and amplification products were cloned and sequenced as previously described [29] . Frequencies were calculated using Poisson assumption as previously described [62] .
|
Lymphoid cells recognize billions of pathogens as a result of gene rearrangements that generate pathogen-specific B- and T-cell receptors . This genetic reshuffling , called V ( D ) J recombination , occasionally misfires and damages genomic integrity . When such aberrations dysregulate proto-oncogenes , cancer ensues . It has become increasingly clear that multiple oncogenes acting in different cellular pathways can cooperate to cause cancer . Nevertheless , in the case of T-cell acute lymphoblastic leukemia , about a third of cases display oncogene activation in the absence of identified aberration , suggesting the presence of additional mechanisms of chromosomal alteration . In the hunt for such mechanisms , episomal circles ( DNA segments that are excised during V ( D ) J recombination ) have recently drawn attention . Moreover , signal joints , short sequences formed after gene rearrangements , once considered harmless , now appear to take part in events that might compromise genomic integrity . Using ex vivo recombination assays and genetically modified mice , we demonstrate that episomal circles may be reintegrated into the genome through recombination occurring between the episomal signal joints and a T-cell receptor target . Furthermore , we show that cryptic recombination sites located in the vicinity of oncogenes constitute hotspots of episomal insertion . Altogether , our results suggest that reintegration of excised episomal circles constitute a potential source of genomic instability and cancer in leukemia and lymphoma .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"oncology",
"immunology",
"molecular",
"biology",
"hematology"
] |
2007
|
In Vivo Reinsertion of Excised Episomes by the V(D)J Recombinase: A Potential
Threat to Genomic Stability
|
Herpes simplex virus ( HSV ) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus . In HSV it consists of the HSV-specific glycoprotein D ( gD ) , and three additional glycoproteins , gH/gL and gB , conserved across the Herpesviridae family and responsible for the execution of fusion . According to the current model , upon receptor binding , gD propagates the activation to gH/gL and to gB in a cascade fashion . Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis , to avoid premature activation and exhaustion of the glycoproteins . We considered the possibility that such control might be carried out by as yet unknown receptors . Indeed , receptors for HSV gB , but not for gH/gL , have been described . In other members of the Herpesviridae family , such as Epstein-Barr virus , integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins . We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells . Evidence rests on loss of function experiments , in which integrins were blocked by antibodies or silenced , and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells . αvβ6- and αvβ8-integrins acted independently and are thus interchangeable . Both bind gH/gL with high affinity . The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes . In the case of αvβ8 , but not αvβ6-integrin , the portal of entry is located at lipid microdomains and requires dynamin 2 . Thus , a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis . We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion .
The glycoproteins of enveloped virions fulfill three major functions to enable virus entry into target cells; the attachment of virions to cells , a step that partly determines the type of cells that the virus targets , hence the viral tropism; the triggering of fusion , i . e . the activation of the fusion machinery , and the execution of fusion . For a number of viruses , a fourth event occurs between these steps , virion internalization by endocytosis , or macropinocytosis . The domains responsible for all these activities are often localized in one or two glycoproteins; this is the case for example for ortho- , paramyxo- and retroviruses . Virion glycoproteins can be considered ready-to-use machines that need to undergo a transition in conformation from the metastable fusion-inactive to the fusion-active form , in order to induce the merging of the two membranes - that of the virion and that of cell - so that lipids are mixed and fusion is executed [1] . A fundamental aspect of the process is that the steps are sequentially ordered and coordinated , to ensure that the glycoprotein transition takes place only after the virus has attached to the cells . Indeed , a premature activation would irreversibly exhaust the fusogenic potential of the virion glycoproteins , and lead to failure to infect . A key question is therefore how the timing of glycoprotein transition and activation is controlled . Essentially , there are two strategies . Either the glycoprotein transition is dependent on the glycoprotein encounter with the cognate cellular receptor , or on the low pH of the endosomal compartment . These levels of control guarantee that the virion fusion machinery is only active after the virus has attached to cells , or , for those viruses which undergo internalization , after they have been endocytosed and the endosomal pH has been lowered . According to this view , two major functions of cellular receptors are determination of viral tropism and triggering of fusion . Herpes simplex virus ( HSV ) , and herpesviruses in general , exhibit a high level of complexity since they encode a multipartite entry/fusion machinery [2] , [3] . Some of the herpesvirus glycoproteins are species-specific; they play a role in the initial steps of virus entry and interact with receptors that belong to variety of molecular families . A prototypic example is HSV glycoprotein D ( gD ) that binds alternatively two major receptors , nectin1 and herpesvirus entry mediator ( HVEM ) [4]–[6] . It functions as a determinant of HSV tropism and as the trigger of fusion capable to activate the downstream glycoproteins gH/gL and gB [7]–[10] . Conformational modification of gD following receptor binding was inferred by structural , biochemical and molecular biology approaches [7] , [8] , [11]–[13] . gD encodes a specific domain – the profusion domain - that interacts with , or at least propagates the activation to gH/gL , and thereafter or simultaneously activates gB in a cascade fashion [9] , [10] , [14] , [15] . Species-specific glycoproteins among human herpesiviruses include Epstein Barr virus ( EBV ) gp42 , which binds major histocompatibility complex II to enable virus entry into lymphocytic cells [16] , [17]; the human cytomegalovirus ( HCMV ) glycoproteins of the 128–131 locus [18] , which confer endothelial and epithelial tropism; the human herpesvirus 6 ( HHV-6 ) gQ1-gQ2 that enable gH/gL interaction with CD134 [19] . In addition to the species-specific glycoproteins , three glycoproteins - the heterodimer gH/gL and gB - constitute the conserved core fusion apparatus across the Herpesviridae family . gB has structural features typical of fusion glycoproteins . In contrast , gH/gL does not resemble any known structure . A number of approaches support the view that gH/gL acts as an intermediate in the activation cascade of HSV entry/fusion glycoproteins . Remarkably , for some herpesviruses , gH/gL or gB serve as major receptor-binding glycoproteins and therefore their activation may be under the direct control of encounter with the receptor . Thus , members of the integrin family serve as receptors for EBV and HCMV gH/gL [20]–[22] , and for HHV-8 gB [23] . EphrinA2 receptor serves as a receptor for HHV-8 gH/gL [24] , [25] . Receptors for HSV gB and varicella zoster virus ( VZV ) gB include paired immunoglobulin-like type 2 receptor alpha ( PILRα ) , myelin associated glycoprotein , and non-muscle myosin IIA [26]–[28]; their role in virus entry remains to be fully elucidated . We have recently discovered two additional functions of cellular receptors for HSV , namely that they serve as routing factors to define the pathway of entry and that they serve as viral sensors capable of initiating the innate response of the cell , and thus coupling virus entry to the innate response . Specifically , HSV enters different cells by different pathways . αvβ3-integrin is a determinant in the choice of the HSV pathway of entry which routes the HSV receptor nectin1 , and consequently HSV , to lipid microdomains [29] , [30] . In this way , αvβ3-integrin enables entry of HSV through a pathway dependent on lipid microdomains , dynamin 2 and acidic endosomes . αvβ3-integrin interacts at low affinity with HSV gH/gL [31] , [32] . Furthermore , αvβ3-integrin also binds Toll-like receptor 2 ( TLR2 ) , which , in turn , binds gH/gL[33] . In this way αvβ3-integrin senses HSV and cooperates with and reinforces the TLR2–dependent response [31] . A signaling cascade is initiated that leads to activation of the transcription factor NF-κB , and to production of intereferon ( IFN ) α and β , the major innate defenses of the cell against HSV [31] . Here , we investigated whether integrins other than αvβ3 play a role in HSV entry . We investigated the αv group of integrins , which are preferentially expressed in epithelial cells , and bind their ligands through the RGD domain . αvβ6-integrin is upregulated in epithelial malignancies . We report that αvβ6- and αvβ8-integrins serve as interchangeable HSV receptors for entry into keratinocytes , other epithelial and neuronal cells . Each integrin acted independently of the other . Both bind gH/gL at high affinity . This interaction profoundly affects the pathway of HSV entry , which takes the way of acidic endosomes . In the case of αvβ8-integrin , the portal of entry is located at lipid microdomains and requires dynamin 2 . In the case of αvβ6-integrin , the portal of entry is located outside the lipid microdomains and does not need dynamin 2 . We propose that the major role of αvβ6- and αvβ8-integrins as gH/gL receptors is to promote HSV endocytosis , and thereby to synchronize virion internalization with the cascade of glycoprotein activation .
The interaction between HSV gH/gL and integrins was explored using surface plasmon resonance spectroscopy of soluble integrins and soluble gH/gL . A kinetic analysis of integrin binding to increasing concentrations of soluble gH/gL indicated that gH/gL interacted with both αvβ6-integrin and with αvβ8-integrin ( Fig . 1 ) . To obtain rate constants of gH/gL binding and to calculate a dissociation constant ( KD ) , kobs was plotted against the concentration of gH/gL . The dependence of kobs on gH/gL concentration was described by a linear function suggesting that the interaction has a single step . The intersection point with the Y-axes corresponds to a dissociation rate constant koff , and the slope to an associate rate constant kon . The rate constants and the resulting KD values ( Table 1 ) are indicative of a high affinity interaction . In contrast , no interaction between integrin αvβ5 and gH/gL was detected . αvβ6-integrin is expressed in epithelial cells and upregulated in epithelial cancer cells . αvβ8-integrin is expressed in some types of epithelial cells , as well as in glial and dendritic cells [34] , [35] . We determined the expression of αvβ6- and of αvβ8-integrins in 293T cells ( epithelial cells transformed by an adenovirus fragment ) , HeLa , colon carcinoma SW480 , keratinocyte HaCaT and in the neuronal SK-N-SH cell lines . By qRT-PCR ( Fig . 2A ) β8-integrin was expressed at higher levels than β6-integrin in 293T , HeLa and SK-N-SH cells , and to comparably high levels in SW480 and HaCaT cells . The epithelial cell lines were analyzed also by flow cytometry; they exhibited the highest fluorescence intensity with the antibody to αvβ6-integrin ( Fig . 2 B ) . A direct comparison between αvβ6- and αvβ8-integrin extent of expression in a same cell line can not be performed by flow cytometry , since reactivity in this assay is strongly influenced by the properties of the antibody being used . The extent of silencing achieved with specific si-RNAs to β6- or to β8-integrin as measured by q-RT-PCR is shown in Fig . 2 C . The extent of silencing was greater than 80% for both integrins in 293T cells . It was around 75 and 50% for β6- and β8-integrin in SW480 cells , and about 40% and 90% for β6- and β8-integrin in HeLa cells . Silencing was specific as there was no off-target effect in any of the cell line . Thus , when β6-integrin was silenced , β8-integrin mRNA level was not decreased . Similarly , when β8-integrin was silenced , β6-integrin mRNA level was not decreased . Silencing was confirmed by flow cytometry analysis in 293T cells and expressed as median fluorescence intensity for β6-integrin ( Fig . 2 D–F ) , and for β8-integrin ( Fig . 2 G–I ) . The lack of off-target was confirmed by flow cytometry . ( Fig . 2 F , I ) . The effect of the double silencing on each of the two integrins could not be differentiated from that of single silencing ( Fig . 2 E and H ) . To define if αvβ6- and αvβ8-integrin play a role in HSV infection , we tested the effect of function-blocking monoclonal antibodies ( MAbs ) to αvβ6-integrin ( MAb 2077Z ) , or αvβ8-integrin ( MAb 37E1 ) , on HSV-1 infection . The neutralizing R1 . 302 MAb to nectin1 was used as a positive control . Cells were preincubated with increasing amounts of MAbs , and infected in the presence of MAbs with the recombinant R8102 which carries a Lac-Z reporter gene . A large body of evidence indicates that the extent of β-galactosidase ( β-gal ) expression directly reflects the extent of infection [4] , [5] . Fig . 3A–E shows that MAbs to αvβ6 or to αvβ8-integrin inhibited R8102 infection in a dose-dependent manner in all cells . The only exception was MAb to αvβ8-integrin which failed to inhibit infection in 293T cells , even though these cells exhibited cell surface expression of this integrin . Thus , both αvβ6 and αvβ8-integrins play a critical role in HSV infection of epithelial , keratynocytic and neuronal cells . As a second approach , silencing of β6- or of β8-integrin with 50 nM si-RNA inhibited infection in the three lines - HeLa and SW480 ( Fig . 4A ) and 293T ( Fig . 4 B–E ) . Thus , two assays concordantly indicate a requirement for αvβ6- and αvβ8-integrins in HSV infection . Because all cell lines tested contained both αvβ6-integrin and αvβ8-integrin , we asked whether each one of the two integrins is required independently one of the other , or whether they play an interchangeable role . In the latter case , the expectation is that in cells silenced for a single integrin , the extent of inhibition of infection is smaller than the extent of silencing . By contrast , in cells simultaneously silenced for both integrins , the inhibition of infection is expected to be higher than that achieved in cells silenced for a single integrin . Fig . 4B–D shows the extent of silencing as percentage of positive cells; their mean fluorescence intensities is shown in Fig . 2 E , H . Indeed , in 293T cells silenced for either or the other integrin , infection was reduced to a lower extent than expression . When the two integrins were simultaneously silenced , infection dropped to about 10% . In a replicate experiment , R8102 infection in singly or doubly silenced cells was quantified by means of β-gal . Fig . 4 E shows that infection was inhibited by about 75% in double-silenced cells , and by about 50% in singly silenced cells . The results indicate that αvβ6-integrin and αvβ8-integrin play each a critical role in HSV infection , each independently of the other . Thus , they appear to act in an interchangeable fashion . To confirm the role of αvβ6-integrin in HSV infection , we performed a gain of function experiment . The myelocytic cell line K562 expresses a very limited number of integrins , predominantly α5β1 , and expresses no detectable αv [36] . A stable cell line in which about 20% of cells transgenically express αvβ6-integrin ( K562αvβ6 ) was obtained from Dr S . Blystone . The wt-K562 and K562αvβ6 cells were infected with RLM5 HSV mutant , which carries the GFP moiety and enables flow cytometry quantification of infection . To authenticate the results , infection was carried in the presence or absence of MAb 2077Z to αvβ6-integrin , MAb L230 to αv-subunit , or polyclonal antibody ( PAb ) R140 to HVEM as a control . Myelocytic cells , including K562 cells , are hardly infected by HSV and likely restrict infection at entry and post-entry steps [37] . Fig . 5 shows a representative experiment , and reports figures relative to the average percentage infected cells , as determined in four independent experiments . It can be seen that the number of infected cells doubled in αvβ6-integrin+ K562 cells , relative to wt-K562 cells ( 10 . 17 versus 5 . 39% ) . Extent of infection in αvβ6-integrin+ K562 cells was strongly inhibited when infection took place in the presence of MAb 2077Z to αvβ6-integrin ( 3 . 12% ) , or MAb L230 to αv-subunit ( 5 . 2% ) , or in the presence of anti-HVEM PAb R140 ( 1 . 98% ) . Although the percentage of infected cells is rather low , in agreement with well-known resistance to HSV infection exhibited by this type of cells [37] , the results clearly indicate that expression of αvβ6-integrin enhances HSV infection of K562 cells; furthermore , the single αvβ6-integrin – in the absence of αvβ8-integrin - was sufficient to induce the increase in infection . The latter finding reinforces the conclusion drawn from the previous series of experiments that each of the two integrins is sufficient to promote HSV infection . The next series of experiments was designed to shed light on the role played by αvβ6- or αvβ8-integrin in HSV infection . First , we defined the step ( s ) in the HSV entry process in which they participate . Cells were exposed to anti-integrin antibodies prior , during and post virus absorption to cells , prior and during virus absorption , or only after virus absorption to cells . The extent of inhibition of infection was then quantified . These experiments could not be performed in 293T cells , as in these cells the anti- αvβ8-integrin MAb fails to inhibit infection ( see , Fig . 3A ) . As shown in Fig . 6 A and B , the highest extent of inhibition was seen when MAbs were present prior , -during and post virus absorption to cells . The decrease seen when Abs were present prior and during , but not post virus absorption was somewhat lower . The presence of Abs after virus absorption decreased infection by about 20% . The results are consistent with the view that blocking αvβ6 or αvβ8-integrins with antibodies inhibits virus infection at different steps in the virus entry process , and suggest a role for integrins at attachment and post-attachment steps . To verify a role for αvβ6- and αvβ8-integrin in HSV attachment , we measured the effect of integrin silencing on HSV absorption to cells at 4°C . Virus absorption was quantified as a decrease in infectious virions present in the inoculum . Fig . 6 C–E shows that virus absorption was inhibited by about 50% in β6- or β8-integrin silenced 293T , HeLa , and SW480 cells , indicating that αvβ6- and αvβ8-integrins contribute to virus absorption to cells . This contribution is small relative to that exerted by heparan sulphate . In cells where heparan sulphate is removed by treatment with heparitinase , in mutants defective in heparan sulphate biosynthesis , or in cells in which infection is competitively blocked by heparin , the decrease in infection is about hundred fold [38] [39] . Notwithstanding this consideration , we asked whether the decrease in HSV absorption seen in cells silenced for β6- or β8-integrins was to be attributed to the decrease in integrins , or reflected an indirect effect of integrin silencing on the extent of cell surface expression of heparan sulphate ( HS ) . We exploited the ability of HSV gB to bind HS , and made use of a soluble form of gB ( gBt ) to quantify the HS binding sites present on the surface of non-silenced versus silenced cells . The properties of gBt , truncated at aa 730 and tagged with a strep tag ( previously named gB730t-st ) were described [33] . Binding to cell surface was quantified by cell enzyme linked immunosorbent assay ( CELISA ) . Preliminarily , we ascertained that the binding of gBt was competitively inhibited by heparin; in contrast , attachment of a soluble form of gH/gL ( gHt/gL ) was not inhibited , as expected ( Fig . 6 F ) [33] . Fig . 6 G – I shows that there was no significant decrease in gBt binding , following β6- or β8-integrin silencing . These results rule out that the decrease in HSV absorption to β6- or β8-integrin-silenced cells ( Fig . 6 C–E ) was due to a silencing-mediated decrease in HS binding sites . For some herpesviruses , e . g . EBV , integrins suffice as receptors for infection of epithelial cells . Here , we asked whether either αvβ6- or αvβ8- integrin suffices as an HSV receptor for entry , or whether their roles are in addition to that of the gD receptors . We made use of the J cells , which are negative for both gD receptors , hence cannot be infected by HSV unless a receptor is transgenically expressed [5] . J cells express endogenous hamster integrins , likely at low levels , hence they are not suitable to test the effect of integrin silencing , or of antibodies to integrins , since the siRNAs and the antibodies were directed to the human orthologs . They were transfected with plasmids encoding for nectin1 , or αvβ6-integrin , or αvβ8-integrin ( 300 ng DNA/well for each plasmid ) , and then infected with increasing amounts of R8102 . Fig . 7 A shows that J cells expressing αvβ6-integrin alone or αvβ8-integrin alone , in the absence of nectin1 , did not enable HSV infection , in contrast to cells expressing nectin1 alone . Thus , neither of the two integrins suffices as HSV receptor in cells negative for gD receptors . Parenthetically , in this assay , in the absence of gD receptors , the candidate gB receptor PILR α [40] did not enable infection . Next , J cells were transfected with low amounts of nectin1 plasmid , plus αvβ6-integrin- or αvβ8-integrin plasmids ( 75 ng DNA/well for nectin1 plasmid , 300 ng DNA/well each for α and for β integrin-subunits ) . It can be seen from Fig . 7 B that αvβ6- or αvβ8-integrin each doubled the efficiency of infection attained with nectin1 alone . Thus , αvβ6- and αvβ8-integrin are not sufficient to substitute for nectin1 , but they do increase nectin1-dependent infection . αv-integrins can interact with their ligands through a RGD motif . Because HSV gH carries an RDG motif at aa 176–178 , we asked whether the interaction of αvβ6- or of αvβ8-integrin with gH occurs through this motif . A mutant form of gH in which the RGD motif was substituted to ADA [41] was used in two functional assays: infection and cell-to-cell fusion . For infection , the ΔgH HSV mutant ScgHZ [42] was grown in cell lines expressing gHwt or gHADA . The cell-expressed gH complements the deletion in the virus , and virions pseudotyped with gHwt or gHADA are generated . J cells transfected with nectin1 plus αvβ6- or αvβ8-integrin , or with nectin1 alone , were infected with virions carrying gHwt or gHADA . Fig . 8 A–C shows that infection of J cells expressing nectin1 alone occurred irrespective of whether virions carried gHwt or gHADA , in agreement with a previous report [32] . In contrast , infection of J cells expressing nectin1 plus αvβ6-integrin was severely impaired when virions carried gHADA . Infection of J cells expressing nectin1 plus αvβ8-integrin was only slightly inhibited when virions carried gHADA . The cell-to-cell fusion assay mimics virus-to-cell entry in that it requires the same four essential virion glycoproteins ( gD , gH/gL , gB ) , and one of the gD receptors nectin1 or HVEM [43] . The effector cells are transfected with the quartet of gH/gL , gD , gB , plus T7-promoter-driven luciferase [44] . The target cells are transfected with the required glycoprotein receptor and T7-polymerase . In our assay the target J cells were transfected with gHwt or gHADA plus the trio of gD , gL , gB . The effector cells were transfected with nectin1 alone , nectin1 plus αvβ6-integrin , or nectin1 plus αvβ8-integrin . Fig . 8 D shows that cell-to-cell fusion was increased by 40% or 30% when J cells expressed αvβ6-integrin or αvβ8-integrin , plus nectin1 , relative to cells expressing nectin1 alone . This occurred with the wt allele of gH . When the gHADA substituted for gHwt there was a dramatic inhibition in the αvβ6-integrin-enhanced fusion , but not in the αvβ8-integrin-enhanced fusion . The results of the two assays indicate that the gH interaction with αvβ6- , but not with the αvβ8-integrin , entails the RGD motif in gH . HSV enters different cells by different pathways . αvβ3 integrin was identified in our laboratory as a cellular determinant in the choice of the pathway of entry , capable of routing HSV to a pathway dependent on lipid microdomains and dynamin 2 , and proceeding to acidic endosomes [30] . Whether integrins other than αvβ3 are determinants in the choice of HSV entry pathways , and whether different integrins route HSV to different pathways is not known . To address these questions , we analyzed the effects of well known entry inhibitors on HSV infection of J cells expressing nectin1 alone , or nectin1 plus αvβ6- , αvβ8- , or αvβ3-integrins . J cells are suitable to address this question , since , when transfected with a gD receptor they enable a pathway of entry independent of lipid rafts and acidic endosome [45] . The significant inhibitors were bafilomycin A ( BFLA ) , a specific inhibitor of the Na-H pump , hence of endosomal acidification , filipin III , an inhibitor of the lipid microdomain platforms , dynasore , an inhibitor of the dynamin 2 GTPase required to seal the endosomal invaginations and generate endosomes , and wortmannin , an inhibitor of phosphoinositide 3-kinase ( PI3K ) . The results were as follows ( Fig . 9 ) . Infection of J cells expressing nectin1 alone was not sensitive to BFLA ( Fig . 9 A ) , as reported [45] , and occurs by fusion at plasma membrane or at a neutral pH compartment . Anyone of the three integrins - αvβ6 , αvβ8 and αvβ3 – rendered the entry pathway sensitive to BFLA ( Fig . 9 A ) . The wortmannin sensitivity closely mirrored that of BFLA , implying an involvement of PI3K when HSV entry occurs through acidic endosomes , but not at the plasma membrane or in a neutral compartment ( Fig . 9 B ) . Filipin III did not inhibit infection in J cells expressing nectin1 alone ( Fig . 9 C ) , but did inhibit infection in J cells expressing nectin1 plus αvβ8- , or αvβ3-integrin ( Fig . 9 C ) . Remarkably , filipin III failed to inhibit infection of J cells expressing nectin1 plus αvβ6-integrin . The effect of dynasore closely overlapped that of filipin III ( Fig . 9 D ) . The filipin III and dynasore sensitivity upon αvβ3-integrin expression was in agreement with our previous findings [30] . Cumulatively , the results indicate that both αvβ6- and αvβ8-integrin greatly impact on the HSV entry pathway . Both switch HSV to acidic endosome entry . αvβ8- , like αvβ3-integrin , routes HSV to a pathway dependent on lipid microdomain and dynamin2; in contrast , αvβ6-integrin routes HSV to a pathway independent of lipid microdomain and dynamin 2 .
The epithelial αv group of integrins , also named as RGD receptors , includes αvβ3- , αvβ5- , αvβ6- , αvβ8-integrins , in addition to the αIIbβ3 . We report that: Cumulatively , the novel features to emerge from this study are that HSV entry into epithelial cells requires the gH/gL interaction with one of the two interchangeable receptors , αvβ6-integrin or αvβ8-integrin . This interaction profoundly affects the pathway of entry , which takes the way of acidic endosomes . In the case of involvement of αvβ8-integrin , the portal of entry is located at lipid microdomains and necessitates dynamin 2 . In the case of involvement of αvβ6-integrin , the portal of entry is located outside the lipid microdomains and does not necessitate dynamin 2 . Two questions arise . Why did HSV evolved to employ gH/gL receptors in addition to the gD receptors ? And , why did HSV choose integrins as gH/gL receptors ? Two characteristics of the entry of HSV , and of herpesviruses in general , are the multipartite nature of the entry apparatus , and the possibility to enter cells through a variety of pathways . The currently accepted model on how the HSV multipartite entry/fusion apparatus mediates fusion of the virion envelope with the cell membranes envisions sequential activation steps in a cascade fashion [7] , [8] , [11] , [12] , [15] . Even though the details of the interactions are still being defined , there is consensus that , upon gD binding to one of its receptors , conformational changes to gD ensue [11] , [12] , such that gD interacts with , or anyway propagates the activation to gH/gL , and thereafter or simultaneously to gB [9] , [10] , [14] . Current results introduce the intervention of the integrin receptors in this process . We propose that , in addition to promoting endocytosis , the interaction of gH/gL with the integrin receptor contributes to induce conformational changes and activation of gH/gL , as seen in EBV [48] ( Fig . 10 ) . Consequently , integrins would act as a trigger point in the activation cascade of the HSV fusion glycoproteins . Indeed , by placing the step of gH/gL activation under the double control of receptor-activated gD and of the integrin trigger point , the virus would ensure that the premature activation of gB is prevented , and , importantly , that virion endocytosis is synchronized with the activation of the fusion apparatus . In particular , when entry occurs at the plasma membranes , the receptor-mediated gD activation propagates to gH/gL and gB , possibly in a very brief timescale , perhaps in a matter of fraction of seconds . In the case of HSV entry by endocytosis , there must be a pause in the activation cascade , or a checkpoint that prevents the premature activation of gH/gL and gB , before endocytosis has taken place . An activating receptor for gH – in this case integrins , which are machines specialized in promoting endocytosis [49] – is ideally suited to fulfill this synchronization ( Fig . 10 ) . The entry pathways exploited by HSV include low pH or neutral pH endosomes , as well as the plasma membrane , which is also a neutral pH compartment . As a consequence , HSV had to evolve a strategy for control of activation of the fusion glycoproteins independent of acidic pH . The integrin trigger point on gH/gL results in a control on fusion independent of acidic pH , and thus confers to the virus the flexibility to use alternative pathways in different cells , and expands the range of cells that HSV infects . Up to now , among herpesviruses , integrins are known to serve as receptors for EBV and HCMV , which engage with integrins through gH/gL , and for HHV-8 , which engages with integrins through gB [20]–[23] . In EBV , the integrin-gH/gL interaction triggers conformational changes to the glycoproteins , likely activating them [48] . Whether all human herpesviruses need integrins in some way to enter certain cell types is an attractive open possibility . All herpesviruses encode a multipartite fusion apparatus , and enter a number of cells by endocytosis . Importantly , for EBV , HCMV and HHV8 , placing the activation of gH/gL ( EBV , HCMV ) or gB ( HHV-8 ) under an integrin trigger point may well result in a synchronization of the final steps of activation of the glycoproteins responsible for fusion execution . While many viruses in other families make use of integrin receptors to promote endocytosis [50]–[52] , most either lack an envelope , e . g . adenoviruses and reoviruses , or do not encode an entry system as complex as that of a herpesvirus . Their usage of integrins thus intrinsically differs from that of HSV , as entry is less dependent on checkpoints to control an activation cascade . We recently found that αvβ3integrin - which functions as a routing factor and drives HSV entry to a pathway dependent on lipid microdomains , dynamin 2 and acidic endosomes [30] , [29] - serves as a sensor of the virus . In cooperation with TLR2 , it elicits a branch of the innate response that contributes to NF-κB activation and production of a specific set of cytokines , mainly IFNα and β [31] . The question naturally arises as to whether the engagement of αvβ6 or αvβ8-integrin as receptors initiates a similar inimical response to the virus . In turn , this raises the question of what selective advantage is provided to HSV by use of an integrin to control fusion . As noted in earlier studies , HSV has evolved to evade the innate response soon after the onset of viral protein synthesis; in particular , infected cell protein 0 ( ICP0 ) and ICP27 provide the first line of evasion [53]–[56] . We conclude that , as highlighted in this work , HSV takes advantage of integrins to enable its endocytic entry and to exert a checkpoint on the cascade of glycoprotein activation . It subsequently counteracts the innate response , when virus entry is completed , by aid of the immediate early proteins .
293T , HeLa , SK-N-SH and J ( a derivative of BHK-TK− cells lacking any HSV receptor ) [5] cells were grown in Dulbecco's modified Eagle's medium ( DMEM ) containing 5% to 20% fetal bovine serum ( FBS ) . Colon carcinoma SW480 , HaCaT and K562 cells were grown in L15 , DMEM 4 . 5% glucose and Iscove's modified Dulbecco's medium , respectively . The growth medium for K562αvβ6 ( a gift from Dr S . Blystone ) contained 750 µg/ml neomycin G418 . F6 cells were a stably transformed Vero cell line expressing HSV-1 gH under the control of HSV-1 gD promoter [42] . R8102 , a HSV-1 recombinant carrying LacZ under the control of the α27 promoter [5] and RLM5 , a HSV-1 ( F ) recombinant expressing green fluorescent protein ( GFP ) [57] were described . In the gH deletion mutant ( ΔgH HSV ) SCgHZ the gH gene was replaced with LacZ gene , the virus was grown and titrated in the complementing F6 cells [42] . 293-B6 AVAP [58] and 293-B8 AVAP cells [59] ( a gift of Dr . Stephen Nishimura , University of California at San Francisco ) , which secrete , respectively , truncated αvβ6 and αvβ8 integrin conjugated to alkaline phosphatase ( AP ) , were grown in DMEM ( Sigma ) supplemented with 10% fetal bovine serum and 1% nonessential amino acids . Insect Sf9 cells were grown in Sf900 II medium ( Invitrogen ) and infected at a multiplicity of 2 with equal ratios of baculoviruses expressing truncated forms of αv and β5 cloned in frame respectively with a fos or jun dimerization domain [60] ( a gift of Dr . Glen Nemerow , Scripps Research Institute ) . Soluble truncated integrins were purified as previously described [48] . Soluble gHt/gL and gBt , carrying One-StrEP-Tag epitope for affinity chromatography purification , were described previously [33] , [41] . One-StrEP-tagged green fluorescent protein ( GFPt ) was provided by IBA GmbH ( Göttingen ) and used as negative control . Plasmids encoding HSV-1 gD , gB , gL and gH all under the control of the cytomegalovirus ( CMV ) promoter were described [61] . gHADA carries the indicated substitutions in the RGD motif [41] . pCAGT7 contained the T7 RNA polymerase gene under the control of the CAG promoter and pT7EMCVLuc plasmid expressed the firefly luciferase under the control of the T7 promoter [62] . EGFR2NΔ ( epithelial growth factor receptor 2 Δ ) , named Erb-2 , carriers the extracellular domain and transmembrane sequence of rat HER-2/neu , and is deleted of the tyrosine kinase domain [63] . Plasmids encoding nectin1 [5] , Renilla luciferase ( Promega ) , αv- , β6- and β8-integrin were described [36] , [64] . MAb 2077Z to the αvβ6 integrin heterodimer is a function-blocking antibody from Chemicon . MAb 37E1 to αvβ8 integrin , MAb R1 . 302 to nectin1 and PAb to HVEM were gifts from Nushimura S . L [65] , M . Lopez [5] and G . H . Cohen and R . Eisenberg [66] . MAb L230 is a function-blocking antibody directed to αv integrin [67] . For surface plasmon resonance analysis MAb LS-C44264 to placental alkaline phosphatase ( LifeSpan BioScience ) was used to capture integrins αvβ6AP , and αvβ8AP the non-blocking monoclonal antibody LM142 to αv integrin ( Millipore ) was used to capture integrin αvβ5 . The kinetic parameters of the interaction between gH/gL and integrins were measured with a Biacore 2000 instrument ( Biacore AB ) . Antibodies were immobilized on a research-grade CM-5 sensor chip by amino coupling with 1 , 200 relative units ( RU ) as a target for immobilization and used to capture soluble integrins . The first flow cell ( FC1 ) was always used as a reference ( no antibody immobilized ) , and the signal from FC1 was automatically subtracted by Biacore software from data obtained from the other three flow cells . Measurements were made at 25°C . Integrins were injected at a flow rate of 10 to 20 µl/min; gH/gL was injected immediately after integrin capture by using the low-dispersal injection setting ( kinject ) at a flow rate of 50 µl/min . After each run , the sensor chip surface was regenerated by injection of 6 M guanidine chloride in 25 mM HEPES-NaOH , pH 7 . 2 ( 50 µl at a flow rate of 100 µl/min ) , followed by additional washing . The baseline remained stable during 30 to 40 runs . Integrins and gH/gL were centrifuged before use and diluted in running buffer if necessary ( 10 mM HEPES-NaOH , pH 7 . 4 , 150 mM NaCl , 0 . 005% surfactant P20 ( Biacore , GE Healthcare ) . Each sample was degassed before injection . BIAevaluation 4 . 1 software was used for the Biacore trace alignments and to zero the baseline . All traces were in full accordance with a 1∶1 Langmuir model . Formation and dissociation sections of Biacore traces fit a single-exponential function which was accepted as a model . Least-squares fitting of data and determinations of standard errors of the fitted parameters were conducted using the program KaleidaGraph ( Synergy Software , Reading , PA ) . One target plus siRNAβ6 or β8 ( Dharmacon , ON-TARGET plus , smart pool ) were trasfected into 293T , HeLA or SW480 cells , 50 nM for each siRNA , by means of Dharmafect I , according to Dharmacon protocol for adherent cells . Control-silenced cells were transfected with siRNA to E . coli-polA_0054 ( CGC GUG AUA UGC GAC GCG AUA AAG ) synthesized by IBA Gmbh . Total RNA was purified by means of Total RNA Isolation kit ( Macherey-Nagel ) and reverse transcribed with high-capacity cDNA reverse transcription kit ( Applied Biosystem ) . Real-time PCR primers were the inventoried TaqMan gene expression assay ITGB6 ( Hs00168458_m1 ) , ITGB8 ( Hs00174456_m1 ) ITGAV ( Hs00233808_m1 ) ( Applied Biosystem ) . Quantitative Real Time PCR ( q-RT-PCR ) was performed as described [31] . 293T , HeLa , SW480 , HaCaT and SK-N-SH cells in 96 wells were preincubated with increasing amounts of MAb R1 . 302 to nectin1 , MAb 2077Z to αvβ6-integrin , MAb 37E1 to αvβ8-integrin , or with mouse IgGs for 60 min at 37°C . R8102 ( 3 pfu/cell ) was added to the MAb-containg medium for additional 90 min . Viral inoculum was removed , and cells were overlaid with DMEM containing MAbs for 6–8 h . Control-silenced , β6-integrin-silenced or β8-integrin-silenced 293T , HeLA or SW480 cells were infected with R8102 ( 3 pfu/cell ) for 90 min at 37°C . The viral inoculum was removed and cells were overlaid with DMEM 1% FBS for 6–8 h . The extent of infection was assessed through β-galactosidase ( β-Gal ) activity at 405 nm , by means of o-nitrophenyl-β-D-galactopyranoside ( ONPG ) , or by in situ staining with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside [5] . K562αvβ6 were preincubated or not with 40 ug/ml of MAb 2077Z to αvβ6-integrin , MAb L230 to αv-integrin , or PAb R140 to HVEM for 1 h at 37°C . K562 and K562αvβ6 cells were infected with RLM5 ( 20 pfu/cell ) in 3 ml for 90 min . The viral inoculum was removed and cells were overlaid with DMEM containing or not the indicated antibodies for 6 h . Control-silenced , β6-integrin-silenced , β8-integrin-silenced or double-silenced 293T cells were infected with RLM5 ( 3 pfu/cells ) for 90 min at 37°C . Viral inoculum was removed; the extent of infection was assessed through EGFP expression in infected cells , 6 h after infection . Expression of αvβ6 or αvβ8–integrins in 293T , HeLA and SW480 cells was determined by FACS . In 293T cells ( control-silenced and integrin-silenced ) integrin expression was determined 72 h after transfection with siRNActrl , siRNAβ6 , siRNAβ8 or a mixture of both . Cells were washed once with PBS ( phosphate buffered saline ) and once with PBS containing 10% FBS and allowed to react with MAb 2077Z to αvβ6-integrin , MAb 37E1 to αvβ8-integrin heterodimers for 1 h at 4°C . Cells were washed three times with PBS and allowed to react with phycoerythrin conjugated secondary antibody ( Becton-Dickinson Pharmingen ) . Where indicated , cells were infected with RLM5 , and infection was quantified through EGFP expression at 6 h after infection . The inhibition of HSV infection by MAb 2077Z to αvβ6-integrin , MAb L230 to αv-integrin and PAb to HVEM in K562 and K562αvβ6 cells was quantified through enhanced green fluorescent protein ( EGFP ) expression from the RLM5 . Cytofluorimetric analysis was performed using a FACScalibur cytometer ( BD ) , equipped with an argon laser on a minimum of 10 , 000 cells per sample , acquired in list mode . The stock solutions of filipin III ( 2 . 5 mM ) , dynasore ( 100 mM ) , bafilomycin A ( BFLA ) ( 160 mM ) and wortmannin ( 2 M ) ( all from Sigma Aldrich ) in dimethyl sulfoxide were stored at -20°C . Cells were exposed to the inhibitors for 1 h at 37°C and then infected with R8102 ( 3 pfu/cell ) for 90 min in the presence of inhibitors . The viral inoculum was removed , and the cells were overlaid with medium containing inhibitors for 6–8 h . For filipin III and dynasore , cells were preincubated with the compounds at 37°C for 30 min or 60 min , respectively , and infected for 30 min ( 30 pfu/cell ) in the same medium . Viral inoculum was removed; infected cells were overlaid without inhibitor and harvested 6–8 h after infection . 293T cells in T150 flasks were transfected with gHwt or gHADA plus gL . After 4 h , cells were infected with a gH−/+ stock of SCgHZ ( 5 pfu/cell ) [42] . Cell-absorbed virions were inactivated by a 1 min rinse with 40 mM sodium citrate-10 mM KCl-135 mM NaCl ( pH 3 ) . The monolayers were then rinsed twice with PBS and overlaid with medium containing 1% fetal calf serum . Cells were incubated overnight at 37°C . Extracellular virions were harvested , pelletted by high speed centrifugation , and titrated in F6 cells . J cells were transfected with plasmids encoding nectin1 ( 300 ng DNA/24 well ) , αv plus β6 , or αv plus β8 integrins ( 300 ng each/24 well ) by means of Lipofect 2000 ( Life Technologies ) . Alternatively , J cells were transfected with low amount of nectin1 plasmid ( 75 ng DNA/24 well ) , plus or minus αv+β6 or αv+β8-integrins ( 300 ng DNA/24 well ) . The total amount of transfected plasmid DNA was made equal ( 675 ng/24 well ) by the addition of Erb-2 plasmid DNA . 48 h after transfection , cells were infected with increasing MOI ( 2 . 5–30 pfu/cell ) of R8102 , or of SCgHZ virions complemented with gHwt or with gHADA , for 90 min at 37°C . After infection cells were overlaid with DMEM and harvested 16–18 h after infection . The luciferase-based cell-cell fusion assay was performed as described [44] , with small modifications . Effector J cells were transfected with plasmid encoding gL , gD , gB , gHwt or gHADA . Target J cells were transfected with Renilla luciferase , low amount of nectin1 ( 75 ng DNA/24 well ) , plus or minus αv+β6 , or αv+β8 ( 300 ng DNA for each plasmid/24 well ) . The total amount of plasmid DNA transfected was made equal by the addition of Erb-2 plasmid DNA . 24 h after transfection , effector and target cells were co-cultured for additional 24 h . Fusion was quantified by means of the T7 promoter-driven reporter luciferase gene; the amount of lysate was quantified by Renilla luciferase . Quantification was performed by means of Dual luciferase report assay ( Promega ) . The extent of fusion was expressed as relative luciferase units ( R . L . U . ) ; 100% represents the extent of fusion induced by gHwt in cells expressing nectin1 alone . Cells grown in 96-well were incubated for 1 h at 4°C with One-StrEP tagged gBt , gHt/gL , or GFPt ( 2 µM ) in DMEM containing 5% FBS and 30 mM HEPES [68] , washed three times with the same buffer , and further incubated for 1 h at 4°C with HRP ( horse radish peroxidase ) -conjugated MAb to One-StrEP tag ( Strep-Tactin ) ( IBA GmbH , Göttingen ) . Following three additional rinsings , cells were reacted with o-phenylenediamine ( Sigma-Aldrich ) at 0 . 5 mg/ml; the optical density was read at 490 nm . For heparin inhibition studies glycoproteins were preincubated for 1 h at 4°C with 5 µg/ml heparin ( Sigma-Aldrich ) , prior to addition to cells .
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In order to infect their hosts and cause disease , viruses must enter their host cells . The human pathogen herpes simplex virus ( HSV ) - and herpesviruses in general - are equipped with a complex , multipartite entry apparatus , made of four glycoproteins – gD , gH/gL , gB . These glycoproteins must be activated in a timely , coordinated manner . According to the current model , the flux of activation goes from receptor-bound gD , to gH/gL and gB . The premature activation , and hence exhaustion of the glycoproteins must also be prevented . We report on a checkpoint at the gH/gL level . Specifically , αvβ6- and αvβ8-integrins serve as receptors for HSV entry into keratinocytes and other epithelial and neuronal cells . Both bind gH/gL with high affinity . The interaction profoundly affects the pathway of HSV entry , promoting HSV endocytosis into acidic endosomes . For αvβ8-integrin , the portal of entry is at lipid microdomains and requires dynamin 2 . We propose that , by placing the activation of gH/gL under control of an integrin trigger point , HSV can synchronize virion endocytosis with the cascade of activation that culminates in the execution of fusion between the virion envelope and cellular membranes .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2013
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αvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion
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Much is known about the mode of action of drugs and resistance mechanisms under laboratory growth conditions , but research on the bacterial transcriptional response to drug pressure in vivo or efficacious mode of action and transient resistance mechanisms of clinically employed drugs is limited . Accordingly , to assess active alternative metabolism and transient resistance mechanisms , and identify molecular markers of treatment response , the in vivo transcriptional response of Burkholderia pseudomallei 1026b to treatment with ceftazidime in infected lungs was compared to the in vitro bacterial response in the presence of drug . There were 1 , 688 transcriptionally active bacterial genes identified that were unique to in vivo treated conditions . Of the in vivo transcriptionally active bacterial genes , 591 ( 9 . 4% coding capacity ) genes were differentially expressed by ceftazidime treatment . In contrast , only 186 genes ( 2 . 7% coding capacity ) were differentially responsive to ceftazidime treatment under in vitro culturing conditions . Within the genes identified were alternative PBP proteins that may compensate for target inactivation and transient resistance mechanisms , such as β-lactamses that may influence the potency of ceftazidime . This disparate observation is consistent with the thought that the host environment significantly alters the bacterial metabolic response to drug exposure compared to the response observed under in vitro growth . Notably , this study revealed 184 bacterial genes and ORFs that were unique to in vivo ceftazidime treatment and thus provide candidate molecular markers for treatment response . This is the first report of the unique transcriptional response of B . pseudomallei from host tissues in an animal model of infection and elucidates the in vivo metabolic vulnerabilities , which is important in terms of defining the efficacious mode of action and transient resistance mechanisms of a frontline meliodosis chemotherapeutic , and biomarkers for monitoring treatment outcome .
Understanding the mode of action and potential resistance mechanisms of frontline chemotherapeutics provides information about how clinically used drugs exert their efficacious effect . Typically the lethal effect of a drug is investigated in the laboratory under artificial conditions and is defined as the drugs mode of action [1] . Here we expand on this concept by assessing the bacterial transcriptional response , and thus metabolic response to drug treatment and transient resistance mechanisms in vivo , and use the term efficacious mode of action to distinguish between analyses performed using bacteria from in vitro or in vivo sources . An important implication is the laboratory-defined mode of action is often used to substantiate a bacterial protein or metabolic pathway for inhibitor design and to inform potential drug resistance mechanisms . The primary drawback of using in vitro only information is that the defined in vitro mode of action may not be the same as the efficacious in vivo mode of action or in vivo specific metabolism or mechanisms that influence susceptibility and resistance . This possibility results from the bacterial alternative metabolism or use of coding redundancy as a result of the host environment , which supersedes the pressure of drug exposure . The well-documented intrinsic resistance of Burkholderia spp . to drug treatment is primarily attributed to the extensive efflux capability of the pathogen [2] . However , Burkholderia spp . are also known for extensive coding capacity , which provides alternative or redundantly encoded components or metabolic capabilities available to the bacteria during stress and treatment pressure [3] . Since an appreciation of the impact of coding redundancy on in vivo drug susceptibility is just emerging , there is a need to understand the metabolic activities of the bacteria under disease conditions in the host environment [3] . Further , to validate the therapeutic value of a putative drug target it is critical to evaluate essentiality during infection and disease dissemination in a host [3] . Throughout our own drug discovery program we have encountered conditional lethal drug targets , that differ significantly in their essentiality between in vitro and in vivo growth environments [3 , 4] . Ceftazidime , a third-generation β-lactam cephalosporin , is the front line standard of care therapeutic when treating acute melioidosis [2] . The mode of action of ceftazidime has been reported to be inhibition of the penicillin-binding protein 3 ( PBP-3 ) , FtsI , which when inhibited leads to filamentation and eventual cell lysis [5–7] . This was followed up later by deletion of the gene encoding PBP-3 resulting in ceftazidime resistance in vitro [8] . This alone , in the mode of action of ceftazidime is paradoxical , since cell division is an essential cellular process regardless of growth environment suggesting that an alternative encoded gene product can perform the function of the primary PBP-3 molecular target . In addition , resistance to ceftazidime in vivo has been associated with a single-nucleotide polymorphism ( SNP ) within the promoter region of the β-lactamase gene penA [9] . Together , these data indicate that the mode of action of ceftazidime and potential resistance mechanisms may well be different than its efficacious mode of action because of the dependence of the bacteria on the host growth environment . Our group has also demonstrated conditional essentiality of a drug target in vitro versus in vivo [3] . This can be problematic when testing compound libraries through the conventional drug discovery pipelines . Novel molecular drug target identification against B . pseudomallei is also challenging due to the genome being comprised of two chromosomes and the resultant coding redundancy within the genome[10] . This leads to questioning essentiality of specific drug targets if multiple isoforms are encoded within the genome and observed differences in expression levels between isoforms under in vitro and in vivo growth environments . In order to better understand the efficacious mode of action , and the impact of the host disease environment on bacterial transcription and metabolism , and the essentiality of drug targets , mapping and comparing of the whole bacterial transcriptome in the host environment during drug treatment is necessary . Accordingly , the transcriptional response of B . pseudomallei in the murine model of infection during ceftazidime treatment was compared to untreated and ceftazidime exposed B . pseudomallei growth under laboratory conditions using next generation sequencing . This is the first report of an in vivo whole bacterial transcriptional profiling from infected tissues and the unique transcriptional response of B . pseudomallei treated with ceftazidime in an animal model of infection , and resulting efficacious [in vivo] mode of action and transient resistance mechanisms . In addition , this analysis revealed bacterial molecular markers of treatment response . These studies are particularly important and promise to have an impact on rational targeted drug discovery for B . pseudomallei because it elucidates the transcriptionally active genes as well as potential drug resistance mechanisms . Further , the unique in vivo bacterial molecular markers revealed in this study promise to provide diagnostics to monitor the bacterial response to treatment .
B . pseudomallei 1026b [11] was grown to an OD600 of ~0 . 6 , frozen at -80°C in 10% glycerol and was used as the standard bacterial stock for these studies . For each evaluation bacteria were prepared fresh by growth from the standard stocks on Luria-Bertani ( LB ) Agar , Miller ( BD ) grown at 37°C for 48–72 h . Bacteria recovered from the LB plates were inoculated in 50 mL LB Broth . Broth cultures were then incubated for 18 h at 37°C passed 1:100 and incubated for an additional 6 h at 37°C . Cultures were then treated with 2X MIC ( 4μg/ml ) ceftazidime or LB broth alone and incubated for an additional 2 h at 37°C . 1mL of each culture was centrifuged at 12 , 000rpm for 5 minutes , supernatant removed and bacteria resuspended in 1mL TRIzol ( Invitrogen , Carlsbad , CA ) . Resuspended cultures were immediately stored at -80°C for further bacterial RNA isolation . 5–6 week old BALB/c female mice ( Charles River Laboratories , Wilmington , MA ) were challenged by intranasal infection with approximately 5 , 000 CFU/mouse B . pseudomallei 1026b[11] ( N = 30 mice; 12 receiving treatment and 18 untreated ) . Animals were anesthetized with a mixture of 100 mg/kg ketamine ( Aurora Veterinary Supply , Aurora , CO ) and 10 mg/kg xylazine ( Aurora Veterinary Supply ) delivered intraperitoneally . The bacteria were diluted to the appropriate concentration in PBS to achieve an inoculum concentration of 2 . 5x105 CFU/mL . The inoculum was then delivered in a 20 μL volume dropwise in alternating nostrils . Ceftazidime ( Sigma Aldrich , St . Louis , MO ) was formulated for injection in PBS ( pH 7 . 4 ) at a concentration of 40 mg/mL and 200mg/kg was delivered inraperitoneally beginning at 36 hours post infection followed by a second dose at 48 hours post infection . Mice from the untreated group ( Grp1 ) were euthanized at 36 , 48 , and 60 hours post infection and mice from the treated group ( Grp2 ) sacrificed at 48 and 60 hours post infection . The number of viable bacteria in lung and spleen was determined for mice in each group at each timepoint ( N = 9 Grp1;N = 6 Grp2 ) by plating serial 10-fold dilutions of homogenates onto LB agar and incubating for 48 h at 37°C . Lung and spleen were also homogenized in TRIzol from the remaining mice ( N = 9 Grp1;N = 6 Grp2 ) and stored at -80°C for further bacterial RNA isolation . In vitro and infected mouse lung tissue samples were thawed and nucleic acid was isolated by organic partition . Samples were treated with DNAse ( Fermentas , Burlington , Ontario ) for 30 minutes and purified by phenol/chloroform/isoamyl alcohol ( 25:24:1 ) ( Fisher Scientific , Pittsburgh , PA ) extraction and ammonium acetate precipitation . Biological replicates ( not pooled ) were submitted to the CSU Next Generation Sequencing core for sample processing and sequencing . Briefly , RNA sample quality was determined on an Agilent 2100 Bioanylizer and samples with a RIN value greater than 8 passed the criteria for sequencing . Host transcripts were removed using MICROBEnrich ( Life Technologies , Carlsbad , CA ) , sample libraries were prepared using the Ion Total RNA-Seq kit v2 ( Life Technologies ) , and multiplexed on a P1 chip using Ionxpress RNA-Seq 1–16 kit ( Life Technologies ) . Whole bacterial transcriptome sequencing was performed using the Ion Proton Next Generation Sequencer ( Life Technologies ) . Data was received from the core in FASTQ format . Data files were uploaded to and analyzed using Galaxy [12–14] . FASTQ files were subjected to quality trimming by use of sickle with a minimum PHRED quality threshold greater than 20 and read length greater than 20bp . Trimmed reads from FASTQ files were aligned to Burkholderia pseudomallei 1026b genome ( NCBI RefSeq NC_017831 . 1 & NC_017832 . 1 ) using Bowtie2 and gene expression determined using Cufflinks [15 , 16] . Expression output was normalized in FPKM format ( fragments per kilobase of exon per million reads ) [17 , 18] . Replicate mean values were calculated and data was further reduced to FPKM values greater than 2 . The Accession number for the data in this study is PRJNA291046 and can be found at BioProject/NCBI under submission SUB863226 . Three genes ( BP1026B_II2144 , BP1026B_II0025 , and BP1026B_I0955 ) that were up or down regulated in both in vitro and in vivo ceftazidime treated conditions were selected and subjected to qRT-PCR to validate the normalized data generated from RNA-Seq and subsequent analysis . BP1026B_I3469 ( 16s rRNA ) was used as a reference gene . Primer pairs used in qRT-PCR are listed in supplementary S1 Table cDNA was prepared from total RNA samples using Transcriptor First Strand cDNA Synthesis kit ( Roche Applied Science ) . The resultant cDNA was used in downstream real-time PCR assays on a Roche LightCycler480 . Samples were added to primer , LightCycler480 SYBR green I master mix ( Roche ) , and water to final volume of 20μl . Real-time PCR cycle parameters were as follows: Pre-incubated at 95°C for 5 minutes , followed by 45 cycles of 95°C for 10 sec , 60°C for 10 sec , and 72°C for 10 sec . All biological replicate samples were quantified independently in technical triplicate . All use of vertebrate animals at Colorado State University is conducted under AAALAC approval and has an OLAW number of A3572-01 . Animals are housed in a state-of-the art ABL-3 facility that is supervised by full-time staff veterinarians and a large number of support staff . The CSU animal assurance welfare number is A3572-01 under file with the NIH . Veterinary care is consistent with the recommendations of the American Veterinary Medical Association ( AVMA ) Guidelines .
To assess concordance and unique differences in the transcriptional response of in vivo and in vitro sourced B . pseudomallei to ceftazidime treatment , bacteria obtained from infected tissues of infected animals treated with ceftazidime were compared to laboratory cultured and treated bacteria . As a comparative control mid-log cultures of B . pseudomallei 1026b were grown at 37°C under ambient conditions in rich medium and were exposed to 2X MIC ceftazidime for 2hrs . As indicated , there is no significant reduction in the number of viable bacteria after 2 hours of exposure to ceftazidime under laboratory conditions ( Fig 1A ) . This drug exposure condition was chosen for this study because it is known from historical studies to result in a drug treatment specific transcriptional response before a more complex mixed response , which includes the cidal response [19–21] . The murine model of acute respiratory melioidosis was achieved by intranasal infection of Balb/c mice with ~5 , 000 CFU B . pseudomallei strain 1026b [4] . Treatment with 200mg/kg ceftazidime was delivered intraperitoneally at 36 and 48 hours post infection . Consistent with previous reports [4] , respiratory infection of B . pseudomallei strain 1026b in the lungs grew rapidly resulting in a total lung burden of 7 . 2 Log10CFU/mL at 36 hours post infection ( Fig 1B ) . After 48 hours of infection or at 12 hours of treatment there was a reduction in culturable bacteria in the lung of 1 . 02 Log10CFU/mL and 60 hours of infection and 24 hours of treatment there was a total bacterial reduction of 1 . 1 Log10CFU/mL . The bacterial burden in the spleens was also enumerated to monitor dissemination and treatment efficacy , which demonstrated that there was a significant reduction in the spleen as a result of treatment ( Fig 1B ) . To further confirm that the infection and efficacious dose used in this in vivo study was consistent with previous studies , we monitored the untreated control group which succumbed to infection at 60 hours and the treatment group which had an observed 20–40% survival rate at day 42 ( Fig 1C ) . There is increasing need to understand how drugs with efficacy that are in clinical use exert a lethal effect on the bacteria and how the bacteria respond to exposure . To determine the differences between the effects of ceftazidime on in vitro laboratory grown bacteria and bacteria in infected tissues , we compared the global transcriptional response of in vitro grown B . pseudomallei 1026b and B . pseudomallei 1026b obtained from infected lungs in response to ceftazidime treatment using enrichment RNA-sequencing . The bacterial RNA from each sample was sequenced and the resulting FASTQ files were trimmed using sickle , aligned to the B . pseudomallei 1026b genome using Bowtie2 , and cufflinks was used to determine gene expression . An average of 6 . 7 million reads were mapped per sample with each read length averaging 75bp . Whole genome mapping of the transcriptionally active ORFs identified from in vitro and in vivo grown bacteria revealed a fairly equal distribution between chromosome 1 or 2 regardless of origin with ratios of 64:36% and 60%:40% , respectively ( Fig 2A and 2B ) . The global distribution of transcripts from these conditions does not support that there is a significant chromosome bias or preference for basic metabolism and adaptive responses as suggested previously [22] . The overall distribution of transcriptionally active ORFs was also assessed according to infection condition [in vitro versus in vivo] and by treatment and is shown in Table 1 . To determine the common global metabolic activity of in vitro and in vivo grown bacteria , the transcriptionally active open reading frames were categorized into different metabolic function groups based on cluster of orthologous groups ( COG ) annotation assignments . This global analysis revealed that approximately 1/3 of the response fell into the category of unknown hypothetical while the next three categories most represented were amino acid transport and metabolism , carbohydrate transport and metabolism , and transcription ( Fig 2C ) . The RNA-Seq data was validated using qRT-PCR analysis of selected differentially expressed genes . The validation was performed comparing the crossing point ( Cp ) values to log2 FPKM values obtained from RNA-seq , which revealed an inverse relationship between the Cp and FPKM values . An inverse correlation between Cp and FPKM values for these genes is considered a standard validation of the RNA-seq data set [23] . This independent assessment revealed a strong concordance between the RNA-seq and quantitative PCR data ( Fig 3 ) . A bacteria’s response to drug exposure and treatment is known to directly reflect the drugs mechanism of action [24] . However , the growth or environmental conditions during exposure and treatment also contribute and have a significant impact on the global transcriptional responses . As a result , the overlapping transcriptional responses from the direct activity of the drug and the environmental growth conditions can make it difficult to discern which responses to assign to the drugs mode of action and which responses to the environmental conditions . To identify the common bacterial response to ceftazidime the global in vitro bacterial transcriptional response to exposure to ceftazidime and the global in vivo bacterial transcriptional response during ceftazidime treatment in the acute model of melioidosis were subjected to concordance analysis to identify transcriptionally active features common to both in vitro and in vivo treated bacteria . This comparative analysis revealed 1 , 234 transcriptionally active open reading frames common to bacteria from in vitro conditions and from in vivo bacteria from infected tissues . Transcriptionally active ORFs or genes were defined as those present in all biological replicates for that condition and with an FPKM group mean greater than 2 . This represents 19 . 7% of the total coding capacity of the B . pseudomallei 1026b genome . Genes annotated in known COG pathways during treatment in vitro and in vivo were more likely to be found on chromosome 1 at 36% of the total response compared to only 12% on chromosome 2 . Unknown hypothetical transcriptionally active genes were found evenly distributed between the two chromosomes at 24% and 28% , respectively ( Fig 4B ) . Genes encoding unknown hypothetical proteins represented 52% of the transcriptionally active ORFs identified under this condition ( Fig 4A ) , which is consistent with our observations in other studies and other reports of bacterial transcriptional responses to alternative conditions and stress [25] . While , the significance and biological role of genes encoding proteins with unknown function is difficult to define because of lack of annotation or biological information , genome context reveals some information . Examples of the most transcriptionally active ORFs in this category were BP1026B_I1021 , BP1026B_I0763 , and BP1026B_I0849 . BP1026B_I1021 is a putative regulator of the nar-operon that encodes a respiratory nitrate reductase , which is known to be associated with denitrification and anaerobic nitrite respiration . BP1026B_I0763 encodes a hypothetical LysR-type transcriptional regulator family , which has been shown to be involved in regulation of diverse sets of genes involved in adaptive metabolism and virulence [26] , and BP1026B_I0849 encodes a hypothetical protein containing the CreA regulatory domain , which is associated with a transcriptional regulator component of the regulatory domain controlling carbon source utilization [27] . COG groups for translation , ribosomal structure and biogenesis , and posttranslational modification , protein turnover , chaperones were the next groups most represented during treatment both in vitro and in vivo . Genes within the rpl operon , which encodes proteins of the large ribosomal subunit , and genes encoding tRNA synthases were represented in the ribosomal structure and biogenesis COG group . Heat shock proteins , clp and hsl proteases , and dnaK and bicP chaperones were among the most transcriptionally abundant within the COG category of posttranslational modification , protein turnover , and chaperones . The number of transcriptionally active genes unique to in vitro growth , not differentially regulated during treatment , represents 112 genes or 2% of the coding capacity of B . pseudomallei and were mostly assigned to unknown hypothetical proteins ( 58% of 112 ) and energy production and conversion ( 12% of 112 ) ( Fig 4C ) . Sixty percent of the genes that are uniquely expressed during in vitro growth mapped to chromosome 1 as compared to 40% to chromosome 2 ( Fig 4D ) . Of the ORFs categorized as hypothetical unknown function , BP1026B_I2494 , BP1026B_I1886 , and BP1026B_II2237 were the most transcriptionally active in this group . BP1026B_I2494 encodes a 119bp ncRNA that maps between a multidrug efflux transporter and unknown hypothetical protein , which the latter is suspected to play a role in regulation . BP1026B_I1886 encodes a 31aa protein that is located by the transcriptional regulator encoded by osmT , and segregation and condensation protein b , which is involved in chromosome separation . BP1026B_II2237 encodes a 203bp ncRNA flanked by a transposase and unknown hypothetical protein . Genes most represented within the energy conservation and conversion COG group are part of the nuo-operon and genes encoding for ATP synthase subunits . Both are involved in energy generation . Genes in the nuo-operon encode the NADH dehydrogenase I , a key component of the respiratory chain , which is important for converting energy from reduced NADH . Coupled to the NADH dehydrogenase I process is the energy consuming process of ATP synthesis , which is consistent with the observed transcriptional activity of ATP synthase encoding genes . Together , the genes involved with this process are known to be essential for basic respiration and generation of energy in bacteria . There were 168 differentially regulated genes unique to in vitro treatment grouped in categories of unknown hypothetical ( 50% of the 168 ) , transcription ( 7% of the 168 ) , and energy production and conversion ( 5% of the 168 ) ( Fig 4E ) . Differentially expressed genes unique to in vitro treatment are distributed more on chromosome 1 ( 66% ) than on chromosome 2 ( 34% ) ( Fig 4F ) . Unknown hypothetical ORFs BP1026B_I1647 , BP1026B_I1551 , and BP1026B_II1658 were among the most transcriptionally active genes in untreated samples and ORFs BP1026B_I3414 , BP1026B_II0244 , and BP1026B_I3157 were the most active in treated samples in vitro . Many transcriptional regulators within the transcription COG category were differentially expressed during treatment in vitro . These included tetR that encodes an efflux regulator , lysR , which is associated with regulation of virulence , motility , and quorum sensing and the multidrug resistance regulator marR . The COG category energy production and conversion is the last major category that is differentially expressed during treatment in vitro . Most notable repressed in this group were the genes cyoD , which encodes the 12-kDa membrane protein ubiquinol oxidase subunit IV , and ppa encoding an inorganic pyrophosphatase which plays a role in lipid metabolism . Conversely , several genes in the succinate dehydrogenase ( sdh ) operon are induced during treatment . To understand how B . pseudomallei responds to treatment in vivo we first analyzed the metabolism unique to in vivo infection . Overall distribution of active ORFs or genes was more evenly assigned to COG pathways as compared to that observed in vitro . A total of 1 , 504 genes ( 24% coding capacity ) were transcriptionally active and are unique to in vivo infection . The largest represented group was unknown hypothetical genes at 374 genes ( 25% ) . The next COG categories with the most transcriptionally active ORFs was carbohydrate transport and metabolism represented by 131 genes ( 9% ) , and amino acid transport and metabolism with 126 genes ( 8% ) ( Fig 4G ) . The overall baseline transcriptional response unique to in vivo treatment was similarly distributed between the two chromosomes with 54% on chromosome 1 and 46% on chromosome 2 ( Fig 4H ) . Carbohydrate transport and metabolism makes up the largest annotated class that is transcriptionally active during in vivo infection . Within this category several genes encoding ABC transporters and more specifically araG and araH of the L-arabinose transport operon . Five genes were transcriptionally active that are EmrB/QacA family drug resistance transporters and twenty-eight genes that encode membrane transport proteins in the major facilitator superfamily ( MFS ) . Most transcriptionally active genes within this COG category were involved in more than one metabolic pathway within carbohydrate transport and metabolism . The top three transcriptionally active genes found were grouped in the hypothetical unknown category was BP1026B_II0381 , BP1026B_II1410 ( 80bp ncRNA ) , and BP1026B_I1586 ( 164bp ncRNA ) . BP1026B_II0381 flanks 16s rRNA and another hypothetical unknown . BP1026B_II1410 flanks an encoded Lrp regulator , which is involved in putative regulation of amino acid metabolism and related genes . BP1026B_I1586 flanks a putative bacteriophage gp31 , which encodes a protein analogous to chaperonin GroES . Amino acid transport and metabolism COG category makes up the second largest annotated class that is transcriptionally active during in vivo infection . Within this category there are many operons that are important for basic metabolism in vivo . BP1026B_I0247-8 and BP1026B_II1815-7 are involved in tryptophan biosynthesis . BP1026B_I3366-7 , hisB and hisC in this operon , are involved in histidine biosynthesis . BP1026B_II1824-6 , leuC and leuD in the leu-operon , are involved in leucine biosynthesis . BP1026B_1410–11 , the hom-1-thrC operon , are involved in threonine biosynthesis . Lastly , there are 23 genes , including hisP , dppA , braC , and oppA ) that are classified as ABC transporters . Notably , genes that encode penicillin-binding proteins ( PBPs ) and the penA β-lactamase were uniquely transcriptionally active in vivo . Altogether there were seven genes encoding PBPs that were transcriptionally active unique to in vivo infection one being BP1026B_II1292 . The PBP 3 encoded by BP1026B_II1292 is known to be a target of ceftazidime and when harboring a deletion increases resistance in vitro [8] . The penA encoded β-lactamase also contributes to ceftazidime resistance and is uniquely expressed in vivo . This mechanism of ceftazidime resistance is attributed to an increase in expression due to point mutations caused by ceftazidime [28] . There were an additional 591 ORFs or 9 . 4% of the coding capacity identified as differentially expressed in the presence of ceftazidime in the in vivo infection . Similarly to the unique to in vivo conditions , the in vivo differentially regulated ORFs categorized dominantly to the COG categories of unknown hypotheticals , amino acid transport and metabolism , and carbohydrate transport and metabolism ( Fig 4I ) . In addition to these COG groups , the COG group of transcription was also significantly represented at 8% total response . Transcriptional distribution within the genome shifted from a more balanced response observed during baseline metabolism in vivo to a more dominant response on chromosome 1 ( 63% of total ) observed in differentially expressed genes in response to treatment in vivo ( Fig 4J ) . Unknown hypothetical genes were the most represented group within the differentially expressed genes in vivo at 178 or 30% of total . BP1026B_I2307 , BP1026B_I0989 , and BP1026B_I2303 were all the most transcriptionally active in untreated samples in vivo while BP1026B_I1657 , BP1026B_II0389 , and BP1026B_II1993 were most represented in the ceftazidime treated samples . All three encode putative proteins that ranged from 38 to 46aa in length . Both BP1026B_I2307 and BP1026B_I2303 flank 16s and 23s rRNA respectively . BP1026B_I0989 is downstream of a gene that encodes for the protein N-formylglutamate amidohydrolase . Only BP1026B_II0389 had a flanking annotated gene and that encodes a copper responsive transcriptional regulator . Carbohydrate transport and metabolism was also uniquely represented with 45 genes or 0 . 7% of the coding capacity . Within this category we found seven genes differentially expressed that are ABC transporters , one gene that is a EmrB/QacA family drug resistance transporter , and twelve genes that encode membrane transport proteins in the MFS . BP1026B_I1984 , a gene encoding beta-hexosaminidase , was found to be differentially expressed and is involved in beta-lactam resistance [29] . The pgl-zwf operon , involved in pentose phosphate metabolism and the paa-operon , involved in phenylacetic acid degradation were both differentially expressed and involved in carbohydrate metabolism . Amino acid transport and metabolism and transcription both round out the top COG categories represented unique to in vivo treatment at 40 genes active per group . Many of the genes within the amino acid transport and metabolism category are involved in many different metabolic pathways within the category . Similar to what we had observed in bacteria grown under other conditions , ABC transporters were well represented within the data set with twelve genes differentially expressed . Out of the transcription COG category there were six different family transcriptional regulator types that were differentially expressed unique to in vivo treatment . Those six were AraC ( 15 total ) , LysR ( 14 total ) , GntR ( 3 total ) , lclR ( 3 total ) , and LuxR and PadR being represented by one gene respectively . Importantly , the transcriptional response of in vivo bacteria in the presence of ceftazidime revealed the activity of genes that encode potential resistance mechanisms that are uniquely expressed in vivo upon ceftazidime treatment . The operon BP1026B II2141-II2142-II2144-II2145 was identified to be differential expressed in vivo by ceftazidime treatment . This operon encodes a hypothetical protein of unknown function , a potential regulator in stress response and adaptation encoded by BP1026B_II2144 , the DNA-binding response regulator irlR2 ( BP1026B_II2142 ) that has been associated with imipenem resistance through the regulation of OprD porin protein , which is involved in entry of carbapenem antibiotics , and BP1026B_II2145 that encodes a class D β-lactamase that is known to confer resistance to beta-lactam drugs through inactivation . The presence of pseudogenes and their role in bacterial physiology remains largely unknown . It is thought that translation of these genes that encode presumably non-functional proteins alter overall energy consumption , and their accumulation in bacterial genomes has been associated with pathogenesis within the host [30] . Analysis revealed that 0 . 4% of the genes ( N = 9 ) that were unique to in vivo were pseudogenes , and these pseudogenes where encoded on chromosome 1 and chromosome 2 similar to expression found under other conditions . To substantiate the assignment of reads to these pseudogenes , BLAST analysis was performed . The reads uniquely mapped to these pseudogenes and did not map to homologous or paralogous gene sequences within the rest of the genome . To identify genome distributed molecular markers of ceftazidime treatment response that can be used to assess whether bacteria are responding to treatment , bacterial molecular markers unique to in vivo infection and molecular markers specific to treatment response where sought . Molecular markers that map across the genome and include both chromosomes in the case of B . pseudomallei are preferable for monitoring viability and treatment response because as a group , their reporting potential is not as influenced by genome-transcriptional behavior . The full compliment of molecular markers that provide the foundation for a set of genome distributed molecular markers for monitoring treatment response consists of markers that indicate viability , are unique , and are responsive to treatment . Identification of bacterial molecular markers of in vivo infection serves as a positive control for viable infectious bacteria . Identification of bacterial molecular markers during in in vivo treatment distinguish if the bacteria are responding to treatment . To identify candidate molecular markers of in vivo infection , the transcriptional profile of bacteria unique to in vivo host growth conditions was analyzed to identify the bacterial molecular features that had the greatest expression as indicated by FPKM intensities . The features identified from this analysis where narrowed to the top 10 features . The top 10 most abundant molecular features group into three categories , genes that encode tRNA and rRNA , genes that encode proteins with unknown function and the serS seryl-tRNA synthetase ( Table 2 ) . Any of these molecular features are potential molecular markers for infection with B . pseudomallei . In addition to finding molecular markers that indicates a viable B . pseudomallei infection , a goal was to determine genome distributed molecular markers specific to ceftazidime susceptibility and treatment in vivo to inform treatment outcome ( Fig 5 ) . Accordingly we utilized the bacterial transcriptional profiles unique to in vivo treatment . This analysis identified 184 genes that were uniquely transcriptionally active during treatment in vivo with an average response of 182 FPKM , which is approximately 100 times the average abundance . The identified 182 gene features informative of in vivo treatment response were further enriched to the most abundant 25 ( Table 3 ) . These transcriptionally active genes were evenly distributed within the genome between both chromosomes and most are unknown hypotheticals ( N = 14 ) . Annotated genes of interest as potential biomarkers are paaF ( BP1026B_I0261 ) , tagD-4 ( BP1026B_II0586 ) , and filR ( BP1026B_I0034 ) . Together , the molecular markers of in vivo infection and the molecular markers discriminant of ceftazidime treatment provide the foundation for diagnostics about response to treatment .
One of the challenging questions in disease treatment and management is , by what mechanism , as a consequence of inhibiting a molecular target , does a clinically used chemotherapeutic exert its lethal effect , and what are all the possible mechanisms of resistance , intrinsic , acquired or transient . It has been reported that the primary target of the clinically used drug , ceftazidime , is the PBP 3 , FtsI protein involved in cell division [7] . Later studies demonstrated that PBP 3 could be knocked out indicating the presence of compensatory activity ( 6 ) . In addition , Burkholderia spp . are known to be intrinsically resistant to the majority of clinically used chemotherapeutics [2] . Much work has been performed on the intrinsic resistance mechanisms of resistance in Burkholderia spp . , for ceftazidime . However , this work has primarily focused on drug efflux and has been performed under in vitro laboratory conditions [2] . Only recently has the details of ceftazidime mode of action and resistance , as well as the role of the penA β-lactamase been studied [31] . While , these studies indicate that FtsI is a molecular target of ceftazidime , and ceftazidime is susceptible to PenA β-lactamase activity , additional factors may contribute to in vivo resistance to treatment . An approach to assessing the global metabolic activity of a bacterium under various growth conditions is via transcriptional profiling . Accordingly , we have used this approach to identify the conserved transcriptional response of B . pseudomallei during in vivo growth in the mouse model of infection and the unique in vivo transcriptional response to ceftazidime treatment . Typically , a drug’s mode of action and resistance mechanisms are elucidated using in vitro studies , which are influenced by the artificial laboratory growth conditions under which they are performed [32] . We have observed that the coding capacity and coding redundancy of B . pseudomallei result in molecular drug targets that are conditionally bactericidal; specifically , some protein targets are only essential under specific growth conditions , which differ between in vitro and in vivo growth conditions [3] . Although informative , it may be difficult to determine a drugs efficacious mode of action and understand the mechanisms of drug resistance strictly from in vitro molecular studies that are routinely performed . Indeed , a pitfall with relying on in vitro only studies are that the molecular targets may not always be essential or even metabolically active in vivo as observed in this study , resulting in incomplete information or missed opportunities . The in vivo transcriptional response provides a starting point to better understand the functional association of unknown hypothetical proteins that are expressed during infection and differentially expressed during treatment . To get a better understanding on how the molecular target profiles differ in vitro compared to in vivo and potential alternative metabolic pathways that may influence susceptibility to ceftazidime we sequenced the transcriptome of B . pseudomallei from in vitro cultures and in vivo infected tissue in the presence of ceftazidime . The transcriptional profiles were compared to determine differences in transcriptional diversity , utilization of coding capacity and protein homolog usage , as a surrogate for assessing the impact on metabolism . The most significant differences observed between in vitro grown bacteria and bacteria in infected tissues were observed in the transcription of hypothetical genes , genes encoding energy production , carbohydrate , amino acid and lipid metabolism , and secondary metabolites . This is attributed to the controlled enriched nutritional and environmental conditions of in vitro laboratory culturing that allow the bacteria to use only a minimal number of metabolic pathways . Similarly , there was greater bacterial transcriptional diversity during treatment in vivo compared to treatment in vitro; the number of significantly differentially expressed genes in vivo outnumbered those observed in vitro ~2:1 . There was ~20% reduction in the number of transcriptionally active genes that encode hypothetical genes . Genes that encode proteins involved in alternative transcriptional regulation , proteome remodeling and protein turnover and secretion where differentially regulated in in vivo bacteria as compared to in vitro grown and treated bacteria . The significant difference in transcriptional activity in hypothetical genes represented approximately 33% of the total data set indicating the use and importance of the coding capacity that provides critical evidence to better understanding how B . pseudomallei responds during treatment ( under stress ) in the host and the potential for discovery of novel molecular targets and molecular markers . The transcriptional response of in vivo bacteria revealed the activity of genes that encode potential transient resistance mechanisms to ceftazidime . The operon BP1026B II2141-II2142-II2144-II2145 operon that encodes a regulator , an OprD porin protein associated with imipenem resistance and a class D β-lactamase ( known to confer resistance to β-lactam drugs through inactivation ) , demonstrates that significant differences in bacterial transcriptional activity occur in vivo that may have a significant impact on susceptibility to treatment and outcome . In addition to the PBP BP1026B_II1292 identified as a target for ceftazidime treatment [8] , there are six others that are uniquely transcriptionally active in vivo . These other PBPs could serve as potential non-susceptible targets that in fact sequester drug decreasing its effectiveness . Interestingly , these multiple PBP targets are only transcriptionally active in vivo suggesting their effect on drug sequestration wouldn’t be evident when testing in vitro . The identification of this operon as well as several PBPs and a β-lactamase that are uniquely active in vivo substantiates that there maybe potential transient resistance mechanisms uniquely active during infection that would not be identified by in vitro studies . Interestingly , genes that encode pseudogenes were also identified . The transcriptional activity of genes that encode non-functional proteins can result from transcriptional coupling as part of a polycistron or transcribed from their own promoter . In this instance , the majority of the transcriptionally active pseudogenes identified is located in regions of the chromosome with greater number of genes encoding hypothetical unknown proteins , and are flanked by hypothetical genes . This raises the possibility that pseudogenes where once hypothetical genes that underwent evolutionary decay . While the potential impact of pseudogenes on bacterial metabolism is unclear , there expression in B . pseudomallei is consistent with reports that demonstrate other bacterial pathogens express genes encoding non-functional proteins in in vivo environments [30] . Disease treatment and outcome monitoring is becoming increasing important with the emergence of drug resistance . In fact , disease management in a clinical setting would be improved if treatments were accompanied with early indication diagnostics of treatment outcome . This is particularly true for emerging pathogens such as B , pseudomallei that cause fatal acute infections and that are naturally resistant to treatment because of the short opportunity for effective treatment . In addition , the ability of B . pseudomallei to establish a chronic infection complicates disease management . Therefore , there is a need for diagnostic molecular markers capable of monitoring disease progression and treatment outcome , and to report the activity of potential resistance mechanisms . The current methodologies for detection of Burkholderia pseudomallei infection relay heavily on serological tests and PCR assays that are low in sensitivity and specificity of detection [33] . There is growing interest in using RNAseq data sets as tools in discovery of new biomarkers that are relevant to host-pathogen interaction and response to treatment [34] . These data underscore the importance to study the bacterial response to drug treatment in in vivo conditions . It is clear that the growth environment and nutrient conditions have a profound impact on the bacterial response and may influence to a great degree the susceptibly to treatment . Molecular target availability in vivo is essential to assess efficacy of current clinically used drugs and compounds progressing through drug development . Researchers could be missing out on many potential drug candidates due to the limited transcriptional diversity observed in vitro . Further , this report highlights the potential of transient or phenotypic resistance mechanisms , and provides a better understanding of how the bacteria respond to treatment in vivo , which may account for the observed differences between bacterial treatment response in vitro versus in vivo .
|
The mode of action and resistance mechanisms for many clinically used drugs are elucidated under defined artificial laboratory growth conditions , and do not necessarily represent the efficacious mode of action or predict the transient resistance mechanisms that are active during infection . Here , we report the unique transcriptional response of B . pseudomallei from host tissues and demonstrate the efficacious mode of action and transient resistance mechanisms of the frontline meliodosis chemotherapeutic , ceftazidime .
|
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2017
|
Transient In Vivo Resistance Mechanisms of Burkholderia pseudomallei to Ceftazidime and Molecular Markers for Monitoring Treatment Response
|
Despite the endemic nature of Echinococcus granulosus and Echinococcus multilocularis infection in regions of the United States ( US ) , there is a lack of data on echinococcosis-related mortality . To measure echinococcosis-associated mortality in the US and assess possible racial/ethnic disparities , we reviewed national-death certificate data for an 18-year period . Echinococcosis-associated deaths from 1990 through 2007 were identified from multiple-cause-coded death records and were combined with US census data to calculate mortality rates . A total of 41 echinococcosis-associated deaths occurred over the 18-year study period . Mortality rates were highest in males , Native Americans , Asians/Pacific Islanders , Hispanics and persons 75 years of age and older . Almost a quarter of fatal echinococcosis-related cases occurred in residents of California . Foreign-born persons accounted for the majority of echinococcosis-related deaths; however , both of the fatalities in Native Americans and almost half of the deaths in whites were among US-born individuals . Although uncommon , echinococcosis-related deaths occur in the US . Clinicians should be aware of the diagnosis , particularly in foreign-born patients from Echinococcus endemic areas , and should consider tropical infectious disease consultation early .
Human echinococcosis , a neglected disease caused by the larval stages of the cestode parasites Echinococcus granulosus ( hydatid cyst disease ) and E . multilocularis ( alveolar hydatid disease ) , affects an estimated 2–3 million people and results in an annual monetary loss of over $750 , 000 , 000 worldwide [1] , [2] . The infection is a zoonosis , normally maintained in dogs and sheep in close association with humans ( E . granulosus ) , or in a wild cycle , such as in foxes or wild canines and rodents ( E . multilocularis ) . Humans are an aberrant intermediate host for the disease , developing life-threatening tissue cysts , predominately in the liver and lung , following accidental ingestion of eggs in infected dog , fox and wild canine feces . Once a human is infected , treatment is complicated and carries significant risks . Approaches include the use of antihelmintic drugs , surgery , and/or medico-surgical procedures such as PAIR ( puncture , aspirate , injection of a scolicide , re-aspirate ) [3] . Hydatid cyst disease , accounting for over 95% of human echinococcosis , predominates in poor , pastoral communities that raise sheep and other livestock , and keep dogs for guarding and herding because of the complex two-host lifecycle [4] . Although alveolar hydatid disease is less common in humans , it is more pathogenic , more difficult to treat , and has a higher mortality than cystic hydatid disease . Although the majority of the disease burden is located outside of the United States , both E . granulosus and E . multilocularis are endemic to regions of the United States [5]–[9] . E . granulosus ( senso stricto or G1 strain ) has been recorded in a few foci such as certain communities in Utah and California and E . granulosus ( G8 or G10 strains ) is more common in Alaska than the lower 48 states . E . multilocularis is endemic in wildlife across much of the mid-western United States and Alaska . In the United States , human echinococcosis is not a nationally notifiable infectious condition [10] . Despite the occurrence of human echinococcosis in the United States and its potential for causing fatal disease , there is a lack of information about echinococcosis-related deaths in the United States . Information on echinococcosis mortality is important to better understand the burden of disease and evaluate the effectiveness of public health interventions . Population-based mortality data has been used to investigate other infectious diseases , but these data have not yet been used in published echinococcosis research . We examined national mortality data to assess the burden and demographics of echinococcosis-related mortality in the United States from 1990 to 2007 .
De-identified , publicly available multiple-cause-of-death data from US death certificates from the National Center for Health Statistics ( NCHS ) were analyzed for the years 1990–2007 [11] . These death certificates contain basic demographic information for each decedent , including age , sex , race/ethnicity , and state of residence . In addition to designating underlying causes , the physician or coroner completing the death certificate may list up to 20 conditions that are believed to have contributed in some way to the death of an individual . Each of these conditions is coded on the basis of the International Classification of Diseases ( ICD ) system for the year in which the death occurred ( ICD , Ninth Revision [ICD-9] for the period 1990–1998 and ICD , Tenth Revision [ICD-10] for the period 1999–2007 ) . We defined echinococcosis-related cases as US resident deaths having an ICD-9 code of 122 . 0–122 . 9 or an ICD-10 code of B67 . 0–B67 . 9 listed as an underlying or contributing cause on the death record . Mortality rates and 95% confidence intervals ( CIs ) were calculated using bridged-race population estimates derived from US census data and were subsequently age-adjusted with weights from the 2000 US standard population data . Mortality rates for race/ethnicity ( non-Hispanic white , non-Hispanic black , Hispanic , Asian/Pacific Islander , and Native American ) , sex , age , year , and state were calculated with aggregated data from all years of our study to ensure stable rates . All calculations were performed with SAS software , version 9 . 2 .
We identified 41 echinococcosis-related deaths among US residents during the period 1990–2007 ( Table 1 ) . Age-adjusted mortality rates were higher in males ( 0 . 012 per 106 ) than in females ( 0 . 005 per 106 ) , with males more than 2 times as likely to die from echinococcosis than were females ( adjusted rate ratio = 2 . 2 , 95% CI 1 . 3–3 . 9 ) . Native Americans ( 0 . 062 per 106 ) , Asian/Pacific Islanders ( 0 . 032 per 106 ) , and Hispanics ( 0 . 014 per 106 ) had the highest age-adjusted echinococcosis mortality rates , with adjusted rate ratios of 8 . 9 ( 95% CI 6 . 1–12 . 9 ) , 4 . 6 ( 95% CI 3 . 2–6 . 7 ) , and 1 . 9 ( 95% CI 1 . 3–2 . 9 ) , respectively , compared to whites . No echinococcosis-related deaths were recorded in non-Hispanic blacks . The majority of echinococcosis-related deaths ( 35 , 85% ) occurred in persons over 35 years of age , with the highest rates noted in persons 85+ years . Echinococcus species was unspecified in 36 ( 88% ) cases and was identified as E . granulosus and E . multilocularis in 3 ( 7% ) and 2 ( 5% ) cases , respectively . Site of infection was unspecified in 23 cases ( 56% ) , infection of liver and lung were recorded in 17 ( 42% ) and 1 ( 2% ) cases , respectively . Echinococcosis-related deaths fluctuated throughout the 18-year study period , ranging from 0–5 deaths annually , with 26 ( 63% ) and 15 ( 37% ) cases reported in the first and second halves of the study period , respectively . Twenty-three states reported echinococcosis-related fatalities , with California ( 9 , 22% ) having the highest number of deaths . A majority of echinococcosis-related deaths ( 30 , 73% ) occurred in foreign-born persons . Mean age at death differed slightly between foreign-born ( 57 . 9 ) and U . S . -born persons ( 64 . 5 ) , and gender differences were observed ( 70% and 45% males , respectively ) .
Although uncommon , fatal echinococcosis occurs in US residents and disproportionally affects selected demographic groups . Mortality rates were highest in males , Native Americans , Asians/Pacific Islanders , Hispanics , and persons 75 years of age and older . There was a weak declining temporal trend over the study period , and almost a quarter of all echinococcosis-related deaths occurred in residents of California . Foreign-born persons accounted for the majority of echinococcosis-related deaths; however , almost half of deaths in whites and both deaths in Native Americans were among US-born individuals . The apparent increased risk of echinococcosis-related mortality in males may reflect higher rates of occupational exposures from the tending of livestock . The increased mortality risk in Asians/Pacific Islanders and Hispanics , all of whom were foreign born , presumably reflects greater exposure to Echinococcus species in their country of origin prior to emigration and upon return trips to visit friends and relatives . The observed increased risk of echinococcosis in persons over 75 years of age may be a result of increased prevalence of comorbid diseases and declining immune function . The higher number of echinococcosis-related deaths in California is expected , given the large population and sizable number of immigrants from Latin America and Asia . Liver was the most common reported site of infection , which is expected , as liver involvement is the most common manifestation of echinococcosis . Incomplete information and lack of sufficient numbers precluded an assessment of possible associations between demographic factors and site of infection . The occurrence of echinococcosis-related deaths in US-born persons reflects either local Echinococcus species transmission or travel-related infection . Because death certificates do not have information on travel , we are unable to determine whether US-born decedents acquired infections domestically or abroad . Several important limitations are associated with the use of multiple-cause-of-death data that require consideration . Although these data are population based and contain large numbers of observations , death certificates likely underreport causes of death and may contain errors , which have been attributed to a variety of factors [12] . Mortality rates may be distorted because of errors in population estimates , particularly for race/ethnicity . Because estimates of the at-risk population factor into the denominator for rate calculations , such errors can lead to biased estimates . In addition , the small number of echinococcosis-related deaths and the lack of species-specific information make interpretation difficult . Finally , although inferential statistics are not designed for use with population-based data , we have used such methods to demonstrate that error does exist . We urge caution in the strict interpretation of our values . Fatal echinococcosis may be more common in the United States than currently appreciated . Echinococcosis causes a mortality burden in the United States that may be modified by increased prevention and control efforts , including vaccine development for adult cestode carriers and livestock [13] . Given the presence of echinococcosis mortality in US-born persons , and the risk of travel-related exposure , hygiene precautions should be advised for individuals traveling to Echinococcus species endemic areas . Clinicians should be aware of the diagnosis , particularly in foreign-born patients from Echinococcus endemic areas , and should consider tropical infectious disease consultation early .
|
Human echinococcosis is a parasitic disease that affects an estimated 2–3 million people and results in an annual monetary loss of over $750 , 000 , 000 worldwide . It results in the development of life threatening tissue cysts , primarily in the liver and lung , following accidental ingestion of eggs in infected dog , fox or wild canine feces . Echinococcus parasites have a complex , two-host lifecycle ( such as in dogs and sheep ) in which humans are an aberrant , dead-end host . The vast majority of cases of human echinococcosis occur outside of the United States ( US ) ; however , cases within the US do occur . In this study , the authors examined death certificate data of US residents from 1990–2007 in which echinococcosis was listed as one of the diagnoses at death . The analysis demonstrated 41 echinococcosis-related deaths over the 18-year study period with foreign-born persons accounting for the majority of the deaths . This study helps quantify echinococcosis deaths among US residents and adds further support to the importance of funding echinococcosis prevention research .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"public",
"health",
"and",
"epidemiology",
"epidemiology",
"public",
"health"
] |
2012
|
Human Echinococcosis Mortality in the United States, 1990–2007
|
Buruli ulcer ( BU ) is a necrotizing disease of the skin , subcutaneous tissue and bone caused by Mycobacterium ulcerans . It has been suggested that the immune response developed during the recommended rifampicin/streptomycin ( RS ) antibiotherapy is protective , contributing to bacterial clearance . On the other hand , paradoxical reactions have been described during or after antibiotherapy , characterized by pathological inflammatory responses . This exacerbated inflammation could be circumvented by immunosuppressive drugs . Therefore , it is important to clarify if the immune system contributes to bacterial clearance during RS antibiotherapy and if immunosuppression hampers the efficacy of the antibiotic regimen . We used the M . ulcerans infection footpad mouse model . Corticosteroid-induced immunosuppression was achieved before experimental infection and maintained during combined RS antibiotherapy by the administration of dexamethasone ( DEX ) . Time-lapsed analyses of macroscopic lesions , bacterial burdens , histology and immunohistochemistry were performed in M . ulcerans-infected footpads . We show here that corticosteroid-immunosuppressed mice are more susceptible to M . ulcerans , with higher bacterial burdens and earlier ulceration . Despite this , macroscopic lesions remised during combined antibiotic/DEX treatment and no viable bacteria were detected in the footpads after RS administration . This was observed despite a delayed kinetics in bacterial clearance , associated with a local reduction of T cell and neutrophil numbers , when compared with immunocompetent RS-treated mice . In addition , no relapse was observed following an additional 3 month period of DEX administration . These findings reveal a major role of the RS bactericidal activity for the resolution of M . ulcerans experimental infections even during immunosuppression , and support clinical investigation on the potential use of corticosteroids or other immunosuppressive/anti-inflammatory drugs for the management of BU patients undergoing paradoxical reactions .
Buruli ulcer ( BU ) is a necrotizing disease of the skin , subcutaneous tissue and bone [1] , [2] . The pathogenesis of the disease is associated with local and regional cytotoxic/immunosuppressive activities of the lipidic toxin mycolactone , produced by the environmental pathogen Mycobacterium ulcerans [3]–[7] . The clinical forms of BU disease are characterized by an initial nonulcerative lesion , often a nodule or a papule or the more disseminated forms plaques and oedema . Each of these forms can evolve to an ulcer and metastasize with the development of new cutaneous lesions or osteomyelitis [1] , [2] . Established BU lesions are characterized by extensive necrotic , acellular areas with clumps of extracellular bacilli surrounded by a band of inflammatory cells , usually neutrophils and macrophages [8]–[10] . Although an extracellular localization of the bacilli is frequently seen in histological sections , M . ulcerans presents an intramacrophage growth phase in its life cycle before shedding to the extracellular compartment , and this supports the observation of intracellular bacilli at the peripheries of necrotic areas [11] . It has also been shown in the mouse model that , in addition to the site of infection , the draining lymph nodes ( DLN ) are colonized with bacilli , leading to extensive cell apoptosis , nodular tissue damage , and consequently depletion of M . ulcerans-specific T cells , further compromising the host immune response [12] . BU is a difficult-to-treat disease , however , improvement in case management has been achieved with the introduction of combined antibiotherapy with rifampicin and streptomycin ( RS ) , a regimen recommended in 2004 by the World Health Organization ( WHO ) [13] . Successful results for the treatment of nonulcerative and small ulcers have been described [14]–[17] , but variation in efficacy has been reported for advanced and disseminated lesions , for which surgery is still required in combination with antibiotherapy to achieve healing [15]–[19] . Subsequent to RS treatment , both in humans and in the mouse model , the immunosuppressive state at the M . ulcerans foci of infection wanes over time , a process characterized by an increase in inflammatory infiltrates , phagocytic activity and development of organized lymphoid structures [9] , [20]–[22] , which , in turn , is associated with a rapid decline of viable bacteria [20] , [21] . Additionally , during antibiotherapy in experimental infections it has been shown that the structure of the DLN is preserved , contributing for the establishment of a cellular immune response at the site of infection [21] . Together , these observations implicate the host immune antimicrobial mechanisms in the process of mycobacterial killing during RS treatment . Despite the efficacy of the RS antibiotic regimen , acid fast-bacilli ( AFB ) persist at the site of infection for extended periods of time [9] , [14] , [20]–[26] . Although these AFB are non-viable , as suggested by the non-reactivation of experimental infections after corticosteroid administration , mice maintain an inflammatory response with active phagocytes at the site of infection [21] . These observations in the mouse model , although not related with apparent pathology , are in line with the descriptions of paradoxical reactions occurring in some BU patients submitted to antibiotherapy . The so-called paradoxical reactions are characterized by exacerbated inflammatory responses and a surplus of degraded bacteria , which persist at the initial sites of treated lesions or in new cutaneous lesions [23] , [26] , [27] . These inflammatory responses are associated with a clinical worsening that follows an initial improvement of the lesion or even the appearance of fluctuant , erythematous and painful new lesions during or after antibiotic treatment [17] , [23] , [27] , [28] . The occurrence of paradoxical reactions has also been described in M . tuberculosis-infected patients undergoing treatment [29]–[31] . In the case of M . tuberculosis infections , most presentations of paradoxical reactions are mild and do not require specific treatment or alteration in the antibiotic regimen [32] , [33] . However , most severe cases , such as those that involve the central nervous system and pleural cavity , require treatment [33] , [34] . Although the treatment of paradoxical reactions is not consensual [35] , in part due to the lack of clinical trials , the use of corticosteroids seems to improve their resolution and the drug is usually used by clinicians [29] , [33] , . The use of corticosteroids has already been proposed for BU patients , in order to avoid or limit the extent of surgical management [27] . Corticosteroids are potent immunosuppressors and anti-inflammatory compounds , which act upon leukocyte circulation , function and migration to the sites of infection and tissue damage [36]–[38] . Considering the unknown contribution of the host effector immune mechanisms to the M . ulcerans killing observed during RS antibiotherapy , and its implications for the possible management of exacerbated inflammatory responses leading to paradoxical reactions through immunomodulation , we used the mouse model of M . ulcerans infection to address the impact of immunosuppression induced by dexamethasone ( DEX ) on the efficacy of RS treatment . For that , we evaluated the macroscopic progression of the lesions , bacterial burdens , histological alterations and occurrence of reactivation of infection after long-term DEX administration .
This study was approved by the Portuguese national authority for animal experimentation Direcção Geral de Veterinária ( ID: DGV 594 from 1st June 2010 ) . Animals were kept and handled in accordance with the guidelines for the care and handling of laboratory animals in the Directive 2010/63/EU of the European Parliament and of the Council . Eight-week-old female Balb/c mice were obtained from Charles River ( Barcelona , Spain ) and were housed under specific-pathogen-free conditions with food and water ad libitum . M . ulcerans 98-912 ( Institute of Tropical Medicine ( ITM ) collection , Antwerp , Belgium ) , a mycolactone D producing strain , was isolated in China from a case of ulcer and is highly virulent for mice , as previously described [6] , [7] , [8] . Preparation of the inoculum was performed as previously described [21] . Mice were inoculated in the left hind footpad with 0 . 03 ml of M . ulcerans suspension containing 5 log10 AFB , determined according to the method described by Shepard and McRae [39] . The right hind footpad was used as a control . Rifampicin and streptomycin ( RS ) were obtained from Sigma-Aldrich ( USA ) . The dose and mode of administration were as previously described [21] , [40] . Briefly , rifampicin was given orally by gavage at a dosage of 10 mg/kg of body weight and streptomycin was given by subcutaneous injection , at a dosage of 150 mg/kg of body weight . The treatment was initiated at the second week post-infection and was performed 6 days per week during 10 weeks . Antibiotic vehicles were given to control mice . Dexamethasone ( DEX ) ( Sigma-Aldrich ) was administrated by intraperitoneal injection at a dosage of 5 mg/kg of body weight , as previously described [21] . The administration was initiated at day 6 before M . ulcerans infection and lasted for 3 months after the end of antibiotic treatment , given 6 days per week . DEX vehicle was given to control antibiotic treated mice . Since DEX induces atrophy of the lymphoid organs ( thymus , spleen and lymph nodes ) in rodents [37] , the kinetics of splenocytes was monitored as a readout of the immunosuppressive state . Approximately a ten to twenty-fold reduction in the total number of splenocytes was observed during the entire period of DEX administration to infected or infected and RS treated mice ( Figure 1 ) . After infection , as an index of lesion development , footpad swelling of infected mice was determined over time , as previously described [8] . M . ulcerans growth in footpad tissues of infected mice was evaluated by colony forming units ( CFU ) at 9 , 12 , 14 , 21 , 42 , 82 and 168 days post-infection . For the preparation of footpad suspensions , tissues were homogenized and decontaminated as previously described [8] , [21] , and serial dilutions were plated on 7H9 agar . CFU's were counted after 6–8 weeks of incubation at 32°C . Mouse footpads were harvested , fixed in buffered formalin and embedded in paraffin . Light-microscopy studies were performed on tissue sections stained with haematoxylin and eosin ( HE ) or Ziehl Neelsen ( ZN ) , as previously described [8] . For immunohistochemistry , footpad tissue sections were deparaffinised and hydrated . Antigen retrieval was performed with EDTA 1 mM pH 8 or Borate buffer 0 . 02 M pH 7 for 30 min for the staining of T cells or neutrophils , respectively . Endogenous peroxidase activity was blocked with 0 . 3% hydrogen peroxide for 30 min and unspecific binding prevented by fetal bovine serum for 1 h , followed by 30 min blocking of avidin/biotin activity ( Avidin/Biotin Blocking kit , Vector Laboratories , Inc . ) . Purified rat anti-CD3 ( T cell marker , AbD Serotec ) or purified rat anti-Ly-6G ( neutrophil marker , BD Pharmingen ) was added to the sections at a concentration of 1∶100 or 1∶1000 , respectively , and incubated overnight at 4°C . Rabbit biotinylated anti-rat IgG antibody ( Vector Laboratories , Inc . ) was added at a concentration of 1∶200 for 1 h at room temperature , followed by 30 min of streptavidin-peroxidase polymer ( Sigma-Aldrich ) . Staining was performed with DAB Peroxidase Substrate Kit , 3 , 3′-diaminobenzidine ( Vector Laboratories , Inc . ) . Tissues were counter stained with haematoxylin and images were obtained with an Olympus BX61 microscope . The quantification of CD3+ T cells and Ly-6G+ neutrophils in the tissue sections was determined by counting the stained cells in the inflammatory area , using the software ImageJ . The values were represented as the mean cells per mm2 of inflammatory area of 5 images per section of total of 2 sections per footpad . Images were taken with a 20× objective lens . Single cell suspensions of the spleens from the different groups of mice were obtained and erythrocytes lysed with 0 . 87% ammonium chloride solution for 2 min at room temperature . Cells were counted using a haemocytometer . Differences between the means of experimental groups were analyzed with the two-tailed Student's t test , with a 95% level of significance , using the GraphPad Prism version 5 . 0 software . Differences with a P value<0 . 05 were considered significant .
To investigate the impact of corticosteroid-induced immunosuppression in the effectiveness of antibiotherapy against M . ulcerans infection , we used the experimental mouse model , treated or not with RS , in combination with DEX administration . As previously described [21] , emergence of ulceration in the footpad of mice infected with virulent M . ulcerans 98-912 ( control-infected mice ) occurred at day 21 post-infection ( Figure 2A ) , while RS administration in infected mice ( RS mice ) , starting at day 12 post-infection , resulted in the continuing reduction of footpad swelling ( Figure 2A ) and viable bacteria in the subcutaneous tissue ( Figure 2B ) , with complete clearance at the end of 10 weeks of treatment . To assess the protective role of host immunity in the early control of M . ulcerans proliferation , mice were administered with DEX from day 6 before infection until the end of the experimental period . Our results show that immunosuppressed mice ( DEX mice ) were more susceptible to infection , with faster progression of footpad swelling/ulceration ( P<0 . 001 from day 8 to 14 post-infection ) and higher bacterial loads ( P<0 . 001 ) as compared to control-infected mice ( Figure 2A and 2B ) . To characterize the anti-M . ulcerans activity of the antibiotics in immunosuppressed hosts , DEX mice were subjected to the same antibiotic regimen as RS mice ( DEX-RS mice ) . At the start of RS treatment , DEX mice presented a higher bacterial load as compared with control-infected mice ( 6 . 2 log10 CFU and 5 . 1 log10 CFU , respectively ) ( Figure 2B ) . During RS treatment , the progression of footpad swelling in the DEX-RS group followed the same trend as in RS mice , with a gradual decrease to basal levels , by the end of the RS administration period ( Figure 2A ) . However , DEX-RS mice showed a delayed kinetics of bacterial clearance as compared to immunocompetent RS treated mice , with 2 . 6 log10 CFU at 42 days post-infection , time-point when CFU were already not detectable in the RS group ( Figure 2B ) . Nevertheless , despite this delay , DEX-RS mice were able to clear the infection after a 10-week period of antibiotic regimen ( Figure 2B ) . Moreover , the extension of DEX administration for 3 months after the completion of antibiotherapy did not result in disease reactivation ( Figure 2A ) nor in the detection of viable bacilli ( Figure 2B ) , showing that the RS regimen is effective , even in corticosteroid-immunosuppressed hosts . DEX-treated mice showed an increased susceptibility to infection by M . ulcerans strain 98-912 in terms of bacterial proliferation and emergence of ulceration . However , DEX-RS mice were able to clear bacteria , although with a delay , as compared to RS mice . To assess the contribution of immune mechanisms to the clearance of M . ulcerans , we analyzed the histopathology at the site of infection in immunocompetent vs . DEX-treated mice . As previously described [21] , at day 12 post-infection the presence of central necrotic areas with extracellular bacilli surrounded by a predominantly neutrophilic/macrophagic infiltrate ( Figure 3A–D ) are histological features of a progressive subcutaneous infection with virulent M . ulcerans 98-912 . On the other hand , during RS treatment we observed a switch of the inflammatory profile to abundant lymphocytic/macrophagic infiltrates , which was maintained until the end of the experimental period ( 10 weeks post-infection ) ( Figure 3H–I and 3P–Q ) . In comparison to control-infected mice ( Figure 3A ) , footpad tissue of DEX mice presented widespread necrosis ( Figure 3E ) associated with massive clumps of extracellular bacilli ( Figure 3G ) , which is consistent with the higher bacterial burden ( Figure 2B ) . The pattern of the inflammatory response in this group of immunosuppressed mice was similar to control-infected mice , with neutrophils adjacent and/or in necrotic areas ( Figure 3B and 3F ) . In immunosuppressed mice submitted to antibiotherapy ( DEX-RS mice ) after 4 weeks of RS administration ( 42 days post-infection ) , the increased bacterial burdens , as compared with RS mice , was reflected in the higher number of clumps of extracellular bacilli ( Figure 3J–K and 3N–O ) . Despite the higher bacterial burden , inflammatory infiltrates showed a similar profile to immunocompetent RS mice ( Figure 3H–I and 3L–M ) , characterized by an increase of a predominantly mononuclear infiltrate , as compared to non-treated mice ( Figure 3E–F ) . This profile was maintained at the end of treatment ( Figure 3P–Q and 3T–U ) . Given the known immunosuppressive and anti-inflammatory properties of DEX , namely the inhibition of inflammatory cell recruitment , including neutrophils and lymphocytes , to the focus of infection [37] , [38] , [41] , [42] , we next analyzed if there were differences in these cell populations in infected footpads . We observed that after 4 weeks of RS treatment ( 42 days post-infection ) , despite the similar amounts of inflammatory infiltrates observed in slides stained with HE ( Figures 3H–I and 3L–M ) , DEX-RS mice presented a lower number of T cells stained by immunohistochemistry ( Figure 4B ) as compared with RS mice ( Figure 4A ) . The quantification of T cells confirmed the histological observations , with a median distribution of 369 cells/mm2 of inflammatory area in RS mice , whereas the DEX-RS group only showed 110 cells/mm2 ( Figure 4E; P<0 . 001 ) In addition , in the DEX-RS group , most of the staining for the neutrophilic marker Ly-6G was observed in the remaining necrotic tissue with cell debris , and only few intact cells were stained in the peripheries of the lesion ( Figure 4D ) , when compared with RS mice for which intact neutrophils were mainly found at the peripheral areas ( Figure 4C ) . The quantification of these cells showed a distribution of 506 vs . 279 cells/mm2 of inflammatory area in the RS and DEX-RS group of mice , respectively ( Figure 4F; P<0 . 01 ) . These data show that corticosteroid-induced immunosuppression , associated with increased M . ulcerans proliferation , results in increased necrosis at infection foci . Following RS administration , bacterial clearance ensues in immunosuppressed mice , although with slower kinetics , which is associated with lower T cell numbers .
The recent regimen with RS , introduced by the WHO in 2004 , has been proven effective in BU patients with nonulcerative or small ulcers , but variation in efficacy is reported for more advanced lesions [14]–[19] . Improvements to this protocol have been tested , such as the introduction of a fully oral antibiotic regimen replacing streptomycin by clarithromycin , since streptomycin cannot be administered to pregnant women or intolerant patients , and demands daily intramuscular administration [16] , [26] , [43]–[46] . Regardless the antibiotic protocol , there have been reported cases of clinical worsening of the lesions after an initial period of improvement . These so-called paradoxical reactions have been attributed to an exacerbated inflammatory response to mycobacterial antigens resulting from effective antibiotic activity [17] , [23] , [26]–28 . In fact , several studies in humans and in mice have shown an increase of the local immune response during the antibiotic treatment , with abundant lymphocyte/macrophage infiltrates , in some cases forming organized lymphoid structures , and with the phagocytosis of bacilli [9] , [20]–[22] . In addition , dead bacilli persist at the site of the treated lesion , which allows the maintenance of the inflammatory response [21] . Therefore , it is important to understand how far the immune response can be modulated , in order to regulate the paradoxical reactions without compromising the efficacy of the antibiotic treatment [9] , [21] , [22] . To address this question , we used a mouse model of M . ulcerans infection to characterize the contribution of the host immune response to the RS-associated clearance of M . ulcerans , as well as to study the impact of immunosuppression in the efficacy of the RS regimen . For that , mice were systemically immunosuppressed with the synthetic corticosteroid DEX and treated with RS during a period of 10 weeks . We observed that , even in a state of induced immunosuppression , RS-treated mice are able to clear the infection , although with a delayed kinetics , with no relapse following more 3 months of DEX administration . DEX is one of the most powerful corticosteroid immunosuppressant drugs , with activity on leukocyte circulation , function and migration to the sites of infection [36]–[38] . In our model , this drug proved to be immunosuppressive , since its continuous administration , initiated 6 days before the inoculation of M . ulcerans , rendered mice more susceptible to infection by the virulent isolate 98-912 , with a faster progression of macroscopic pathology and increased bacterial burdens . Accordingly , we observed a high reduction in the total number of splenocytes in DEX and DEX-RS mice during the entire period of DEX administration . Moreover , the administration of DEX also induced a reduction of local T cells ( CD3 positive ) during antibiotherapy . It is known that M . ulcerans infection induces the activation of IFNγ-specific T cells that are later depleted , locally and regionally , due to the cytotoxic activity of mycolactone [12] . This Th1 type of immune response was proven to be important for protection against M . ulcerans strains of lower virulence , as shown by the higher susceptibility of mice deficient in either T cells or IFNγ [7] , [12] . Therefore , the lower number of T cells in the footpads of mice treated with DEX is expected to contribute to the host susceptibility to infection in the present model . On the other hand , T cell survival is allowed during RS administration , in association with the decline of viable bacilli . The fact that DEX-RS mice presented a delayed clearance of viable bacteria in the footpad lesions suggests a role of the immune response in the efficacy of the antibiotic regimen . Such a type of immune participation is suggested in another experimental model of antibiotherapy in mice infected with Mycobacterium avium complex , where treatment with sparfloxacin and ethambutol is enhanced by combination with an inhibitor of the cortisol receptor [47] . However , it is also important to stress that , at the beginning of the RS regimen , immunosuppressed mice already presented a higher bacterial burden associated with more severe histopathology , which may also hamper the diffusion of the antibiotics to the core of the lesion . Nevertheless , despite the higher bacterial load and suppressed local inflammatory responses in the footpad of immunosuppressed mice ( with lower numbers of T cells and neutrophils ) , the antibiotic regimen was able to clear the infection after 10 weeks of administration . Indeed , no relapse was observed after an additional 3 months of DEX administration . This points out that the bactericidal activity of the drug is the main factor in the resolution of the infection . In addition to the analysis of the treatment efficacy in mice administered with DEX , it would have been interesting to test specifically if corticosteroids or other immunosuppressive/anti-inflammatory drugs could control paradoxical reactions during or after antibiotherapy . However , there is currently no proper animal model to study paradoxical reactions . Corticosteroids are being successfully used in the management of other types of paradoxical reactions , for instance in patients with tuberculosis presenting severe forms that must be treated , and when surgery of the affected area is unwanted or difficult/risky to perform , such as in the central nervous system [29] , [34] . The fact that the antibiotic treatment was efficient in our model , even in mice administered with DEX , suggests that the use of corticosteroids in BU patients undergoing severe paradoxical reactions may not represent a risk of reactivation/treatment failure . However , more studies are needed to address this point , especially when regarding the management of more severe lesions , where culture positivity is sometimes detected at the end of antibiotic treatment [16] , [17] . On the other hand , monitoring the persistence of AFB in lesions is also a feature to be considered for the management of BU patients with paradoxical reactions submitted to corticotherapy , since the end of the immunosuppressed-induced state could be followed by an exacerbated up-regulation of the immune response . Although the use of corticosteroids in our mouse model does not compromise the efficacy of antibiotic treatment , we should stress that in humans the use of these drugs should be considered with caution . Several side effects are associated with corticosteroids , such as the development of metabolic alterations like hyperglycemia or adrenal atrophy , or even impaired wound healing , but these effects are dependent on the type of corticosteroid used , doses and the time of administration [48] , [49] . Patients receiving corticosteroids are also at risk of developing opportunistic or reactivating infections , like strongyloidiasis , tuberculosis , fungal infections and cytomegalovirus [48] , [50] . However , a randomized placebo-controlled clinical trial in South Africa on the systemic use of corticosteroids to control paradoxical tuberculosis-associated immune-reconstitution inflammatory syndrome in HIV-infected patients receiving antitubercular and antiretroviral therapy , showed beneficial activity in ameliorating the symptoms with minimal side-effects , when a low and short-term therapy with prednisone was used [51] . The authors advise , though , that excluded diagnosis of multidrug-resistant tuberculosis or Kaposi's sarcoma should be performed before starting corticosteroids [51] . Therefore , a possible use of corticosteroids in BU patients or other alternative management strategies justifies clinical investigation and deserves consideration , depending on the severity of the case , potential side effects and evaluation of the risk/benefit ratio . Like in tuberculosis patients , paradoxical reactions in BU patients are transient , but in some cases these result in a considerable enlargement of the lesions and a prolonged period to achieve healing [28] . A strategy to avoid or improve such outcome during or after antibiotherapy would be desirable . Although we did not test other immunosuppressive/anti-inflammatory drugs , our study may also open possibilities to study the management of more severe paradoxical reactions with drugs that , for instance , could be applied locally , thus minimizing systemic effects and avoiding the need of surgery . In summary , corticosteroid-induced immunosuppression during experimental M . ulcerans infection , although delaying bacterial clearance , does not ultimately compromise the efficacy of the WHO recommended RS regimen . This observation may be explained by a major role of the bactericidal activity of RS that overlaps the activity of the local immune response . This study justifies future clinical studies on the potential use of corticosteroids or other immunosuppressive/anti-inflammatory drugs in the management of BU patients undergoing paradoxical reactions .
|
Buruli ulcer ( BU ) is an infectious disease caused by the environmental pathogen Mycobacterium ulcerans that affects the skin , subcutaneous tissue and bone , presenting extensive tissue necrosis . Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin ( RS ) for 8 weeks . Histological analysis of biopsies taken from the lesions of treated patients reveals an augmented inflammation that is suggested to contribute to the antibiotics' efficacy . However , in some patients , this inflammatory process developed during RS treatment may cause disease worsening , the so-called paradoxical reactions . By using a mouse model of M . ulcerans footpad infection , we show that mice co-administered with RS and the immunosuppressive/anti-inflammatory corticosteroid dexamethasone ( DEX ) are efficiently cured by the end of antibiotic treatment , although with a slight delay in bacterial clearance , pointing to a contribution of immune effector mechanisms . Additionally , no disease reactivation was observed after an additional period of 3 months of DEX administration . These findings have an important impact for the management of antibiotic-treated BU patients with paradoxical reactions , since the use of corticosteroids in mouse experimental infection do not cause treatment failure or disease reactivation , and therefore represents a potential strategy to control exacerbated immune responses during BU antibiotic treatment .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"infectious",
"diseases",
"buruli",
"ulcer",
"neglected",
"tropical",
"diseases",
"immunology",
"biology",
"microbiology",
"host-pathogen",
"interaction",
"immune",
"response"
] |
2012
|
Corticosteroid-Induced Immunosuppression Ultimately Does Not Compromise the Efficacy of Antibiotherapy in Murine Mycobacterium ulcerans Infection
|
How muscles are used is a key to understanding the internal driving of fish swimming . However , the underlying mechanisms of some features of the muscle activation patterns and their differential appearance in different species are still obscure . In this study , we explain the muscle activation patterns by using 3D computational fluid dynamics models coupled to the motion of fish with prescribed deformation and examining the torque and power required along the fish body with two primary swimming modes . We find that the torque required by the hydrodynamic forces and body inertia exhibits a wave pattern that travels faster than the curvature wave in both anguilliform and carangiform swimmers , which can explain the traveling wave speeds of the muscle activations . Notably , intermittent negative power ( i . e . , power delivered by the fluid to the body ) on the posterior part , along with a timely transfer of torque and energy by tendons , explains the decrease in the duration of muscle activation towards the tail . The torque contribution from the body elasticity further clarifies the wave speed increase or the reverse of the wave direction of the muscle activation on the posterior part of a carangiform swimmer . For anguilliform swimmers , the absence of the aforementioned changes in the muscle activation on the posterior part is consistent with our torque prediction and the absence of long tendons from experimental observations . These results provide novel insights into the functions of muscles and tendons as an integral part of the internal driving system , especially from an energy perspective , and they highlight the differences in the internal driving systems between the two primary swimming modes .
During the undulatory swimming of fish , a backward-traveling wave of body bending is formed to push against the water and generate propulsion . Muscle is the executor of the neural control and the source of mechanical power in fish swimming . Therefore , how muscles are used is a key question in understanding the control and mechanics of fish swimming and has been a focus multidisciplinary research over the past decades . Experimentally , muscle activation during swimming is measured using electromyography ( EMG ) for various fish species ( [1–5] , for a review see [6] ) . During steady swimming , a common pattern emerges: the muscle elements are activated as a wave traveling posteriorly , but this EMG wave travels faster than the curvature wave . Consequently , the phase difference between the curvature and EMG waves varies along the body , which is known as “neuromechanical phase lags” . Nonetheless , the details of the muscle activation pattern vary among species . For anguilliform swimmers such as eels , the speed difference is not large , and the duration of the muscle activation on one side of the body is approximately half of the undulation period [3] . For carangiform swimmers such as carp , the propagation speed of the EMG onset is much higher than that of the curvature wave , whereas that of EMG termination is even higher , resulting in a decrease in duration towards the tail [4] . The EMG activity , along with the muscle contraction kinetics , the strain and the volume of the active muscle , can determine the absolute muscle power output along the body . With this approach , Rome et al . [5] showed that for scup , the power is generated mostly by the posterior part of the body . To understand the muscle activation patterns and underlying mechanical principles of internal driving , researchers previously studied the internal torque and the corresponding power required . The sign of the torque has been used to predict which side of the muscle should be activated . Using theoretical models , namely resistive force theory [7] , elongated body theory [8] , and 3D waving plate theory [9 , 10] , previous studies obtained torque waves that travel faster than the curvature waves and qualitatively explained the neuromechanical phase lag . However , since positive and negative torques both occupy half of the period throughout the body , the decrease in the EMG duration in carangiform swimmers remains an obscure phenomenon . Another approach used to understand the internal driving in the coupled system is to use neural control signals as an input and observe the kinematics emerging from the coupling of internal driving , the body , and the external fluid . Using resistive force theory and 2D computational fluid dynamics ( CFD ) with a prescribed uniform muscle activation , McMillen et al . [11] and Tytell et al . [12] studied lamprey-like swimmers and showed that the same muscle forces can generate body bending with different wavelengths , corresponding to varying magnitudes of the neuromechanical phase lags , depending on passive body properties such as stiffness . However , since the kinematics emerge from the coupling of many components , this type of approach may generate kinematics that do not match the experimental observations; therefore , the approach may cause difficulties for systematically studying the features of muscle activation and for explaining the differences in the muscle activation between species . These previous modeling studies were all based on either theoretical models with strong assumptions or 2D CFD models , which cannot capture 3D flow around the top and bottom of the fish body and the jet left behind and 3D shapes for carangiform swimmers [13] . Therefore , while the qualitative explanations from the models are reasonable , the errors in these predictions are difficult to estimate . To study the features of muscle activations among different species and elucidate the underlying mechanical principles , we use 3D CFD simulations to investigate the torque patterns and power output patterns for a typical anguilliform swimmer and a typical carangiform swimmer . By combining the simulation results with experimental observations , we aim to explain the features and their variations in the EMG patterns among fish with different swimming modes .
The carangiform body is modeled based on the actual anatomy of a mackerel , whereas the anguilliform body is created from a lamprey computed tomography ( CT ) scan ( see [14] for details ) . Except for the caudal fin , the fins are neglected for the swimmers . The lengths of the fish bodies ( L ) are used as the unit length in the simulations . The bodies are meshed with triangular elements , and some sharp and small structures from the scan are removed to avoid instability in the CFD computation . After obtaining the surface data of the two fish , we reshaped the fish and remeshed the surface grid such that our code could accommodate the boundary between the fish and the fluid . The sharp and thin tail of the mackerel was modeled as a zero-thickness membranous structure . The number of surface mesh points was 3962 for the eel and 2127 for the mackerel ( including 1962 for the mackerel’s body and 165 for the tail ) . The body mass ( M ) was computed by assuming a uniform distribution of density equal to the fluid density and was 1 in nondimensional units . M = 0 . 0019 for the eel and M = 0 . 0101 for the mackerel . The kinematics for undulatory locomotion are generally in the form of a posteriorly traveling wave with the largest wave amplitude at the tail . To describe the deformation of the fish bodies , centerline curvatures κ are prescribed in the form of κ ( s , t ) = A ( s ) sin ( ks − ωut ) , where s is the arc length measured along the fish axis from the tip of the fish head , A ( s ) is the amplitude envelope of curvature as a function of s , k is the wavenumber of the body undulations that corresponds to wavelength λ , and ωu is angular frequency . We use the undulation period as the unit of time; thus , ωu = 2π . The amplitude envelope A ( s ) for the anguilliform kinematics has the form A ( s ) = amaxes−1 , where amax is the tail-beat amplitude . For carangiform kinematics , the amplitude envelope has the form A ( s ) = a0 + a1s + a2s2 . The parameters for A ( s ) were adjusted to fit the envelope of the movement of real fish observed in experiments [8 , 15] . The parameters used were amax = 11 . 41 , and k = 2π/0 . 59 for the anguilliform swimmer and a0 = 1 , a1 = −3 . 2 , a2 = 5 . 6 , and k = 2π/1 . 0 for the carangiform swimmer . To avoid generating spurious forces and torques in the interaction between the fish bodies and fluid , we added rotation and translation in the body frame of the swimmers to ensure that the movement of the bodies without external forces satisfied two conservation laws: linear momentum conservation and angular momentum conservation ( see S1 Appendix for details ) . The resulting kinematics are shown in S1 Fig . The in-house immersed boundary method code that is used is capable of simulating 3D incompressible , unsteady , and viscous flows in a domain with complex embedded objects including zero-thickness membranes and general 3D bodies [16 , 17] . The flow is computed on a nonuniform Cartesian grid in x′y′z′ coordinates . The fluid domain has a size of 8 . 5 × 5 × 5 , and a total of 620 × 400 × 400 ≈ 99 million points are used . The grid is locally refined near the body , with the finest spacing being 0 . 005 × 0 . 005 × 0 . 005 . The fish models are placed in the center of the computational domain , and the body centerlines are in the z′ = 0 plane . A homogeneous Neumann boundary condition is used for the pressure at all boundaries . The flow speeds of the inlet flow at the front boundary is set as the swimming speed in the trial runs such that the model swimmers move only minimally in the computational domain . A zero-gradient boundary condition is used at all other boundaries . At the surface of the swimmers , nonslip boundary conditions are enforced . The time interval for the integration is 5 × 10−4 . Because the deformations of the bodies are prescribed , there are 6 degrees of freedom for the overall movement of the swimmers , the same as that of a rigid body . We computed the 3 degrees of freedom in the 2D plane of undulation from the fluid-structure interaction , namely , forward translation , lateral translation , and yaw motion . The velocity components of the swimmers are numerically integrated at the same time interval as the CFD based on Newton-Euler equations with forces and torques from the CFD . Those 3 degrees of freedoms related to the vertical direction are neglected but the force magnitude in the direction perpendicular to the plane of motion is on average less than 1/10 of the force magnitude in the plane of motion . Because the bodies of the swimmers are deforming , the governing equation for the angular degree of freedom is d ( Iω ) /dt = Ttot , where I is the moment of inertia , ω is the angular speed of the body , and Ttot is the total torque computed by integrating the contributions from the hydrodynamic forces on the surface of the swimmer . Since the deformation is prescribed , I and I ˙ are known . Therefore , ω can be obtained by numerically integrating ω ˙ = ( T tot - I ˙ ω ) / I while integrating other equations for the translational movement of the body and the flow of the fluid . We set an initial swimming speed of 0 . 3 at the beginning of the simulation and waited at least two full cycles for the swimmer to reach steady swimming . All the data presented are collected from two periods . Because the swimming direction is not perfectly aligned with the -x′-axis of the computation grid , a new coordinate system is used such that the swimming direction is aligned with -x , y is the lateral direction , and the z-axis is the vertical direction . The Reynolds number is defined as Re = UL/νk , where U is the swimming speed , and νk = 1/15000 is the kinematic viscosity . The force , internal torque , and power distributions along the fish body as a function of time are computed from the simulation . The force per unit length on the fish body , F , is calculated as follows: take an arc length Δs along the body centerline , and integrate all forces from every mesh point in Δs; then , divide the total force by the arc length Δs . Considering the hydrodynamic forces , we compute the internal torque required to overcome the hydrodynamic forces and body inertia . The body elasticity and the other internal resistive forces are initially ignored and will be discussed later . The torque can be found by analyzing the force balance on either side of the body from the point of interest [18] and using the concept of inertial force . When the effect of acceleration on the torque of a segment is considered as inertial force ( −mba ) , the effective external force can be considered F − mba , where mb is the body mass per unit length , a = v ˙ is the acceleration of the body segment , and the body is in static equilibrium . Then from the torque balance equation on either side of the body from the point of interest , we obtain the torque at the point of interest: T posterior ( s , t ) = - e z ∫ s 1 r × ( F - m b a ) d l or T anterior ( s , t ) = e z ∫ 0 s r × ( F - m b a ) d l , which is consistent with [9] . Although T = Tposterior = Tanterior theoretically , the relative error becomes significant at the ends where torques are small . To minimize the numerical error , we use a weighted average of the torques computed from both sides , namely , T = sTposterior + ( 1 − s ) Tanterior . The internal power by the torque and the power transferred to the fluid per unit length are computed as P T ( s , t ) = T κ ˙ [8] and PF ( s , t ) = −F ⋅ v , respectively , where κ ˙ is the time derivative of curvature , and v is the velocity of the body segment . The difference between the total power computed by integrating the internal power and the external power along the body is within the numerical error ( < 3% ) . We varied the kinematics ( amplitude and wavelength ) and the body shape ( height and width ) by 10% to examine the sensitivity of the results . We found that the force and torque patterns are qualitatively the same in these tests . Simulations with a smaller mesh size result in forces and torques within the numerical error . The detailed of the test parameters and the results are provided in S2 Appendix .
The free swimming speeds ( U ) are 0 . 285±0 . 004 and 0 . 245±0 . 005 in nondimensionalized units for the eel and the mackerel , respectively . The corresponding Strouhal numbers are 0 . 63 and 0 . 65 . These values are consistent with previous numerical studies at similar Reynolds numbers ( Re ≈4000 ) ( e . g . , [14] ) . For both fish , double row vortices are shed behind the tail , similar to previous numerical results ( see Fig 1 ) . The velocity field behind the mackerel clearly shows a backward flow , while a mean flow behind the eel in the fore-aft direction is not easily detected . As expected from the input kinematics and body shapes , the forces are relatively uniformly distributed on the eel but concentrated on the tail of the mackerel ( Figs 2 and 3A & 3C , S1 and S2 Videos ) . The fore-aft and lateral forces both show posteriorly traveling wave patterns similar to those of body bending , except at the head where the surface orientation rapidly changes . For the eel , the peaks in the force components near 0 . 7 body length correspond to an increase in the body height ( in z direction ) at that position . For the mackerel , the separation of the thrust and drag is clear: the tail generates most of the thrust , and the anterior part of the body generates drag at all times . Because the phase of the force , especially the lateral force , is essential in determining the phase of the torque [18] , we compare the phases of the lateral forces from the simulation with those of the velocity and the acceleration of the segments . In general , if the lateral force from the fluid on a segment is in phase with the negation of the segment velocity , it is a resistive-like force , and if the lateral force is in phase with the negation of the acceleration of the segment , it is a reactive-like force . We find that the phase of the observed lateral force on the body is closer to the phase of the negation of the acceleration except near the snout tips and the tail for the mackerel . In these regions , the phase of the lateral force is close to the negation of the velocity . In general , the forces on the fish are close to the predicted forces from elongated body theory , but discrepancies exist when the shape changes are rapid . Detailed discussions of the hydrodynamics underlying the force pattern are beyond the scope of this paper . The torque required to overcome the hydrodynamic forces and body inertia in both species exhibits a traveling wave pattern moving posteriorly with a higher speed than the curvature wave ( Fig 4 ) . For the eel , the average speed of the torque wave ( vT ) is 1 . 41 in the nondimensionalized unit ( body length/period ) . The traveling wave speed of the torque is even higher in the mackerel ( vT = 2 . 11 ) , exhibiting a nearly standing wave pattern . The torque wave speeds qualitatively match the observation that the EMG speed is much higher in carangiform swimmers [6] . The maximal value of the torque appears at approximately the middle of the body of the eel and slightly posterior to the middle point for the mackerel . As shown in Fig 5 , the power from the torque is mostly positive , indicating the energy output from the muscle , but negative values are observed on the posterior parts of both fish . For the eel , the power is nearly all negative for s > 0 . 7 , similar to the case with a floppy body in a previous 2D study [12] , whereas for the mackerel , the negative power is intermittent on the posterior part . The work over a cycle calculated by simply integrating the power is the minimal work needed , because the dissipation due to the internal resistance is not included; this method implies that the negative power transferred to the body is fully stored and recovered . The peak of this work per cycle is at the anterior part ( ≈0 . 4 ) for the eel and at a more posterior position for the mackerel ( ≈0 . 58 ) , slightly posterior to the peak magnitude of the torque . We find that the work over a cycle is significantly negative on the posterior half of the eel body and slightly negative near the tail of the mackerel . If we assume that no energy-storing and energy-transmitting elements exist , then the work done by the muscles is the integration of only the positive power . We denote this quantity by W+ . The differences between the two types of work per cycle are the greatest for the posterior part of the eel , indicating that power is lost if no spatial energy transfer is performed inside the body of the eel . The distribution of power transferred to the fluid from the body is relatively uniform on the eel but concentrated on the tail of the mackerel ( cyan dashed lines in Fig 5B & 5D ) . The mean total power Ptot averaged over a cycle is 2 . 2 × 10−4 ( in nondimensionalized units ) for the eel and 2 . 6 × 10−4 for the mackerel . If only the positive power is used , the power becomes P tot + = 8 . 6 × 10 - 4 and P tot + = 3 . 4 × 10 - 4 , for the eel and the mackerel , respectively . The significant differences between Ptot and P tot + indicate a great potential to improve energetic efficiency through the spatiotemporal transfer of energy . The torque pattern can be understood by applying the results obtained in a previous study [18]: the torque pattern in undulatory locomotion is determined mainly by the wavelength and phase of the lateral force relative to the lateral movement . The torque wave of the eel has a relatively low wave speed compared to that of the mackerel due to the short wavelength of the undulation . Because the phase of the force for the eel is overall close to the phase of the reactive force , the internal torque and power patterns are also similar to the patterns associated with pure reactive forces ( Fig 6 , left column ) . For the mackerel , the long wavelength of the curvature wave and the concentrated force on the tail result in nearly synchronized torques on the body . Because the force from the tail to the fluid is nearly in phase with the velocity , the rate of change of the curvature ( κ ˙ ) and the torque are also nearly in phase . Consequently , the torque and power patterns are similar to the patterns associated with pure resistive forces ( Fig 6 , right column ) , and the internal power is nearly all positive . Previous bending tests and experiences in the handling of fish indicate that the torque from the viscoelasticity of an eel body is significant but smaller than the torque generated by muscles [19] . For carangiform swimmers , since no muscles exist behind the peduncle region and the curvature is comparable ( albeit greater ) to the rest of the body , the torque from elasticity must be significant at least in the tail region . However , an accurate in vivo measurement of the body viscoelasticity distribution is not available . Therefore , we discuss the trend of the influences of the viscosity and elasticity individually when the elasticity or viscosity is small relative to the torque from hydrodynamics and the body inertia ( Fig 7 ) . We assume that the magnitude of the torque from the body elasticity or viscosity is 40% of the torque at individual positions along the body , namely Te = 0 . 4〈T〉κ ( s , t ) /〈κ〉 or T v = 0 . 4 〈 T 〉 κ ˙ ( s , t ) / 〈 κ ˙ 〉 , where “〈〉” means standard deviation over time . Then , the total torque that needs to be generated becomes T + Te or T + Tv . As shown in Fig 7 , we find that the effect of elasticity on the torque is different along the body , separated by a position ( s ≈ 0 . 5 for the mackerel and s ≈ 0 . 3 for the eel ) where T and κ ˙ are in phase and the power is all positive . Anterior to that point , the torque magnitudes increase , and the torque wave speeds decrease; posterior to that point , the torque magnitude decreases , and the speed of the torque wave increases . For the mackerel , the torque wave can even reverse when the phase shift effect of the elasticity is strong . The reversal of the wave resembles the reversal of the wave of the offset of the EMG observed for carangiform swimmers [6] . As a result of the changes in the torque , the area of the negative power region in the posterior part of the body decreases , and W+ decreases ( Fig 5D ) . This observation is consistent with the findings of previous studies that suitable elasticity can save and restore energy to improve efficiency ( e . g . , [20] ) . For the eel , the effect of the speed increase ends near s = 0 . 7 when the maximal curvature coincides with the minimal torque without elasticity . Therefore , the energy storage and release for the eel is in the middle part of the body ( Fig 5B ) . Since the body viscoelasticity of the eel is weak and this effect is subtle , changes in the wave speed of the middle part of the torque wave or EMG are not obvious . Since the body viscosity requires a torque that is in phase with the time derivative of the curvature , for both the eel and the mackerel , the resulting torques become more aligned with the time derivative of the curvature and hence have wave speeds closer to the speed of the curvature ( Fig 8 ) . Consequently , the negative power regions are reduced because the viscosity of the body always dissipates energy . The effects of the viscosity and elasticity are qualitatively the same as greater contributions , at least to a prefactor of 0 . 8 for Te and Tv ( see S1 Appendix for details ) . Although local elasticity can temporally transfer the energy flow into this region due to the fluid-structure interactions , the spatial transmission of such energy can only be achieved by other structures . In animals , coupled joint articulation by tendons over two or more joints is common and is an effective structure to save and transfer energy by connecting a joint with positive power and another joint with negative power [21 , 22] . For carangiform swimmers , long tendons exist that span many vertebra [23] . Although the force transfer function of these tendons towards the tail has been experimentally confirmed , to our best knowledge , their function in saving energy has not been considered . We hypothesize that these long tendons are used to transfer energy from the posterior part to the middle part of the body when the negative power appears on the posterior part ( Fig 5 ) . This hypothesis can explain the observed decrease in the muscle activation duration among the carangiform swimmers , including some detailed features: the increase in the duration of the negative power from the middle of the body towards the tail matches the decrease in the EMG duration . The start of the positive power is aligned with the sign change of κ ˙ ( the lines in Fig 4B ) , resulting in a low speed that is the same as that of the curvature wave . The end of the positive power is aligned with the sign change in the torque ( the dashed lines in Fig 5B ) , resulting in a high speed that is the same as that of the torque wave . Such differences in wave speed qualitatively match the speed differences of the onset and offset of the EMG . Note that this hypothesis does not contradict the common view that force and energy are transmitted to the tail to interact with the fluid . Torque is still required when the power is negative on the posterior region and can be provided by the muscle in a more anterior position connected by the tendon . This hypothesis is also consistent with the observation that the EMG duration is nearly half of the undulation period on the whole body of anguilliform swimmers , which do not possess long tendons [23] . The energy transfer and savings by a tendon and the shortening of muscle activation can be further elucidated by a simplistic rope model ( Fig 9 ) . We take the positions s1 = 0 . 47 and s2 = 0 . 71 on the mackerel as an example . We assume that the designated muscles ( muscle 1 and muscle 2 in Fig 9A ) are attached to virtual struts with a height Hs . Based on experimental observations on the arrangement of muscles , tendons and vertebral segments in the posterior part of carangiform swimmers [24 , 25] , a pair of muscles on anterior position are connected to the posterior point by tendons ( hereafter referred to as ‘tendon muscles’ ) . A simple relationship between the muscle force Fm and the torque about the points on the centerline from a muscle Tm can be derived: Tm = HsFm . Correspondingly , the change in the muscle length and the curvature has the following relation: ΔLm = Lm − Lm0 = HsκΔs , where Lm0 is the muscle length at rest , and Δs is the arc length between the struts without bending . The power per unit length can be computed as P = F m L ˙ m / Δ s = T κ ˙ . This relationship also holds for the tendon muscles . Because the height of the struts , the arc distance between the struts , and the resting length of the muscle do not affect the power , the exact values of these quantities are not important in this analysis . We consider the case where the phases of T1 = T ( s1 ) , T2 = T ( s2 ) , κ ˙ 1 = κ ˙ ( s 1 ) , and κ ˙ 2 = κ ˙ ( s 2 ) have the relation ϕ κ ˙ 1 ≈ ϕ T 1 > ϕ T 2 > ϕ κ ˙ 2 ( Fig 9B ) . We assume a strategy in which the tendon muscles are only active when the torque required at these two points have the same sign and one of the tendon muscles generates a torque Td that is needed by both joints , namely , Td = sgn ( T1 ) min ( |T1| , |T2| ) . We first assume that any negative power of the muscles is wasted . Then , there are four stages ( see Fig 9B & 9C ) : In the example , because ϕ κ ˙ 1 < ϕ T 1 , only stages I–III are present . The energy saved is 20% of the energy output from muscle 2 without the tendon and accounts for 60% of the total negative power that can potentially be saved . The remaining 40% of the energy is wasted in the tendon muscle due to the lengthening of the tendon-muscle system at the beginning of stage II ( gray curves in Fig 9C ) . If the tendon is elastic , then the energy could be saved during the lengthening and be released during the shortening in the later stage II to further improve efficiency . The muscle activation at point 1 comes from the combination of muscle 1 and tendon muscle , which take a half period on one side of the body , and the muscle activation at point 2 only comes from muscle 2 , which takes less than a half period ( see the bars at the bottom of Fig 9B ) . The low swimming speeds that we observed ( compared with those of real animals ) are likely due to the low Re used in our simulations . However , we argue that the results are qualitatively representative of real adult fish . First , a meta-analysis of previously reported fish swimming data indicates that the transition from the viscous regime to the turbulent regime occurs at a Re of several thousand [26] . Second , even the eel model in our study shows an inertia-dominated mode of swimming . Because the drag coefficient decreases with increasing Re in general , the speed of the simulated swimmer is expected to increase with increasing Re , and the contribution of the resistive force is expected to decrease for a real adult eel . Using 3D numerical models , we provide the most accurate prediction of the torque and power required for hydrodynamic forces during the undulatory swimming of fish . By considering the torque and power transfer by tendons and the body viscoelasticity , we for the first time provide explanations for some long-standing questions in muscle activation patterns: the shortening of muscle activations in carangiform swimmers and reversal of the wave of the offset of EMG . Our study offers an integrative view of the function of the muscles as part of the mechanical system , highlights the differences in the internal driving of two primary swimming modes , and provides insights into the energy transfer and energy saving mechanisms by body elasticity and tendons in undulatory swimming . The numerical models developed and the mechanisms revealed in this study may guide the design of efficient bioinspired robots , especially soft robots with distributed driving systems and elastic bodies [27 , 28] .
|
For undulatory swimming , fish form posteriorly traveling waves of body bending by activating their muscles sequentially along the body . However , experimental observations have shown that the muscle activation wave does not simply match the bending wave . Researchers have previously computed the torque required for muscles along the body based on classic hydrodynamic theories and explained the higher wave speed of the muscle activation compared to the curvature wave . However , the origins of other features of the muscle activation pattern and their variation among different species are still obscure after decades of research . In this study , we use 3D computational fluid dynamics models to compute the spatiotemporal distributions of both the torque and power required for eel-like and mackerel-like swimming . By examining both the torque and power patterns and considering the energy transfer , storage , and release by tendons and body viscoelasticity , we can explain not only the features and variations in the muscle activation patterns as observed from fish experiments but also how tendons and body elasticity save energy . We provide a mechanical picture in which the body shape , body movement , muscles , tendons , and body elasticity of a mackerel ( or similar ) orchestrate to make swimming efficient .
|
[
"Abstract",
"Introduction",
"Model",
"and",
"numerical",
"methods",
"Results",
"and",
"discussion"
] |
[
"medicine",
"and",
"health",
"sciences",
"fish",
"eels",
"swimming",
"classical",
"mechanics",
"fluid",
"mechanics",
"vertebrates",
"animals",
"biological",
"locomotion",
"osteichthyes",
"bioassays",
"and",
"physiological",
"analysis",
"muscle",
"electrophysiology",
"kinematics",
"research",
"and",
"analysis",
"methods",
"connective",
"tissue",
"biological",
"tissue",
"fluid",
"dynamics",
"electrophysiological",
"techniques",
"continuum",
"mechanics",
"hydrodynamics",
"torque",
"physics",
"eukaryota",
"anatomy",
"electromyography",
"tendons",
"physiology",
"biology",
"and",
"life",
"sciences",
"physical",
"sciences",
"organisms",
"motion"
] |
2019
|
3D computational models explain muscle activation patterns and energetic functions of internal structures in fish swimming
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Co-inheritance of α-thalassemia has a significant protective effect on the severity of complications of sickle cell disease ( SCD ) , including stroke . However , little information exists on the association and interactions for the common African ancestral α-thalassemia mutation ( −α3 . 7 deletion ) and β-globin traits ( HbS trait [SCT] and HbC trait ) on important clinical phenotypes such as red blood cell parameters , anemia , and chronic kidney disease ( CKD ) . In a community-based cohort of 2 , 916 African Americans from the Jackson Heart Study , we confirmed the expected associations between SCT , HbC trait , and the −α3 . 7 deletion with lower mean corpuscular volume/mean corpuscular hemoglobin and higher red blood cell count and red cell distribution width . In addition to the recently recognized association of SCT with lower estimated glomerular filtration rate and glycated hemoglobin ( HbA1c ) , we observed a novel association of the −α3 . 7 deletion with higher HbA1c levels . Co-inheritance of each additional copy of the −α3 . 7 deletion significantly lowered the risk of anemia and chronic kidney disease among individuals with SCT ( P-interaction = 0 . 031 and 0 . 019 , respectively ) . Furthermore , co-inheritance of a novel α-globin regulatory variant was associated with normalization of red cell parameters in individuals with the −α3 . 7 deletion and significantly negated the protective effect of α-thalassemia on stroke in 1 , 139 patients with sickle cell anemia from the Cooperative Study of Sickle Cell Disease ( CSSCD ) ( P-interaction = 0 . 0049 ) . Functional assays determined that rs11865131 , located in the major alpha-globin enhancer MCS-R2 , was the most likely causal variant . These findings suggest that common α- and β-globin variants interact to influence hematologic and clinical phenotypes in African Americans , with potential implications for risk-stratification and counseling of individuals with SCD and SCT .
Hemoglobin , the primary component of the red blood cell that carries oxygen to tissues in the body , is comprised of two β-globin chains , two α-globin chains , and a heme molecule bound to each subunit . In African Americans , β-globin protein structural variants , including hemoglobin S ( HbS ) and hemoglobin C ( HbC ) , and α-globin copy number variants , such as the −α3 . 7 deletion , are common . Historically , carrier status ( one copy ) of these common α- and β-globin gene variants has been viewed as having no clinical implications or sequelae [1 , 2] . Nevertheless , several well-powered studies have demonstrated that globin gene variants are associated with multiple erythrocyte parameters ( i . e . hemoglobin , mean corpuscular volume ( MCV ) , and mean corpuscular hemoglobin ( MCH ) [3] ) , and there is evidence that these variants at least partially contribute to the higher prevalence of anemia observed in African Americans compared to those of European ancestry [4–6] . More recently , other important clinical implications of globin variant carrier status have been identified . For example , HbS trait has been firmly established as a risk factor for kidney disease[7 , 8] , and the correlation between hemoglobin A1c ( HbA1c ) and fasting and 2-hour glucose measures can also be altered by sickle cell trait ( SCT ) status . [9] In addition to being required for hemoglobin production in a 1-to-1 ratio , excess free α- or β-globin chains result in reduced red cell survival . However , the implications of and interactions among concurrent mutations in both globin genes are not fully understood . Several studies have investigated the effects of co-inheritance of one copy of the −α3 . 7 deletion on sickle cell disease or SCD ( two copies of HbS ) , reporting that the −α3 . 7 deletion lowers the erythrocyte rheologic effects of hemoglobin S , which likely decreases the severity of many clinical complications of SCD[10–12] , including stroke[13] , priapism[14] , and leg ulcers[15] . However , few studies have reported on the hematologic consequences of co-inheritance of SCT ( one copy of HbS ) and α-thalassemia trait ( one copy of −α3 . 7 ) [16–18] . Moreover , whether presence of the −α3 . 7 deletion also ameliorates more recently recognized clinical sequelae of SCT , such as kidney function or HbA1c levels , is unknown . In addition to copy number variants ( CNVs ) and coding variants , other common DNA polymorphisms located in the α and β -globin gene regions ( chromosome 16p13 . 3 and 11p15 . 4 , respectively ) have been associated with red cell traits in large genome-wide association studies ( GWAS ) . [19 , 20] One such variant associated with erythrocyte traits is rs11248850 [19] , which is located upstream of HBA2-HBA1 within the major α-globin HS-40/ MCS-R2 regulatory enhancer element . [21 , 22] Nevertheless , the exact functional variant ( s ) responsible for this signal have not yet been identified , and the effects of co-inheritance with −α3 . 7 copy number is unknown . In addition , the association of this regulatory variant on clinical complications of SCD has not been explored . Whole genome sequencing ( WGS ) is unique in that it allows for the ascertainment of globin structural variants , noncoding genetic variants , and copy number variants simultaneously . Therefore , in a large , population-based sample of African Americans from the Jackson Heart Study ( JHS ) , which was not selected based upon disease status or genotype , we performed WGS and confirmed previously reported associations and identified novel interactions between α-thalassemia carrier status ( one copy ) , the GWAS sentinel variant rs11248850 , and structural β-globin variants on red cell and clinical phenotypes . We performed functional fine-mapping of the genomic region containing the α-globin GWAS signal and identified the regulatory SNP rs11865131 as the most likely causal variant . Finally , in an independent cohort of SCD individuals , we examined the effect of co-inheritance of α-thalassemia and rs11865131 on important clinical phenotypes .
The demographic and hematologic measures of the 2 , 916 JHS participants , overall and stratified by sex , are summarized in S1 Table . Anemia was present in 26% , microcytosis in 12% , and iron deficiency in 4% of individuals . Women were more likely to be anemic , microcytic , and iron deficient when compared to men . Overall , 67% of JHS participants had two diploid copies of the α3 . 7 CNV , 28% were heterozygous for the −α3 . 7 deletion , and 4% were homozygous for the −α3 . 7 deletion , but only 1% carried extra copies ( 3 or 4 ) ( S2 Table ) . Overall , 9% of the African Americans in JHS were carriers of SCT and 3% were carriers of HbC trait . The associations of α-globin 3 . 7 kb deletion status , HbS , and HbC trait on red cell and other clinically relevant quantitative phenotypes in JHS are shown in Table 1 . Confirming the observations of previous reports , we observed that SCT , HbC trait , and each additional copy of the −α3 . 7 deletion were significantly associated with lower MCV and MCH , but higher RBC count and RDW; the −α3 . 7 deletion was associated with lower hemoglobin/ hematocrit , lower MCHC , and higher HbA1c levels; and HbC trait was associated with higher MCHC . There was no evidence that the α3 . 7 duplication was associated with any red cell parameter ( S3 Table ) . We further confirmed that SCT was associated with lower eGFR and lower HbA1c in the JHS cohort [8 , 9] . However , we observed an unexpected and novel association of the −α3 . 7 deletion with higher HbA1c levels . Consistent with the quantitative trait results , when hemoglobin , MCV , and eGFR were dichotomized and analyzed as binary traits , carrier status for the −α3 . 7 deletion , SCT , and HbC trait were each significantly associated with increased risk of microcytosis . Moreover , we observed that the −α3 . 7 deletion was associated with increased risk of anemia , while SCT was associated with increased risk of CKD ( Table 1; S4 Table ) . We next assessed the association of SCT and HbC trait , stratified by −α3 . 7 deletion copy number status , on red cell phenotypes , kidney function , and HbA1c . Lower hemoglobin levels are typically reported in SCT carriers , but we observed that co-inheritance of at least one copy of the −α3 . 7 deletion ameliorated this phenotype in SCT carriers ( Table 2 ) . Moreover , we only observed a higher risk of anemia for SCT carriers among individuals carrying the normal diploid copy number of the α-globin structural variant ( odds ratio = 1 . 5 , P = 0 . 02 ) . In an interaction model , co-inheritance of −α3 . 7 significantly modified hemoglobin , RBC count , and anemia ( interaction P values were ~0 . 03 ) . Strikingly , we also observed that co-inheritance of the −α3 . 7 deletion attenuated the association of SCT with kidney dysfunction and HbA1c ( Table 2 ) . Specifically , the odds ratio of CKD associated with SCT was reduced from 2 . 6 among deletion non-carriers to 1 . 2 among deletion carriers and the interaction P values for HbA1c , eGFR , and CKD were 0 . 06 , 0 . 04 , and 0 . 02 , respectively . We did not observe any modification of the effect of HbC on red cell traits by the −α3 . 7 deletion ( S5 Table ) . In a recent meta-GWAS of red cell traits conducted in ~70 , 000 individuals of European or South Asian ancestry , a common single nucleotide variant ( rs11248850 , MAF = 0 . 50 in Europeans ) was associated with MCH[19] . This variant , which is intronic to NPRL3 , is located within the well-characterized major α-globin HS-40/ MCS-R2 enhancer element [22] approximately 70 kb upstream from the −α3 . 7 deletion ( Fig 1A and 1B ) . In JHS African Americans , we observed an allele frequency of 0 . 22 for the rs11248850 A ( variant ) allele . The rs11248850 A allele frequency was higher among African American individuals carrying the normal diploid copy number of the α-globin 3 . 7 kb structural variant ( 0 . 27 ) compared to carriers of the −α3 . 7 deletion ( 0 . 14 ) ( S2 Table ) . Nonetheless , the extent of linkage disequilibrium between rs11248850 and the α3 . 7 CNV was quite modest in JHS: the pairwise r2 between rs11248850 and the −α3 . 7 deletion was 0 . 04 , and the pairwise r2 between rs11248850 and the α3 . 7 duplication was 0 . 0004 . Given the established associations of rs11248850 and the α-globin 3 . 7 kb CNV on red cell traits , we investigated whether the common variant could modify the effects of the α-globin CNV . In models adjusted for age , sex , and the first 10 principal components of genetic ancestry , we confirmed the previously reported association of the rs11248850 A allele with higher MCV , MCH , MCHC , and lower RBC count and RDW ( Table 3 ) . Interestingly , upon adjustment for α-globin 3 . 7 kb CNV status , the association of the rs11248850 A allele with red cell traits was largely abolished ( S6 Table ) . To further explore the influence of the -α3 . 7 CNV on the association between rs11248850 and red cell traits , we performed analyses stratified by the presence or absence of the -α3 . 7 deletion ( Table 3 ) . Notably , the association of rs11248850 on RBC traits was almost exclusively confined to -α3 . 7 deletion carriers ( Table 3; interaction P values range from 0 . 0001 to 0 . 03 ) . Although we were unable to definitively determine if this modification occurred primarily in cis or in trans due to insufficient sample size , haplotype association analyses showed that the association of rs11248850 with RBC traits was apparent only when comparing rs11248850 minor allele effects on the background of the −α3 . 7 deletion allele ( S7 Table ) . Given that a GWAS signal may represent tens or hundreds of variants in high linkage disequilibrium ( LD ) , each of which could be causal , we set out to use genetic inheritance and functional assays to identify the most likely causal variant ( s ) underlying the α-globin proximal association . We identified 12 SNPs in high LD ( r2 > 0 . 8 in JHS ) with the sentinel GWAS variant rs11248850 , spanning a ~25kb region of the NPRL3 gene ( Fig 1A and 1B ) . As none of these 13 variants were coding , we investigated the ability of elements containing each variant to regulate transcription in a massively parallel reporter assay[21] . Only elements containing the sentinel SNP rs11248850 or its proxy , rs11865131 ( JHS r2 = 0 . 999 with predominant haplotypes of GG and AA ) , increased transcriptional activity of a reporter gene with a minimal promoter ( Fig 2A ) . Importantly , these two elements are the only tested elements that overlap with open chromatin in erythroid cells , are part of the known MCS-R2 enhancer element , and overlap ChIP-Seq peaks for the key erythroid transcription factors GATA1 and TAL1 ( Fig 1B ) . Furthermore , in the highly homologous mouse locus , elegant chromatin confirmation assays have demonstrated that the MCS-R2 element interacts strongly with both α-globin gene promoters[23] , and targeted genomic deletions of this element result in altered α-globin transcription[24] . In rare experiments of nature , humans specifically lacking only MCS-R2 exhibit decreased α-globin levels and altered red cell traits consistent with α-thalassemia trait[25 , 26]; deletion of MCS-R2 in primary human hematopoietic stem cells caused knockdown of α-globin and restored globin chain balance in cells from β-thalassemia patients[27] . Thus , we reasoned that rs11248850 and rs11865131are strong candidate regulatory variants of the α-globin gene potentially underlying the original GWAS signal . As our MPRA was technically only sensitive to large differences in activity ( >1 log2-fold change [21] ) , we additionally performed individual allele-specific luciferase assays for all possible haplotypes of the two variants in the MCS-R2 element . Interestingly , rs11248850 did not show a significant difference in enhancer activity by allele , but its perfect proxy variant rs11865131 exhibited a significant allelic difference in enhancer activity ( lower enhancer activity with the A allele , P = 0 . 00691 ) ( Fig 2B ) . Consistent with these functional results , EIGEN-PC , a state of the art unsupervised method for identifying functional regulatory variants [28] , predicts that rs11865131 is more likely to be functional than rs11248850 ( 5 . 92 vs . 2 . 20 ) . Interestingly , these results were somewhat contrary to our expectation , given the closer proximity of rs11248850 to the binding sites for GATA1 and TAL1 ( Fig 1C ) , two key erythroid transcription factors , which we have previously shown can be affected by regulatory variants even outside of their core motif [21] . To determine whether the lower transcriptional activity associated with the A allele could be observed endogenously in the genome , we investigated DNase I hypersensitivity ( DHS ) data across 46 cell lines ( previously analyzed in [29] ) , including erythroblasts , and found significant evidence for allelic skew in the expected direction ( P < 0 . 0001 ) ( Fig 2C ) . Indeed , this direction of effect is supported by two predictive algorithms , deepSEA [30] and gkmerSVM [31] , trained on either K562 or CD34+ DHS datasets[21 , 32] . Together , these data suggest that the most likely “causal” variant for this association lies within the MCS-R2 enhancer element , but further work , such as allelic replacement by genome editing across multiple independent cell clones , would be required to definitely prove causality for either rs11865131 or rs11248850 . To further investigate putative regulatory mechanisms by which the best candidate SNP , rs11865131 , could alter regulatory activity , we investigated whether this variant affected any of the 426 transcription factor binding motifs in the HOCOMOCO database[33] . We found that the A allele was predicted to weaken the binding motifs for only ZNF219 , MAZ , ZNF148 , WT1 , and EGR1/2 . In K562 cells , we determined that both MAZ and EGR1 strongly occupied the MCS-R2 element ( Fig 1C ) . Several other transcription factors , such as GATA1/TAL1 , BACH1 , and NFE2 occupied MCS-R2 and had motifs proximal to either rs11865131 or rs11248850 , although these motifs were not predicted to be disrupted by either variant . The possibility that the GATA1/TAL1 , BACH1 , and NFE2 motifs could be important for regulation of this enhancer was supported by evolutionary conservation and by in silico mutagenesis using a gkm-SVM trained on K562 DHS ( Fig 1C ) . To explore if any of these mechanisms were plausible in vivo , we investigated whether knockdown of 4 of these genes ( MAZ , BACH1 , TAL1 , MAFK ) in K562 cells would alter α-globin expression , but observed no significant changes in functional α-globin gene expression ( S1 Fig ) . Therefore , the exact molecular mechanisms underlying the differences in expression observed in the reporter assay are currently unclear , but it is possible that rs11865131 could tune the binding of a TF such as NFE2 or JUN/FOS without disrupting the core motif ( Fig 1C ) . Thus far , our molecular investigation has not uncovered why the rs11865131-A allele , which is associated at the population level with higher MCV , MCH , and the amelioration of clinical phenotypes in SCT , shows decreased regulatory activity , when we expect that the A-allele should instead increase functional α-globin expression . To further investigate this , we turned to expression quantitative trait loci ( eQTL ) studies of whole blood . In most published blood eQTL studies , globin mRNAs were depleted[34] , sample sizes were small[35] , or microarray probe readings for the nearly identical adult α-globin genes ( HBA1 and HBA2 ) were of low quality[36] . Nevertheless , within a whole blood eQTL study of up to 5 , 311 individual adults[36 , 37] , we identified genome-wide significant associations for the rs11865131-A allele with decreased expression of the functional embryonic ζ-globin gene ( HBZ ) , increased expression of the canonically non-functional θ-globin gene ( HBQ1 ) , and decreased expression for 2 proximal non-globin genes , NPRL3 and SNRNP2 ( Fig 1A ) , all of which are highly expressed in cultured adult erythroblasts[38] . Furthermore , when we investigated a recent , large methylation QTL ( meQTL ) study of whole blood[39] , the rs11865131-A allele was also associated with genome-wide significant changes in methylation ( both increases and decreases ) at 4 CpG dinucleotides that were measured within the α-globin region , including a CpG dinucleotide less than 300 base pairs away from the MCS-R2 element ( Fig 1A ) . Taken all together , these analyses suggest that one or both of the SNPs identified within the MCS-R2 element are involved in the complex regulation of a spectrum of genes within the α-globin cluster , although the exact mechanisms remain to be elucidated . α-thalassemia status , which is most often due to the inheritance of the −α3 . 7 deletion , has been reported to influence both the severity of anemia and clinical sequelae of SCD ( i . e . erythrocyte indices , risk of stroke , priapism , leg ulcers; reviewed in [40] ) . Given our finding that the MCS-R2 regulatory variant rs11865131 normalizes red cell parameters in African American carriers of the −α3 . 7 deletion , we explored the extent to which rs11865131 could modify clinical phenotypes in SCD patients with or without α-thalassemia ( two copies of the −α3 . 7 deletion ) . We first confirmed that α-thalassemia status was protective against stroke , priapism , and leg ulcers in HbSS patients , although the protective effect was only significant for stroke[13–15] ( OR = 0 . 45 , P = 0 . 0004 ) ( Table 4 ) . Similar to our novel findings in JHS , we found that rs11865131/rs11248850 was associated with higher MCV and that this association was abolished by adjustment for α-thalassemia status ( S6 and S8 Tables ) . Importantly , when we stratified these associations on the rs11865131 genotype , the protective effect of α-thalassemia on stroke was only present in rs11865131 GG homozygotes ( OR = 0 . 29; P = 9 . 8 x 10−4 ) , and there was no protective association of −α3 . 7 deletion with stroke among rs11865131 A-allele carriers ( OR = 1 . 29; P = 0 . 47; interaction P = 0 . 0049 ) . Similar stratum-specific association results were observed for priapism ( protection in GG homozygotes only ) but not for leg ulcers ( Table 4 ) .
Carrier status for hemoglobin chain variants , including the -α3 . 7 deletion and SCT , are common in African populations due to their strong selective advantage against severe malaria . [41] Although the implications of co-inheritance of α-thalassemia have been widely studied in SCD ( two copies ) , genetic interactions of −α3 . 7 deletion , α-globin regulatory variants , and SCT ( one copy ) for clinical outcomes in the general African American population have not been fully elucidated . In a large community-based cohort of African Americans residing in the southeastern U . S . , we now firmly demonstrate that co-inheritance of the −α3 . 7 deletion attenuates the risk of anemia and CKD in individuals with SCT . In addition , we find that a haplotype containing two common regulatory variants located within the α-globin HS-40/ MCS-R2 regulatory enhancer element normalizes red cell parameters in individuals with the −α3 . 7 deletion . Importantly , the same common haplotype negates the protective effect of α-thalassemia on stroke among HbSS patients from the Cooperative Study of Sickle Cell Disease ( CSSCD ) . These findings suggest a complex relationship between hemoglobin variants and clinical phenotypes in African Americans . Several recent studies have demonstrated that SCT is associated with progressive renal impairment including CKD , albuminuria , and end-stage renal disease . [7 , 8] HbS-dependent sickling of erythrocytes in the hypoxic environment of the renal medulla has been theorized to result in renal injury; however the pathophysiology of renal disease in SCT remains largely unknown . In SCT , co-inheritance of α-thalassemia is a major determinant of intracellular HbS concentration , and lower HbS percentage due to increasing −α3 . 7 deletion copy number has been demonstrated to protect against urinary concentrating defects in individuals with SCT . [42] Our findings of a similar interaction between −α3 . 7 deletion and SCT for the development of CKD offers further biologic plausibility that HbS concentration is the causal determinant of SCT-related nephropathy . Furthermore , it is striking to note that individuals in JHS with coinheritance of −α3 . 7 deletion and SCT are nearly completely protected against CKD , whereas SCT carriers without α-thalassemia have a 2 . 6-fold increased risk of CKD . This finding may have important implications for SCT carrier risk stratification and clinical management . SCT is associated with lower HbA1c measured using standard high-performance liquid chromatography assays[9] , thereby limiting the clinical utility of HbA1c in screening and monitoring of glucose intolerance . In general , African Americans have higher HbA1c levels for the same level of fasting glucose compared to whites . [43] Our findings in JHS suggest that the −α3 . 7 deletion may account at least in part for the higher HbA1c levels among African Americans . Since the glycated residues of HbA1c reside on the N-terminus of the hemoglobin β-chain , the relative increase in the proportion of β-chain synthesis among α-thalassemia carriers may constitute a possible mechanism for the apparent increase in HbA1c . Similar to CKD and anemia , co-inheritance of α-thalassemia attenuated the HbA1c-lowering effect of SCT , suggesting that lower HbS percentage may be either associated with less interference with the HbA1c assay or results in improved erythrocyte survival . α -globin gene expression is highly regulated by several multi-species conserved sequences ( MCS-R ) or enhancers located 30–70 kb upstream of HBA1/2 . [44] In this region , we identified an association signal for red cell traits in our study of African Americans ( previously reported in European populations[19] ) that normalizes red cell parameters in individuals who carry the −α3 . 7 deletion and negates the protective effect of α-thalassemia for stroke in individuals with HbSS . By performing functional fine mapping with reporter assays and open chromatin localization in erythroid precursor cells , we identified rs11865131 within the MCS-R2 element as the most likely and rs11248850 as the second most likely causal variant for this association , but were unable to identify a specific mechanism by which variants comprising this common haplotype were likely to influence α-globin transcript . We were unable to fully resolve the paradox of how the rs11865131-A allele impairs enhancer activity , yet appears to be associated with laboratory and clinical parameters suggesting increased HBA1/HBA2 expression , which ultimately suggests that regulation within this locus is quite complex and not fully understood . As an example of this emerging complexity , we and others recently discovered a low frequency , loss of function missense variant within HBQ1 that was strongly associated with lower MCH[45] , although HBQ1 is largely thought to be a non-functional α-like globin gene . Here , the common rs11865131-A allele was associated with increased HBQ1 but decreased expression of other proximal genes , including the functional embryonic globin gene HBZ and nearby NPRL3 . Regardless of the exact regulatory mechanisms , our study suggests that this haplotype could be an important modifier of CKD , SCD , malaria[46] , or other phenotypes whose severity is modified by the −α3 . 7 deletion . Indeed , we demonstrate that genotypes at rs11865131 can act as a “modifier of the modifier” in SCD patients , by impairing the protective effect of α-thalassemia on stroke risk . Furthermore , this variant may have an outsized effect on and contribute to the phenotypic variability of HbH , where transcriptional regulation of the single functional adult α-globin gene is paramount . [47] The strengths of our study include the large , population-based cohort with WGS , which allowed assessment of α- and β-globin variant carrier status , including the α-globin 3 . 7kb CNV , in an unselected sample of African Americans with hematologic and other clinical data . By comparison with WGS , standard 1000 Genomes imputation from GWAS may not accurately infer genotype calls for the −α3 . 7 deletion , systematically underestimating the number of individuals with this deletion[3] . Our study does have limitations . We did not have hemoglobin electrophoresis data in JHS to determine HbS percentage , and our sample size was not large enough to evaluate subtle phenotypic differences between α-globin 3 . 7 CNV categories , particularly carriers of the α3 . 7 duplication . We also did not have statistical power to separate the diplotype effects of the risk allele rs11865131-A and the −α3 . 7 deletion . Many of our novel interaction findings would not meet a strict multiple testing threshold ( for example , twelve interaction tests are performed between α-globin -3 . 7 kb deletion and sickle cell trait , giving a significance threshold of 0 . 05/12 = 0 . 004 for Table 2 using a Bonferroni correction to adjust for multiple testing ) . Additionally , the current analysis does not assess the potential modifying influence of any additional HPFH/ β-globin single nucleotide variants or CNVs , though their frequency is unlikely to be appreciable in an unselected African American population-based sample . Finally , although we were able to strongly implicate two variants in the MCS-R2 element ( rs11865131 and rs11248850 ) as responsible for altering gene expression in the α-globin cluster , we could not identify a definitive molecular mechanism by which either of these variants could act , and it is certainly possible that the observed enhancer activity may display context dependent activity . In conclusion , in this large African American cohort , we show that co-inheritance of α-thalassemia significantly modified the risk of clinically relevant phenotypes such as CKD and anemia among individuals with SCT . We also demonstrate that α-globin regulatory variant rs11865131 is associated with decreased phenotypic expression of the −α3 . 7 deletion in both the general African American population and among patients with SCD . These findings may have important implications for future research and genetic counseling in African Americans with SCD and SCT .
All Jackson Heart Study participants included in the analysis provided written informed consent for genetic studies . Approval was obtained from the institutional review board of the University of Mississippi Medical Center . The JHS is a prospective community-based study of African Americans in Jackson , Mississippi . [48 , 49] During the baseline examination period ( 2000–2004 ) 5 , 306 self-identified African Americans were recruited from urban and rural areas of the three counties ( Hinds , Madison and Rankin ) that comprise the Jackson , Mississippi metropolitan area . Recruitment was limited to adult African Americans ≥ 21 years old . All participants included in analyses provided written informed consent for genetic studies . Approval was obtained from the institutional review board of the University of Mississippi Medical Center ( UMMC ) . Data on participants’ health behaviors , medical history , and medication use were collected at baseline and subjects underwent venipuncture , including complete blood cell counts , measurements of iron indices , HbA1c , and serum creatinine . HbA1c was measured by NGSP-certified high-performance liquid chromatography ( Tosoh 2 . 2 ) . Serum creatinine ( at baseline and exam 3 ) was measured using the Jaffé method and calibrated to measurements traceable to isotope-dilution mass spectrometry . [50] Estimated glomerular filtration rate ( eGFR ) was calculated using the CKD-EPI ( CKD Epidemiology Collaboration ) creatinine equation . [51] Chronic kidney disease ( CKD ) was defined as a creatinine eGFR < 60 mL/min/1 . 73 m2 at baseline or any follow-up visit[52] . Anemia was defined as hemoglobin level < 13 g/dL in men and < 12 g/dL in women . [53] Red cell microcytosis was defined as MCV < 80 fL . Iron deficiency was defined as ferritin < 15 ng/mL . A total of 3 , 404 JHS participants underwent ~30X whole genome sequencing through the NHLBI TOPMed project . Inclusion in TOPMed was based on consent for widespread genetic data sharing , not phenotypic selection . Details of the sequencing , variant calling , and QC protocols are described in the Supplemental Methods . Genotypes for β-globin variants HbS ( rs334 ) and HbC ( rs33930165 ) and α-globin variant rs11248850 were extracted from the variant call set and used in association analyses . Principal components of genetic ancestry were calculated for each participant from the sequence data . [54] A subset of 3 , 009 JHS TOPMed participants underwent genotyping for the α-globin copy number variation ( CNV ) using the Genome STRiP multi-sample structural variant calling algorithm[55] and were eligible for the current analysis . We further excluded 6 individuals for low-quality CNV calls , 3 individuals who were homozygous and one individual missing data for the rs334 sickle cell variant , and 83 individuals who did not have hematologic phenotypes , leaving 2 , 916 individuals for analysis . The association of carrier status for α and β globin gene variants with hematologic quantitative traits was assessed using linear regression and reported as β regression coefficient ( mean difference in red cell parameter between genotype comparison groups ) and standard errors ( SE ) . For binary traits ( anemia , microcytosis , and CKD outcomes ) logistic regression was used to estimate odds ratios and 95% confidence intervals ( CI ) . Because of the small number of homozygotes for the α3 . 7 duplication ( N = 2 ) , we combined individuals carrying one or two extra copies of the α3 . 7 duplication in association analyses . All linear or logistic regression models were adjusted for age , sex , and the first 10 principal components of genetic ancestry to account for population stratification . To evaluate effect modification or genotype x genotype interactions , we performed association analyses stratified by α globin deletion status , and also introduced a multiplicative interaction term into regression models . All tests of main effect and effect modification were 2-sided and a P value < 0 . 05 was considered statistically significant . Haplotype association analyses were conducted using HAPSTAT ( V3 . 0 ) [56] . We first identified all SNPs in high linkage disequilibrium ( r2 > 0 . 8 ) with the previously reported sentinel GWAS variant rs11248850 from CEU and AFR populations of the 1000 Genomes Project Phase 3 , and performed erythroid-specific functional annotations for each SNP , including genomic location , DNaseI hypersensitivity[57] , histone modifications H3K4me1 , H3K27ac and H3K4me3[58] , and ChIP-seq for erythroid transcription factors GATA1 and TAL1 . [59] Using a massively parallel reporter assay ( MPRA ) , [21] 145 base pair elements centered at each allele of the rs11248850 sentinel SNP and all proxy SNPs were simultaneously tested for regulatory activity in vitro . Activity estimates from this assay are reported as previously described . [21] In the current study , for sites showing significant enhancer activity in the MPRA ( rs11248850 and rs11865131 ) , we additionally conducted allele-specific luciferase reporter assays assessing the function of MCS-R2 ( 436 nucleotides from chr16:163 , 406–163 , 841 in hg19 ) in K562 erythroid cells . The enhancer elements containing all combinations of allelic variants across rs11248850 and rs11865131 were cloned into the pGL4 . 24 minimal promoter ( minP ) containing vector ( Promega ) . Dual luciferase assays in K562 erythroid cells were performed in a manner similar to assays assessing other similar non-coding regulatory variants in hematopoietic cells . [21 , 60 , 61] Allelic differences in enhancer activity were tested using a two-sided Student’s t-test . For the rs11865131 variant with a significant allelic difference , DNase I hypersensitivity ( DHS ) data from multiple cell lines ( the sum of the counts for each allele across 46 heterozygous cell types ) and erythroblasts were used to assess allelic skew in sequenced reads . [29 , 62] EIGEN-PC , deepSEA , and gkm-SVM are algorithms that predict the function of non-coding variants and were used as previously described to in silico predict the effect of common variants . [21 , 28 , 30 , 31] In silico mutagenesis was performed as described in [32] . The ChIP-Atlas resource as well as K562 experiments from the ENCODE project were used to search for DHS and transcription factor ( TF ) occupancy in blood-based tissues . [63 , 64] TF binding motif disruptions were determined using the motifbreakR R package [65] and the HOCOMOCO motif set . [33] PhyloP calculations for 100 vertebrates were accessed using the University of California Santa Cruz ( UCSC ) genome browser [66] . MAZ , BACH1 , TAL1 , and MAFK knockdown using siRNAs and subsequent RNA-seq was performed in duplicate or triplicate and gene expression was quantified as described previously by the ENCODE project ( samples used were ENCFF253LFQ , ENCFF965QZJ , ENCFF133JRN , ENCFF289FNP , ENCFF595QDW , ENCFF848TKB , ENCFF675IJS , ENCFF064FJU , ENCFF464MQB , ENCFF517WDW , ENCFF213TLT , ENCFF634XCE , ENCFF714HMY , ENCFF035JUJ , ENCFF715DXD , ENCFF232WNR ) [64] . eQTL [36 , 37] and meQTL [39] results were obtained from the SMR data repository [67] and all genome-wide significant ( p < 5 . 0x10-8 ) results for cis associations in the α-globin region are reported if identified in any single study . CSSCD has been described elsewhere [68 , 69] . We used the clinical definitions from the CSSCD investigators to define incident stroke[13] , priapism[14] , and leg ulcers[15] in our analyses . The number of α-globin genes was determined by blot hybridization for 2 , 703 HbSS patients; participants with 2 or 3 copies of the α-globin genes were considered α-thalassemic . [70] Age at recruitment , sex , and fetal hemoglobin ( HbF ) levels were available for 2 , 253 of these 2 , 703 participants . CSSCD patients were genotyped on the Illumina 610-Quad array . [71] Genome-wide genotyping data was available for 1 , 140 of the 2 , 253 HbSS patients with α-thalassemia status information and covariates available . We imputed rs11865131 genotypes on 1000 Genomes Project ( phase 3 ) haplotypes ( version 5 , hg19 ) using Minimac3 ( v1 . 0 . 11 ) with high quality ( imputation r2 = 0 . 995 ) . For baseline RBC traits , we tested the association with α-thalassemia status using linear regression correcting for sex , age , and the first 10 principal components of genetic ancestry . For dichotomous complications ( stroke , priapism , and leg ulcers ) , we applied logistic regression , correcting for age , sex , and fetal hemoglobin levels . We further stratified these analyses by presence of the rs11865131 A-allele . Adjustment for the first 10 principal components did not change the conclusions from these analyses of dichotomous complication measures .
|
Recent work has shown that inheriting a single copy of the β-globin gene variant which causes sickle cell disease can be associated with medical risks , such as worsening kidney function . In individuals with sickle cell disease , co-inheritance of other globin gene variants , notably α-thalassemia , can modify an individual’s risk of clinical sequelae such as stroke . In this paper , our results suggest that inheritance of the same 3 . 7kb deletion that causes α-thalassemia in African populations can lower the risk of anemia and chronic kidney disease among African American community-dwelling individuals with sickle cell trait . Another α-globin genetic locus , located upstream within a well-known non-coding regulatory element , was found to modify associations of the α-thalassemia 3 . 7kb copy number variant with red blood cell traits in the African American general population and , in sickle cell disease patients , with risk of stroke . Using functional fine mapping and reporter assays , we localized rs11865131 and rs11248850 as the two most likely causal variants for these phenotypic associations . Additional molecular studies will be required to understand the complex regulatory mechanism by which these variants influence α-globin production .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
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2018
|
Common α-globin variants modify hematologic and other clinical phenotypes in sickle cell trait and disease
|
Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis . This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids . Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase , sister chromatids are separated and segregated onto the daughter nuclei . The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8 . In Drosophila , however , no clear Rec8 homolog has been identified so far . Therefore , we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis . Inactivation of an engineered Rad21 variant by premature , ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin , but also in precocious disassembly of the synaptonemal complex ( SC ) . We demonstrate that the lateral SC component C ( 2 ) M can interact directly with Rad21 , potentially explaining why Rad21 is required for SC maintenance . Intriguingly , the experimentally induced premature Rad21 elimination , as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites , do not interfere with chromosome segregation during meiosis , while successful mitotic divisions are completely prevented . Thus , chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin .
During meiosis , haploid germ cells are generated from diploid parental cells by two consecutive cell divisions without intervening DNA replication . Before the first meiotic division , homologous chromosomes are paired into bivalents and the two sister centromeres in each homolog are constrained to behave as a functional unit . The two homologous centromeres of each bivalent are bi-oriented in the spindle and segregated apart during the first meiotic division . Thereafter sister centromeres become functionally independent , allowing their bi-orientation and separation during the second meiotic division , very much like during mitosis ( for review see: [1] ) . Importantly , error-free chromosome segregation during each meiotic division ( homologs in meiosis I and sisters in meiosis II ) does not just depend on regulated centromere behavior but also on temporal and regional control of sister chromatid cohesion . Sister chromatid cohesion in combination with meiotic crossovers keeps bivalents physically together until the metaphase-to-anaphase transition of the first meiotic division . Crossovers are generated by meiotic recombination between non-sister chromatids of homologous chromosomes . The order of events during initiation of meiotic recombination varies among the organisms . In mice , fungi and plants double strand breaks ( DSBs ) mark the first event of meiotic recombination , and DSBs are required for the intimate pairing ( synapsis ) of homologous chromosomes during the extended prophase of meiosis I . In Drosophila , however , synapsis can occur in the absence of prior DSB formation [2] . A unique proteinaceous structure , the synaptonemal complex ( SC ) , is formed during early stages of prophase I between the homologs . SC formation commences with the establishment of the axial elements ( AE ) which represent a scaffold running alongside the paired sister chromatids within each homolog . Concomitant with pairing of homologs , the AE mature into the lateral elements ( LE ) of the SC . The LE are connected by perpendicularly oriented transverse filaments ( TF ) which form the central element ( CE ) of the SC . In Drosophila melanogaster , meiotic recombination only occurs in females and consequently the SC is only assembled during oogenesis . The protein C ( 2 ) M has been identified as a component of the LE , and the main element of the TF is the elongated coiled-coil protein C ( 3 ) G [3] , [4] . Loss of either protein results in a severely compromised SC structure and high levels of chromosome non-disjunction during the meiotic divisions [3] , [4] . Proper C ( 3 ) G localization requires C ( 2 ) M but not vice versa [3] . After crossover formation , the SC is disassembled and crossovers mature into chiasmata . Despite SC disassembly , paired homologous chromosomes cannot disjoin , because sister chromatid cohesion distal to the crossover sites prevents terminalization of chiasmata . This cohesion between replicated sister chromatids is mediated by the heterotetrameric ring-shaped cohesin complex ( for review see: [5] , [6] ) . Cohesin complex components were originally identified by genetic screens in the yeast Saccharomyces cerevisiae [7] , [8] . The core cohesin complex consists of the two structural maintenance of chromosomes ( SMC ) molecules SMC1 and SMC3 , which form extended intramolecular coiled-coils and heterodimerize via their hinge regions . An α-kleisin subunit connects SMC1 and SMC3 by binding to their head domains , thus forming a tripartite ring-like structure . The α-kleisin Rad21/Scc1/Mcd1 also recruits the accessory subunit Scc3 ( for reviews see: [5] , [6] ) . The cohesin ring most likely embraces the sister chromatids and thereby establishes sister chromatid cohesion topologically [9] . Several eukaryotes are known to express meiosis-specific cohesin components ( for review see: [10] ) . In yeasts the meiosis-specific α-kleisin Rec8 associates with the SMC head domains instead of Rad21/Scc1/Mcd1 [11] , [12] . Apart from Rec8 homologs , vertebrate genomes encode yet an additional meiosis-specific α-kleisin , Rad21L , but a direct role for this variant in sister chromatid cohesion awaits demonstration [13] , [14] , [15] . However , it has been shown in mouse spermatocytes that Rad21L is involved in assembly of the axial elements of the SC [16] , [17] . An involvement of cohesin in SC maintenance has been demonstrated previously in several distantly related eukaryotes [11] , [18] , [19] , [20] , [21] . Mammalian meiotic cohesin complexes contain the specific subunit SMC1β and the Scc3 homolog STAG3/SA3 , while mitotic cells harbor cohesin complexes with SMC1α and either STAG1/SA1 or STAG2/SA2 . Not all imaginable combinations of these subunits may be realized in cohesin complexes occurring in vivo , but immunoprecipitation of complexes present in mouse testis extracts revealed five variant cohesin complexes with differing subunit composition [15] . Cohesion is abrogated at the metaphase-to-anaphase transition by proteolytic cleavage of the α-kleisin cohesin subunit by the cysteine protease separase , thus opening the cohesin ring and liberating the sister chromatids [22] , [23] . In meiosis , two waves of separase activity occur during the two divisions . In meiosis I , separase-dependent cleavage of phosphorylated Rec8 , which is present in cohesin complexes located at the chromosome arm regions , allows chiasmata terminalization and hence homolog separation [24] , [25] , [26] , [27] . Importantly , Rec8 in cohesin complexes located within pericentromeric regions is protected from proteolytic cleavage during meiosis I . Proteins of the Shugoshin ( Sgo ) -family recruit protein phosphatase 2A ( PP2A ) to the centromeric region , thus keeping Rec8 locally in a cleavage-resistant unphosphorylated state [28] , [29] . Consequently , sister centromeres remain paired throughout meiosis I permitting their bi-orientation during meiosis II . A second burst of separase activity destroys pericentromeric cohesion before anaphase II . In several organisms , the α-kleisin Rad21 is expressed not only before mitotic but also before meiotic divisions . Its role in meiotic sister chromatid cohesion has been discussed controversially . In the mouse , Rad21 is clearly expressed in meiotic cells of both sexes [30] . A number of immunolocalization studies have shown the persistence of Rad21 on mammalian meiotic chromatin at least through meiosis I which has been interpreted as Rad21 possibly serving a cohesive function during meiosis [20] , [31] , [32] , [33] , [34] . However , other studies either failed to detect Rad21 in premeiotic S-phase or later stages of rat spermatogenesis [35] , or reported Rad21 to apparently leave chromatin before metaphase I in mouse spermatocytes [36] . Elegant functional studies have recently revealed that premature TEV protease-mediated cleavage of all Rad21 has no obvious effect on chromatid cohesion in mouse oocytes , while analogous premature Rec8 cleavage resulted in premature and complete loss of cohesion both in metaphase I , leading to chiasmata resolution , and also in metaphase II [37] . Thus , Rad21 serves no cohesive function during meiosis , at least not in mouse oocytes , and Rec8 cleavage is sufficient for loss of cohesion in both meiotic divisions . Intriguingly , the Drosophila genome does not contain an obvious Rec8 homolog . However , the SC component C ( 2 ) M [3] was shown to be a divergent member of the α-kleisin family by in-depth bioinformatics analyses [38] . Its meiosis-specific expression , its association with the cohesion subunit SMC3 , and the high level of chromosome missegregation in c ( 2 ) M mutants are consistent with C ( 2 ) M functioning analogous to Rec8 . However , the low level of sister non-disjunction in c ( 2 ) M mutants , as well as C ( 2 ) M localization dynamics during meiosis and the lack of abnormalities after expression of variants predicted to be separase cleavage-resistant , argue against C ( 2 ) M being the bona fide Rec8 homolog [3] , [39] . Two additional genes that seem to be specific to the Drosophila lineage , solo and ord , qualify to encode meiotic cohesins , as both solo and ord null mutants show premature dissociation of homologous chromosomes and of sister chromatids , resulting in high frequencies of meiotic non-disjunction events [40] , [41] , [42] . Also , in ord and solo mutants , the SC is formed , but it disassembles prematurely [41] , [43] . However , neither SOLO nor ORD display similarity with α-kleisins at the primary structure level and there are no reports that either one of the two proteins is a substrate for separase , which is active during the meiotic divisions in Drosophila [39] . Thus , it is at present not clear whether Drosophila harbors , as part of meiotic cohesin complexes , an α-kleisin homolog , which needs to be removed in a separase-dependent manner during the meiotic divisions to allow chromosome/chromatid segregation . Here we have addressed whether Drosophila Rad21/Verthandi takes over the function of Rec8 by acting as a meiotic α-kleisin and whether it is involved in SC maintenance . We find that experimentally induced premature Rad21 proteolysis during oogenesis does not result in premature chromosome/chromatid separation or chromosome non-disjunction , arguing against an essential contribution of Rad21 to meiotic sister chromatid cohesion . However , maintenance of the SC is clearly dependent on Rad21 , which co-localizes with C ( 2 ) M and C ( 3 ) G in nuclei with a fully formed SC . Moreover , our finding that C ( 2 ) M can interact physically with Rad21 allows speculations towards a molecular mechanism for the linkage of the SC to meiotic chromosome cores in Drosophila .
Rad21 provides essential functions during mitosis . To evaluate whether Rad21 also provides important functions during meiosis , we applied a system allowing controlled Rad21 inactivation specifically during oogenesis . We took advantage of Drosophila strains expressing Rad21 variants that can be proteolytically inactivated by TEV protease . These Rad21 variants contain three consecutive TEV protease cleavage sites at position 271 or 550 , as well as a C-terminal myc-epitope tag ( Rad21TEV-myc ) . Rad21 mutant rescue experiments have proven these variants to be functional [44] . Furthermore , TEV protease expression has been shown to result in efficient Rad21TEV-myc cleavage and consequential inactivation . Cleavage in mutant embryos that rely on Rad21TEV-myc as their sole Rad21 species , resulted in completely penetrant premature sister chromatid separation during the first mitosis after onset of TEV protease expression [44] . To express specifically in the female germline a UAS transgene , which encodes a TEV protease variant with improved catalytic properties ( see materials and methods ) , we used the maternal alpha-tubulin GAL4-driver line ( mat-GAL4 ) . The resulting efficiency of Rad21TEV-myc cleavage was assessed with extracts from stage 14 oocytes . Oocytes from sibling females with and without UAS-TEV protease transgene were compared ( Fig . 1A , +TEV and −TEV ) . Immunoblot analyses using antibodies against myc allowed the detection of full-length Rad21TEV-myc as well as the C-terminal cleavage product ( Fig . 1B ) . Quantification revealed that around 95% of Rad21TEV-myc was cleaved in the TEV protease expressing oocytes ( Fig . 1B ) . To assess phenotypic consequences of Rad21TEV-myc cleavage in situ , we immunolabeled ovarioles with anti-myc antibodies . In the absence of TEV protease expression , only weak signals were obtained within the nucleoplasm surrounding the karyosome , the highly condensed chromatin of the oocytes ( Fig . 1C , −TEV ) . However , upon expression of TEV protease , strong anti-myc signals were detected in the nucleoplasm ( Fig . 1C , +TEV ) , indicative of Rad21TEV-myc cleavage product accumulation . The inability to detect uncleaved Rad21TEV-myc localizing on oocyte chromatin in these whole mount preparations could , in principle , be due to accessibility problems . Thus , we also stained chromosome spread preparations of germaria and early egg chambers . While this method did not allow the unambiguous assignment of nuclei to specific stages of oogenesis , we clearly detected nuclear anti-myc signals in cells of germaria . Most importantly , while the signals were diffuse in nuclei without an SC , we obtained strong anti-myc signals co-localizing with the synaptonemal complex ( SC ) component C ( 3 ) G in the typical thread-like pattern in pro-oocytes ( Fig . 1D ) . To confirm the presence of Rad21 in the SC , we also analyzed spread preparations of ovarioles from females expressing a functional Rad21-EGFP variant by double labeling with anti-EGFP and anti-C ( 3 ) G antibodies . We again observed co-localization in nuclei with a fully formed SC , corroborating our results obtained for localization of Rad21TEV-myc . Rad21 is , together with SMC1 and SMC3 , part of the tripartite cohesin ring , embracing sister chromatids after DNA replication ( for review see [5] ) . As the cohesin rings are bound to chromatin in a topological fashion , SMC1 and SMC3 are expected to dissociate from chromatin upon Rad21TEV-myc cleavage . In contrast to Rad21 , SMC1 and SMC3 can be readily visualized on meiotic chromatin by immunostaining of Drosophila ovariole whole mount preparations [45] . There , SMC1 and SMC3 are associated with the lateral elements of the SC [45] . Indeed , while a characteristic pattern of SMC1 can be detected in the karyosome in the absence of TEV protease expression , SMC1 is delocalized upon Rad21TEV-myc cleavage ( Fig . 1E ) . We conclude that in our system Rad21TEV-myc is efficiently cleaved during oogenesis , and that this cleavage leads to premature dissociation of cohesin from meiotic chromatin . Since Rad21TEV-myc cleavage occurs during a developmental stage when the SC is fully formed in the oocyte nucleus ( TEV protease expression driven by mat-GAL4 can be detected starting in region 3 of the germaria; Fig . S1A ) , we addressed possible phenotypic consequences on SC integrity . Immunolabeling of the SC-components C ( 3 ) G and an HA tagged variant of C ( 2 ) M within wild type oocyte nuclei of stage 4–5 egg chambers resulted in the expected ribbon-like SC staining ( Fig . 2 ) [3] , [4] . TEV protease expression in a background without Rad21TEV-myc but with wild type Rad21 did not affect the SC-associated anti-C ( 3 ) G signals that were just like in wild type ovarioles ( Fig . 2A ) . A normal C ( 3 ) G staining pattern was also observed in control ovarioles expressing Rad21TEV-myc in the Rad21 mutant background in the absence of TEV protease ( Fig . 2A ) , and in ovarioles from Rad21+/Rad21− heterozygote individuals ( Fig . 3B ) . However , the characteristic ribbon-like C ( 3 ) G staining was almost completely lost from the oocyte chromatin after TEV protease expression in a background with exclusively Rad21TEV-myc , and C ( 3 ) G accumulated instead in the nucleoplasm ( Fig . 2A ) . We point out that in these same ovarioles at earlier stages within the germarium , C ( 3 ) G staining still revealed the normal ribbon-like structures ( Fig . S2 ) , as expected , because mat-GAL4 driven TEV protease expression is not yet detectable at these early stages ( Fig . S1A ) . To assess whether the localization of the lateral SC component C ( 2 ) M is also affected after premature Rad21 cleavage , we generated flies , which express c ( 2 ) M-HA under genomic control in a rad21 mutant background rescued by Rad21TEV-myc expression . When TEV protease was expressed in these ovarioles , the ribbon-like C ( 2 ) M-HA-staining typical for the SC also disappeared from the meiotic chromatin and C ( 2 ) M-HA distributed throughout the nucleus ( Fig . 2B ) . Thus , our results suggest that the SC disassembles as a consequence of Rad21TEV-myc cleavage . To evaluate whether the observed phenotype is due to a dominant negative effect of the particular cleavage products generated , we analyzed the dependence of the phenotype on the precise position of the TEV cleavage sites within Rad21TEV-myc . Moreover , to rule out effects of the GAL4 driver background , we repeated the experiments with a different driver , nanos ( nos ) -GAL4 . nos-GAL4 expression commences earlier during oogenesis , in region 2a of the germarium ( Fig . S1B ) . Indeed , TEV protease expression directed by this driver resulted in premature SC disassembly at an even earlier stage during oogenesis ( nos-GAL4: stage 3 . 4+/−0 . 6 ( n = 34 ) ; mat-GAL4: stage 5 . 3+/−0 . 6 ( n = 30 ) ; P<0 . 0001; Mann-Whitney U-test; Fig . 3A ) . Moreover , SC disassembly was observed to occur at an earlier stage with Rad21TEV-myc having the TEV cleavage sites after amino acid 271 compared to after amino acid 550 ( position 271: SC disassembly + TEV at stage 3 . 4+/−0 . 6 vs . − TEV at stage 7 . 4+/−0 . 5; position 550: SC disassembly + TEV at stage 5 . 4+/−0 . 6 vs . −TEV stage 7 . 1+/−0 . 6; Fig . 3A ) . Although nos-GAL4 driven TEV protease expression can be detected early in region 2a of the germarium , establishment of the SC was not affected ( Fig . S2 ) . Analysis of spread preparations of germaria revealed clear evidence of initial Rad21 degradation before SC disassembly ( Fig . S3 ) . Therefore , the SC disassembly which is observed during later oogenesis might depend on complete Rad21 degradation . Taken together , our results demonstrate that precocious SC disassembly is a robust phenotype that is observed with different GAL4 drivers and different TEV cleavage site insertion positions within Rad21TEV-myc . Interestingly , premature SMC1 delocalization and SC disassembly also occurred when Rad21TEV-myc was cleaved by TEV protease expression in the presence of one wild type Rad21+ allele ( Fig . 3 ) . While in these cases the SC stayed intact longer than in the Rad21 mutant situation , the difference in SC disassembly timing compared to the control situation was still highly significant ( position 271: stage 5 . 4+/−0 . 8 vs . stage 7 . 4+/−0 . 5; position 550: stage 6 . 1+/−0 . 5 vs . stage 7 . 1+/−0 . 6; Fig . 3A ) . The precocious SC disassembly is not due to the reduced Rad21+ gene dosage , because the dynamics of SC disassembly is like wild type in females heterozygous for Rad21ex without any ectopic Rad21TEV-myc cleavage ( Fig . 3 ) . Moreover , we re-iterate that TEV protease expression in a background with wild type Rad21 has no effect when Rad21TEV-myc is not expressed ( Fig . 2A and 3A ) . As an independent approach to remove Rad21 from developing egg chambers , we applied targeted destruction of GFP-tagged proteins by the deGradFP system [46] . In this system , GFP-fused proteins are recruited to a recombinant SCF ubiquitin ligase generated by expression of a specific single-chain anti-GFP antibody fused to the F-box region of Slmb ( NSlmb-vhhGFP4 ) . The recruitment of GFP fusions by NSlmb-vhhGFP4 results in their proteasomal degradation . We constructed strains in which Rad21 mutants are rescued by the expression of Rad21-EGFP . NSlmb-vhhGFP4 expression driven by mat-GAL4 markedly reduced Rad21-EGFP protein levels ( Fig . S4 ) . nanos-GAL4 driven expression of the NSlmb-vhhGFP4 fusion protein again resulted in premature dissociation of the SC . The difference in SC disassembly timing compared to the control situation ( Rad21ex , Rad21-EGFP homozygous females without NSlmb-vhhGFP4 expression ) was again highly significant ( Rad21ex , Rad21-EGFP homozygous females + NSlmb-vhhGFP4: stage 5 . 6+/−0 . 8 vs . stage 7 . 2+/−0 . 5; Rad21ex , Rad21-EGFP heterozygous females + NSlmb-vhhGFP4: stage 6 . 5+/−0 . 5 vs . stage 7 . 1+/−0 . 5; Fig . 3A ) . This premature SC disassembly after proteasomal degradation of Rad21-EGFP confirms that loss of Rad21 results in SC disintegration . This SC instability therefore does not depend on the presence of Rad21TEV-myc cleavage fragments , which are generated after TEV protease expression , and which might in principle have a dominant effect . Although the majority of the SC components C ( 3 ) G and C ( 2 ) M leaves the oocyte chromatin after forced Rad21 cleavage , some bright staining patches remain ( Fig . 2A , Fig . 3A ) . As residual SC components have been described to remain associated with clustered centromeres after normal SC disassembly [47] , we analyzed whether residual C ( 3 ) G after ectopic Rad21TEV-myc cleavage was colocalized with centromeres . Upon premature Rad21 cleavage , we indeed found colocalization of the centromeric H3 variant Cid/Cenp-A with persisting C ( 3 ) G patches ( Fig . S5 ) , suggesting that association of the SC with centromeric regions might not depend on Rad21-containing cohesin . Taken together , our data imply that the integrity of the mitotic α-kleisin cohesin subunit Rad21 is required for SC maintenance at chromosome arms during Drosophila oogenesis . To address how Rad21 interacts with the SC , we first analyzed whether Rad21 protein might bind to the SC component C ( 2 ) M . We performed co-immunoprecipitation experiments using protein extracts prepared from fly embryos expressing c ( 2 ) M-HA and Rad21TEV-myc . The presence of C ( 2 ) M and Rad21 in early embryos has been demonstrated previously [39] . Indeed , Rad21TEV-myc was co-precipitated with C ( 2 ) M-HA . In control experiments , where we used the same anti-HA antibodies for immunoprecipitation from an extract containing Rad21TEV-myc , but not C ( 2 ) M-HA , we were unable to pull down Rad21TEV-myc ( Fig . 4A ) , ruling out a non-specific association of Rad21TEV-myc with HA-antibodies or beads . To obtain independent support for an interaction between Rad21 und C ( 2 ) M , we conducted in vitro pull-down assays . To this end , we used an in vitro transcription/translation ( IVT ) system to synthesize Rad21 and Flag epitope-tagged C ( 2 ) M in a reticulocyte lysate in the presence of [35S]methionine . Autoradiography of the samples after anti-Flag immunoprecipitation revealed that Rad21 specifically bound to Flag-C ( 2 ) M ( Fig . 4B ) . To delineate the interacting domains of the two proteins , we repeated the assay with in vitro synthesized fragments of both proteins . These experiments revealed that an N-terminal fragment of C ( 2 ) M ( C ( 2 ) MN , aa 1–191 ) is sufficient to precipitate Rad21 ( Fig . 4C ) . Furthermore , the C-terminus of Rad21 ( Rad21C , aa 478–715 ) is sufficient for interaction with Flag-C ( 2 ) M or Flag-C ( 2 ) MN ( Fig . 4C ) . None of the other fragments were able to mediate an interaction in this system ( Fig . 4C ) . In the same assay system , we neither detected an interaction between Rad21 and one of the other SC components , C ( 3 ) G or Corona , nor an interaction between Rad21 and the cohesion proteins ORD or SOLO ( data not shown ) . C ( 2 ) M has been found in a complex with SMC3 [39] . If C ( 2 ) M binds directly to the SMC heads , as it is regarded typical for α-kleisins , one would expect the binding of C ( 2 ) M and Rad21 to the SMC cohesin subunits to be mutually exclusive . Using the IVT system , we analyzed the binding potential of C ( 2 ) M and Rad21 towards SMC1 . In these experiments , C ( 2 ) M was N-terminally fused with a 6× myc epitope tag , co-expressed with Rad21 and/or SMC1 and anti-myc immunoprecipitates were analyzed ( Fig . 4D ) . While Rad21 could be readily co-immunoprecipitated together with myc-C ( 2 ) M , co-immunoprecipitation of SMC1 depended on the presence of Rad21 . Thus , these data suggest that myc-C ( 2 ) M does not bind directly to SMC1 but that Rad21 mediates the association of C ( 2 ) M with SMC1 . Taken together , our immunoprecipitation analyses reveal a novel interaction between the two α-kleisin proteins Rad21 and C ( 2 ) M . Specifically , we show that the C-terminus of Rad21 binds to the N-terminus of C ( 2 ) M , suggesting that this interaction mediates the association of C ( 2 ) M with the core cohesin complex . In vivo , localization of Rad21 and C ( 2 ) M are mutually dependent , consistent with an interaction of these two proteins ( Figs . 1D and 2B ) . Having established that Rad21TEV-myc cleavage results in premature SC disassembly , we wondered whether additional late meiotic processes were affected . If Rad21 is required for cohesion between sister chromatids during the meiotic divisions , one would expect precocious separation of sister chromatids in the Rad21 mutant situation , and consequently chromosome missegregation . Classical genetic non-disjunction assays are not possible in our system , because TEV protease expression in our experiments inactivates the essential maternal Rad21 contribution and therefore results in complete female sterility . In multiple experiments , after mat-GAL4 driven TEV protease expression causing Rad21TEV-myc cleavage , no larvae hatched from the eggs laid by those females . Immunofluorescence analysis of these embryos revealed massive defects already during the very early zygotic divisions . Fragmented and unequally sized DNA masses could be observed , organizing multiple and/or multipolar spindles ( Fig . S6 ) . Most embryos appeared to have arrested in a metaphase-like state . Thus , the sterility of these females precluded scoring of genetic markers in adult progeny . As an alternative approach , we applied fluorescent in situ hybridization ( FISH ) to detect chromosome-specific regions in metaphase I-arrested oocytes and analyzed them with reference to precocious chromosome separation . We used an X-chromosome-specific probe ( 359 bp repeat ) and a chromosome 4-specific probe ( AATAT ) 6 ( Fig . 5A ) . The observed phenotypes were assigned to two different categories: ( 1 ) normal meiotic figures exhibiting 2+2 FISH signals , and ( 2 ) precocious separation of chromatids as indicated by supernumerary FISH signals ( >2 FISH signals for at least one of the probes; Fig . 5A ) . In the wild type situation , 98% of the oocytes showed normal meiotic figures , as indicated by the two signals for the different chromosomes ( Fig . 5A and B ) . 2% of the wild type oocytes contained more than 2 signals for one of the two probes ( n = 45 ) . After TEV protease-mediated Rad21TEV-myc cleavage , the distribution of phenotypes was similar: 95% of the oocytes displayed a normal arrangement , and 5% of the analyzed oocytes had an elevated number of FISH signals indicative of premature chromatid separation ( n = 40 ) . Also , oocytes from c ( 2 ) M mutant females which have been shown previously to display high levels of meiosis I non-disjunction [3] , [39] , showed no increase of premature chromatid separation ( 5% of the c ( 2 ) M mutant oocytes exhibited supernumerary FISH signals ( n = 62 ) . Finally , we analyzed oocytes of individuals in which Rad21TEV-myc cleavage was performed in the c ( 2 ) M mutant background ( Fig . 5B ) . In this constellation , again , only 5% of the oocytes were assigned to the ‘supernumerary FISH signals’ category ( n = 61 ) . On the contrary , very similar analyses in ord mutants using a probe directed against the same repetitive region of the X-chromosome revealed a high proportion ( 46% ) of prometaphase oocyte nuclei with three or four FISH signals , indicating loss of cohesion [48] . Thus , our data do not suggest any additional contribution of Rad21 to chromatid cohesion at this developmental stage during oogenesis . To analyze meiotic divisions directly , we also performed FISH on very early embryos shortly after egg deposition . In this experiment , we observed in the majority of cases a correct 1∶1∶1∶1 distribution of FISH signals among the four meiosis II products in the Rad21 mutant situation , indicative of normal segregation in both meiotic divisions . In only one out of 83 cases , we detected a clear example of missegregation ( in meiosis I ) with a signal distribution of 0∶0∶2∶2 ( Fig . 5C ) . Taken together , after efficient cleavage of Rad21TEV-myc in the oocytes , the effects on meiotic chromosome segregation , if any , were very mild . These findings indicate that Rad21 is not required for sister chromatid cohesion in the oocyte nuclei , in contrast to ORD and SOLO [40] , [41] . ORD and/or SOLO may function to maintain sister chromatid cohesion and thereby explain that Rad21 is not required during the meiotic divisions for normal chromosome segregation . To evaluate this possibility , we analyzed the localization of a functional Venus-SOLO variant [42] . In wild-type egg chambers , Venus-SOLO is localized in the vicinity of centromere clusters [41] . Upon Rad21TEV-myc cleavage , Venus-SOLO persisted in a dot-like pattern co-localizing with Cid/Cenp-A and C ( 3 ) G remnants ( Fig . S7 ) . In contrast , the ribbon-like C ( 3 ) G staining characteristically present in early egg chambers in wild type [4] ( Fig . 2 ) was largely dissipated in the oocyte nucleoplasm , confirming that premature SC disassembly after Rad21TEV-myc cleavage occurred also in the Venus-SOLO background as expected ( Fig . S7 ) . The observed pericentromeric presence of SOLO even after Rad21TEV-myc cleavage is consistent with the notion that SOLO might render Rad21 dispensable during the meiotic divisions . Alternatively , the apparently normal meiotic chromosome segregation observed after TEV protease-mediated Rad21TEV-myc cleavage before the meiotic divisions might also reflect the presence of a low , but sufficient , amount of residual non-cleaved Rad21TEV-myc . If sister chromatid cohesion during the meiotic division was indeed provided by Rad21 containing cohesin , separase-mediated Rad21 cleavage would be predicted to be essential for normal chromosome segregation during meiosis . To evaluate the significance of separase-mediated Rad21 cleavage during meiosis , we expressed a variant of Rad21-myc , in which the predicted separase cleavage sites ( EXXR at positions 172–175 and 471–474 ) were destroyed by exchange of the arginines with alanines ( Fig . 6A ) . This variant , dubbed Rad21NC-myc ( non-cleavable ) , is predicted to be highly toxic in mitotically proliferating cells . Indeed , after expression of Rad21NC-myc in the proliferating eye imaginal disc , adults with severely reduced eyes were obtained ( Fig . S8 ) . After expression during oogenesis , Rad21NC-myc was observed to be co-localized with C ( 3 ) G in the SC during the early stages ( Fig . 6B ) , indicating that this mutant is still capable to associate with chromatin . However , those females expressing Rad21NC-myc during oogenesis were almost completely sterile . Importantly , abnormalities were only apparent after normal completion of meiosis . All the late meiotic figures observed in early embryonic progeny were normal ( 13 clear MII anaphase/telophase figures among 230 analyzed embryos; Fig . 6C ) . FISH analysis demonstrated that X-chromosome segregation during meiosis is not perturbed by Rad21NC-myc expression ( Fig . 6C ) . Apart from late meiotic figures , also all of the remnants of the polar bodies displayed a normal morphology and the expected three X chromosome FISH signals . In contrast to the meiotic divisions , however , mitotic divisions during early embryogenesis were severely affected by the maternally expressed Rad21NC-myc . In many embryos , strong defects were apparent already during mitosis 1 , as only a single DNA mass was observed in the interior of the embryos ( Fig . 6C ) . During this as well as later mitoses , prominent anaphase bridges were detected and X chromosome FISH revealed chromosome stretching ( Fig . 6C ) , as expected after expression of a Rad21 variant that can no longer be cleaved by separase to initiate a normal anaphase . The observed drastic effect of Rad21NC-myc on mitotic , but not meiotic , chromosome segregation further confirms that Rad21 is not functioning as an essential α-kleisin component of meiotic cohesin .
Rad21/Scc1 has been established as the α-kleisin subunit of cohesin in mitotic cycles from yeast to man . Even though Rad21/Scc1 is expressed during meiosis , a cohesive role in the meiotic divisions has been ruled out for murine female meiosis [37] . In Saccharomyces cerevisiae , Scc1 levels decline sharply when cells enter meiosis , while Rec8 abundance increases dramatically [11] . scc1 mutants have mild meiotic phenotypes and separase-dependent Rec8 cleavage is required for meiotic chromosome segregation [11] , [26] . Likewise in Schizosaccharomyces pombe , Rec8 , but not Rad21 , localizes throughout chromatin during prophase of meiosis I and Rec8 cleavage is required for both meiosis I and meiosis II chromosome segregation [12] , [27] . Thus , the emerging view is that during entry into the meiotic program a switch occurs from Rad21 containing cohesin complexes to Rec8 containing cohesin complexes , which are responsible for establishing and maintaining sister chromatid cohesion throughout meiosis . This initially simple picture has become more complicated in vertebrates with the discovery of Rad21L , whose possible function in sister chromatid cohesion remains to be addressed [13] , [14] , [15] , [17] . The situation is even more puzzling in Drosophila , because an unambiguous Rec8 homolog appears to be missing and unrelated proteins like ORD and SOLO with no obvious homology to α-kleisins functionally qualify as cohesion proteins . Originally , C ( 2 ) M was assigned as the Drosophila Rec8 homolog based on its meiotic expression profile and its membership in the α-kleisin protein family [38] . However , we have shown that C ( 2 ) M accumulates on chromatin only after completion of premeiotic S-phase , appears to dissociate long before pro-metaphase I , and that the mutation of putative separase cleavage sites had no effect on C ( 2 ) M function , which is inconsistent with a behavior expected for a meiotic cohesin component [39] . Moreover , c ( 2 ) M mutants display high levels of non-disjunction only in meiosis I , and not in meiosis II , and SMC1/SMC3 is able to localize to meiotic chromatin in the absence of C ( 2 ) M [3] , [45] . Thus , it remains an open question whether Drosophila expresses a meiotic α-kleisin , which needs to be removed in a stepwise fashion during the two meiotic divisions . In the present study , we have investigated whether Rad21 might function also as a meiotic kleisin in Drosophila , in addition to its established role as mitotic cohesin subunit . As a precedent , a recent study has shown that the protist Tetrahymena thermophila uses only one α-kleisin both in mitosis and meiosis [49] . If Rad21 fulfilled a cohesive function during the meiotic divisions in Drosophila , one would expect to observe after ectopic Rad21 cleavage a dissociation of paired homologous chromosomes and premature sister chromatid separation during the extended pachytene stage of meiosis I and , in addition , missegregation of chromosomes in both meiotic divisions . We have performed immunostainings against the constitutive Drosophila kinetochore component Cenp-C and we did not notice an elevated number of Cenp-C spots in the oocyte nuclei of early egg chambers after Rad21TEV-myc cleavage , arguing against premature chromatid separation . This conclusion is also supported by our FISH results where Rad21TEV-myc cleavage was not observed to cause increased missegregation during the meiotic divisions . In contrast , increased numbers of centromere signals and increased missegregation were clearly detected in ord and solo mutants , which lack proteins specifically required for meiotic chromatid cohesion [40] , [41] , [48] . We consider the explanation that the normal meiotic chromosome segregation observed after TEV protease-mediated premature cleavage of Rad21TEV-myc might be due to putative residual non-cleaved Rad21TEV-myc to be highly unlikely . The same experimental strategy has proven to be extremely efficient in case of mitosis [44] . When Rad21TEV-myc is the sole Rad21 species in mitotically proliferating cells , ectopic Rad21TEV-myc cleavage results in a completely penetrant premature separation of sister chromatids in the first mitosis following TEV protease expression . It could be argued that a meiosis-specific factor might shield Rad21TEV-myc from TEV protease-mediated cleavage . However , in mouse oocytes , TEV protease-mediated inactivation of the meiotic α-kleisin was demonstrated to be efficient , arguing against a conserved shielding mechanism [37] , [50] . In addition , we point out that TEV protease-mediated cleavage of Rad21TEV-myc before the meiotic division destroyed the maternal contribution of this mitotic α-kleisin so effectively that embryonic mitoses were completely defective . Our hypothesis that Rad21 does not act as an essential meiotic α-kleisin during Drosophila female meiosis not only rests on the evidence obtained by TEV protease-mediated premature Rad21TEV-myc cleavage , but also on the unperturbed meiotic chromosome segregation observed after expression of Rad21NC-myc , in which the separase consensus cleavage sites were destroyed by site-directed mutations . An assay for the direct biochemical analysis of Rad21 cleavage by separase is still lacking in the Drosophila system . Therefore , it remains to be shown whether Rad21NC-myc is indeed resistant to separase-dependent proteolysis . However , the consequences resulting from expression of this variant in mitotically proliferating cells are perfectly consistent with the presence of separase-resistant cohesin rings that cannot be opened at the metaphase-to-anaphase transition in mitosis to liberate , and allow segregation of , the replicated sister chromatids . After expression during oogenesis , Rad21NC-myc is incorporated into meiotic chromatin and it is present in amounts sufficient to inhibit early embryonic mitoses . Thus , if separase-dependent removal of Rad21-containing cohesin was a crucial step during meiotic chromosome segregation , severe phenotypic consequences should not be restricted to early embryonic mitoses . Meiotic chromosome segregation would be predicted to be affected as well . However , we did not observe any meiotic abnormalities like chromosome bridges or missegregation of the X-chromosome in the FISH analyses . Proper SC assembly depends on all three known meiotic α-kleisins in C . elegans and on both Rad21L and Rec8 in mouse spermatocytes [16] , [21] , [51] . Also in yeasts , which express only one meiotic α-kleisin , Rec8 , SC integrity depends on Rec8 [11] , [52] . However , no role for the mitotic α-kleisin Rad21/Scc1 in maintaining SC integrity has been found so far . Thus , our observation that the SC disassembles prematurely upon Rad21TEV-myc cleavage in Drosophila , demonstrates for the first time the dependence of SC maintenance on intact , Rad21-containing cohesin . However , this premature SC disassembly does not result in chromosome missegregation later in meiosis . We assume that the premature SC disassembly induced in our experiments occurs not early enough to interfere with crossover formation . A normal presence of chiasmata might therefore explain the absence of chromosome segregation defects . While our results clearly demonstrate that Rad21 is required for maintenance of the SC , we did not see an effect on establishment of the SC , not even when we used nos-GAL4 to drive TEV protease expression early in the germarium . We suspect that nos-driven inactivation of Rad21TEV-myc is not fast or complete enough to profoundly affect SC maintenance in germarial stages , and only after a certain lag period enough TEV protease has accumulated to cleave sufficient Rad21TEV-myc , which then triggers SC disassembly . An intriguing result of our experiments is the premature disassembly of the SC even when Rad21TEV-myc is ectopically cleaved , or when Rad21-EGFP is degraded , in the presence of one Rad21 wild type allele . Because SC disassembly follows wild type kinetics in Rad21 heterozygous females in the absence of ectopic Rad21 inactivation , the observed early SC disassembly after ectopic inactivation is not due to reduced Rad21 gene dosage . Also , because cleavage at different positions and ectopic degradation of Rad21-EGFP resulted in premature SC disassembly , a dominant negative effect of the Rad21 fragments on SC structure is highly unlikely . One possible explanation can be based on a model that more than one cohesin ring is required at each linkage position to tether the SC to the chromosome cores . If just one out of two ( or more ) interconnected cohesin rings is opened by TEV protease action , or Rad21-EGFP proteolysis , linkage at this point would be abrogated despite the presence of uncleaved Rad21 in interconnecting rings . In support of this model , interaction studies between cohesin subunits led to the proposal of a “handcuff model” postulating interconnected cohesin rings [53] . The interaction between C ( 2 ) M and Rad21 , which this work has revealed , suggests a model how the SC might be linked to cohesin within the chromosome cores . We propose that a direct interaction between the α-kleisin proteins C ( 2 ) M and Rad21 may provide a structural framework within the SC . We point out that so far we have been unable to confirm this interaction , which we have detected by co-immunoprecipitation from embryonic extracts and in vitro translation reactions , also by co-immunoprecipitation from ovary extracts , presumably because of technical difficulties ( expression levels , insolubility of the SC associated proteins ) . While our demonstration of the C ( 2 ) M-Rad21 interaction is , to our knowledge , the first published report of an association of different α-kleisins , a homodimerization of human Rad21 has been demonstrated using yeast two-hybrid assays and immunoprecipitation experiments [53] . The reported localization of C ( 2 ) M as an LE component of the SC is also consistent with a direct connection to cohesin , which localizes to the chromosome cores [3] , [45] , [54] . Electron microscopy ( EM ) studies have mapped the N-terminus of C ( 2 ) M to the inner edge of the LEs [54] . According to our data , we would also expect the C-terminus of Rad21 to localize to this region of the SC . Staining of chromosome squash preparations indeed revealed a clear co-localization of Rad21TEV-myc and C ( 3 ) G ( Fig . 1D ) , very similar to what has been observed previously for SMC1 [45] . Within the resolution limits of light microscopy , however , we cannot address the question , where Rad21TEV-myc exactly localizes within the SC . Analysis of the SC in Rad21TEV-myc expressing flies via immuno-EM will help to resolve this issue . Our inability to detect uncleaved Rad21TEV-myc localizing to meiotic chromatin in whole mount preparations may be due to epitope masking by a component , which might have been lost during the extensive washing steps with detergent-containing buffer in the chromosome squash preparations . Taken together , we put forward a model in which at least two types of cohesin complexes are required during Drosophila oogenesis . Firstly , cohesin connecting the chromosome cores to the components of the SC contains the α-kleisin Rad21 , possibly composed of multiple interconnected rings . Secondly , cohesin complexes holding together sister chromatids , either contain a very loosely conserved α-kleisin , which awaits to be discovered , or one of the non-kleisin cohesion proteins SOLO or ORD . In the latter case , it will be interesting to find out whether these proteins are substrates of separase .
Flies expressing variants of Rad21 , which are TEV protease cleavable and C-terminally fused to ten copies of the human c-myc epitope tag , in a Rad21 mutant background , have been described [44] . Expression of these variants is driven by the ubiquitously active α-tubulin 84B promoter . The nanos-GAL4 driver line ( y1 w*; P{w[+mC] = GAL4-nos . NGT}40 ) , the maternal triple driver ( MTD-GAL4 ) [55] , as well as the c ( 2 ) MEP2115 stock [3] , [39] were obtained from the Drosophila stock center ( Bloomington , Indiana ) . The maternal alpha-tubulin GAL4 ( mat-GAL4 ) driver line has been described previously [56] . As source for TEV protease , we constructed transgenes encoding a modified enzyme ( NLS-V5-TEVS219V ) , which possesses an N-terminal nuclear localization signal ( NLS ) followed by a V5 epitope tag and a valine instead of a serine residue at position 219 , resulting in inhibition of self-cleavage and in about twofold higher activity levels [57] . We have exclusively used NLS-V5-TEVS219V in this study and , for simplicity , refer to it as TEV protease throughout . To allow TEV protease expression during oogenesis , the NLS-V5-TEVS219V coding sequence was cloned into pUASP1 [58] . Transgenic strains were established after injection into w1 embryos using established procedures . To obtain flies expressing a C ( 2 ) M variant tagged at its C-terminus with six copies of the hemagglutinin tag ( 6×HA ) under control of the c ( 2 ) m genomic regulatory sequences , we replaced the 10×myc tag in a progenitor plasmid of the construct pCaSpeR-gC ( 2 ) M-myc [39] by the coding sequence for 6×HA . Briefly , a BamHI-XbaI-fragment containing the 3′-terminal part of c ( 2 ) m including the 10×myc encoding sequence was subcloned into pBSSK+ ( Stratagene ) . An AgeI site was introduced immediately upstream the c ( 2 ) m stop codon by inverse PCR using the oligonucleotides C ( 2 ) M7 ( 5′- GGTGAGACCGGTTGAATATTTTTAGATAATTTTTTTCAAG-3′ ) and C ( 2 ) M8 ( 5′-CGTTCAACCGGTCTCACTCAGCATAAGATTG-3′ ) to yield pBSSK+ - BamHI-C ( 2 ) M- ( AgeI ) -XbaI . This step also removed the sequence encoding 10×myc . Next , an XhoI-BamHI fragment containing the 5′-terminal region of c ( 2 ) m including flanking genomic sequences was cloned into pBSSK+-BamHI-C ( 2 ) M- ( AgeI ) -XbaI resulting in pBSSK+-gC ( 2 ) M- ( AgeI ) . The sequence encoding the 6×HA tag was obtained from the plasmid pUASP-HA-Sse [59] and cloned into the unique AgeI site of pBSSK+-gC ( 2 ) M- ( AgeI ) . Finally , the complete insert was transferred as a 4 . 2 kb NotI-Asp718 fragment into the pattB vector [60] . Transgenic lines were generated by germline transformation of pattB-gC ( 2 ) M-HA into y1 , w1 , M[vas-int]ZH2A; M[3x3P-RFP , attP']ZH51D embryos [60] . To obtain lines carrying a functional Rad21-EGFP transgene , a construct similar to Rad21-10myc was generated . Briefly , the EGFP coding sequence was amplified using the primers SH257 ( 5′-CGTCTGTTCGAAAACCCAAAAATTGGCGGCGGCATGGTGAGCAAGG-3′ ) and SH258 ( 5′-CGTCTGTTCGAACTACTTGTACAGCTCGTCCATGC-3′ ) and cloned into the naturally occurring BstBI-site upstream of the Rad21 translational stop codon in the Rad21 cDNA clone LD14219 ( BDGP ) . After introducing an additional Acc65I site in the polylinker upstream of the Rad21 coding sequence , the complete Rad21-EGFP fragment was cloned as an Acc65I fragment into the modified pCaSpeR vector used for generating Rad21TEV-myc lines [44] . This vector allows expression of genes inserted in the unique Acc65I site under control of the ubiquitous active α-tubulin 84B promoter . Transgenic lines were established after P-element mediated germ-line transformation using pCaSpeR{w+ , αtub-Rad21-EGFP} and injection into embryos derived from parents with the genotype Rad21ex3/TM3 , Ser . For the construction of a putative separase-resistant variant of Rad21 , Rad21NC-myc , we employed a PCR-based strategy to exchange the codons 175 and 474 specifying arginines within the separase consensus sequences EXXR into codons specifying alanine residues . We have chosen these two sites because they align well with the known separase cleavage sites in Scc1/Rad21 from humans and yeasts [23] . As template for the PCR reactions , the Rad21 cDNA-based plasmid clone pUAS-Rad21-10myc [44] was used . A first 625 bp fragment comprising the Rad21 5′-UTR up to the region encoding the mutated first separase consensus cleavage site ( EIIA ) was PCR-amplified using the primers SH341 ( ATAAGGCCGGCCACGAGACAGTTTTAGGTGATG ) and SH342 ( GAAGGTATACTGCAGGCTATAATTTCAGGCGTTTCTGC ) . A second 910 bp fragment corresponding to the Rad21 region from the mutated first putative separase cleavage site ( EIIA ) up to the mutated second separase consensus cleavage site ( EVLA ) was PCR-amplified using the primers SH343 ( TTATAGCCTGCAGTATACCTTCAAATATTAATGATAAAA ) and SH344 ( TTCGCAGCTAGCACTTCCGGAGCTTCCAAACT ) . A third 1208 bp fragment corresponding to the Rad21 region from the mutated second separase consensus cleavage site ( EVLA ) , through the C-terminal fused c-myc tag , was PCR-amplified using the primers SH345 ( GGAAGTGCTAGCTGCGAATCATAAATCTCTAGGG ) and SH346 ( GTAGGCGCGCCATTAAAACAGATTTACATTCAACTT ) . The three PCR-generated DNA fragments partially overlap in the regions encoding the mutated separase consensus cleavage sites . After purification using the PCR purification kit ( Thermo Scientific ) , the three PCR products were pooled and served as template for a final PCR using the flanking primers SH341 and SH346 . The final 2696 bp PCR-product was cloned as an FseI/AscI fragment into a modified pUASP-vector containing unique FseI and AscI sites within its multiple cloning site . Transgenic lines were generated by P-element mediated germline transformation of w1-embryos using established procedures . For expression of this Rad21 variant in the developing eye , the ey-GAL4 [61] driver line was used and for expression during oogenesis the MTD-GAL4 driver line [55] . For the construction of the transgene P{w+ , UASP-NSlmb-vhh-GFP4} , the EcoRI – XbaI insert fragment was isolated from P{w+ , UAST-NSlmb-vhh-GFP4} [46] , and inserted into the corresponding sites of pUASP1 [58] . Transgenic lines were generated by P-element mediated germline transformation of w1-embryos using established procedures . For deGradFP dependent destruction of Rad21-EGFP during oogenesis , we generated w*; P{w+ , UASP-NSlmb-vhh-GFP4}II . 1/nos-GAL4; Rad21ex3 , P{w+ , αtub-Rad21-EGFP} III . 1/Rad21ex3 , P{w+ , αtub-Rad21-EGFP} III . 1 females by standard crossing schemes . As controls , we also generated w*; P{w+ , UASP-NSlmb-vhh-GFP4}II . 1/nos-GAL4; Rad21ex3 , P{w+ , αtub-Rad21-EGFP}III . 1/+ as well as w*; P{w+ , UASP-NSlmb-vhh-GFP4}II . 1/+; Rad21ex3 , P{w+ , αtub-Rad21-EGFP}III . 1/Rad21ex3 , P{w+ , αtub-Rad21-EGFP}III . 1 females . For the immunoblotting experiments shown in Fig . 1 , ovaries of 4–5 day old females fattened with yeast were dissected in 1× PBS and stage 14 oocytes were isolated and homogenized in SDS gel sample buffer . Protein samples were separated on Tris-glycine based polyacrylamide gels and blotted onto nitrocellulose membranes . For detection of myc epitope tags , HA epitope tags , V5 epitope tags , FLAG epitope tags and α-tubulin , the mouse monoclonal antibodies 9E10 [62] , 12CA5 [63] , anti-V5 ( Invitrogen ) , anti-FLAG ( Sigma ) and DM1A ( Sigma ) were used , respectively . A guinea pig polyclonal antibody against Rad21 [39] and a rabbit antibody against EGFP [64] have been described . For detection of bound antibodies on immunoblots , the horseradish peroxidase based system from p . j . k was used according to the manufacturer's recommendations . For the immunoprecipitation experiments shown in Fig . 4A , embryos were collected on apple juice agar plates for 1 . 5 h at 25°C . After dechorionization , the eggs were homogenized in 4× volume of lysis buffer ( 50 mM HEPES at pH 7 . 5 , 60 mM NaCl , 3 mM MgCl2 , 1 mM CaCl2 , 0 . 2% Triton X-100 , 0 . 2% Nonidet NP-40 , 10% glycerol , 2 mM Pefabloc , 2 mM Benzamidin , 10 µg/ml Aprotinin , 2 µg/ml Pepstatin A , 10 µg/ml Leupeptin ) . The extracts were centrifuged and the supernatants were used for immunoprecipitation with anti-HA agarose beads ( Roche ) . After 3 hours incubation at 4°C under rotation , the beads were washed 5× with lysis buffer and transferred into mobicol columns ( MoBiTec ) . Bound proteins were eluted by adding 3× SDS sample buffer ( 6% SDS , 0 . 3 M β-mercaptoethanol , 30% glycerol , 0 . 3% bromophenol blue , 0 . 15 M Tris/HCl , pH 6 . 8 ) and boiling of the sample . The immunoprecipitates as well as samples of the input fractions and supernatants after precipitation were analyzed by immunoblotting . For the in vitro interaction assays , proteins were synthesized using the TNT SP6 coupled reticulocyte lysate system ( Promega ) allowing coupled in vitro transcription and translation . To obtain the coding region of the SC components ( C ( 3 ) G , C ( 2 ) M , Corona ) and the cohesion proteins ( SMC1 , ORD , SOLO ) , RNA from ovaries was isolated and cDNA was synthesized using RevertAid H Minus M-MuLV Reverse Transcriptase ( Fermentas ) according to the manufacturer's recommendations . As templates for the TNT reactions , the reading frames of the respective genes were cloned into the expression vector pCS2 ( F/A ) [64] or derivatives thereof , allowing an N-terminal translational fusion with three copies of the FLAG epitope tag or with six copies of the myc epitope tag . For co-expression , equal amounts of plasmid constructs were added to the components of the TNT kit . Synthesized proteins were labeled by incorporation of [35S]methionine . For immunoprecipitation , anti-Flag or anti-myc agarose beads ( Sigma ) were used . Ovaries were dissected in 1× PBS and fixed at room temperature for 20 min in a mixture of 300 µl heptane and 150 µl ovary fixation solution ( 1× PBS , 0 . 5% Nonidet NP 40 and 2% para-formaldehyde ) . Fixed ovaries were blocked for 1 h in PBS containing 0 . 2% Tween ( PBTw ) and 10% normal goat serum ( NGS ) . Spread preparations of chromosomes were done as previously described [45] . Rabbit antibodies against Cenp-C [65] and against C ( 3 ) G [66] have been described and were used at a 1∶3 , 000 dilution . For some experiments , we used an anti C ( 3 ) G antibody we have raised in guinea pigs by immunization with a bacterially expressed C ( 3 ) G fragment corresponding to the C-terminus ( aa 565–743 ) . A rat antibody against Cid/Cenp-A ( 4F8 , [67] ) was diluted 1∶200 . For SMC1 staining , a polyclonal antiserum was raised in rabbits using a bacterially expressed protein fragment corresponding to the N-terminal 133 amino acids of SMC1 . The affinity purified antibody was used at a 1∶400 dilution . Antibodies against the HA epitope tag ( Roche ) , the myc epitope tag ( Sigma ) , and the V5 epitope tag ( Invitrogen ) were used at 1∶10 , 1∶10 and 1∶500 , respectively . All primary antibodies were diluted in PBTw +10% NGS . After washing twice in PBTw , secondary goat antibodies conjugated with Alexa 488 or Cy3 ( Molecular Probes ) were applied for 2 h in PBTw containing 5% NGS , followed by additional washes in PBTw . DNA was stained with Hoechst 33258 ( 1 µg/ml ) . Fluorescence images were acquired with a Leica SP5 confocal system ( Leica Microsystems , Germany ) or a Zeiss Axioplan 2 epifluorescence microscope . All images were processed using ImageJ v1 . 41 ( National Institutes of Health , USA ) . For scoring SC disassembly , we have recorded the stages of those egg chambers showing complete SC disassembly , as indicated by the presence of only dot-like C ( 3 ) G signals within the oocyte chromatin and strong C ( 3 ) G staining of the oocyte nucleoplasm . The assignment of the stages was done based on size of the egg chambers as determined by equatorial focal planes . Because these planes only rarely allowed illustration of the oocyte nuclei , non-equatorial , and consequently smaller , sections containing the oocyte nuclei are shown in figs . 1 , 2 , 3 , S5 and S7 . To assess the significance of the differences in SC disassembly timing ( Fig . 3A ) , we tested pairwise between the respective control situations ( no TEV or no NSlmb-vhh-GFP4 expression ) and the Rad21 degradation situations ( TEV or NSlmb-vhh-GFP4 expression in Rad21TEV-myc , Rad21ex or Rad21-EGFP , Rad21ex background , respectively ) using the Mann-Whitney U test ( STATISTICA , StatSoft , Tulsa , OK , USA ) . The X chromosome-specific 359 bp repeat was amplified by PCR with Drosophila genomic DNA as template [68] . The PCR product was digested overnight with a mixture of the restriction enzymes AluI , HaeIII , Tru1I , MspI , RsaI , and Sau3AI . Digested DNA was precipitated , dissolved in water , denatured at 100°C for 1 min and chilled on ice . The AATAT repeat specific for chromosome 4 was synthesized as a single-stranded oligonucleotide ( ( AATAT ) 6; Metabion international AG , Germany ) . 3′-Tailing of the single stranded DNAs with the reactive nucleotide Aminoallyl dUTP analog was done by using Terminal deoxynucleotidyl Transferase ( Roche ) at 37°C for 2 h in a reaction mixture containing 200 mM Na-Cacodylate ( pH 7 . 2 ) , 100 µM DTT , 1 mM CoCl2 , 50 µM Aminoallyl dUTP ( ARES DNA Alexa Fluor 555/647 labeling kit , Molecular probes ) and 5 µM unlabeled dTTP . Reactions were stopped by adding 5 mM EDTA . Aminoallyl-conjugated probes were precipitated , dissolved in water and labeled with Alexa Fluor 555 or Alexa Fluor 647 in labeling buffer for 2 h in the dark , followed by quenching of the reactions with 150 µM hydroxylamine . Labeled probes were precipitated and dissolved in elution buffer . FISH was done on stage 14 oocytes as described in [69] with some modifications . Oocytes were fixed in heptane/oocyte fixation solution , rinsed three times in 2× SSCT ( 0 . 3 M NaCl , 30 mM sodium citrate , 0 . 1% Tween 20 ) , sequentially washed with 2× SSCT-20% formamide , 2× SSCT-40% formamide , and 2× SSCT-50% formamide for 10 min each followed by incubation in fresh 2× SSCT-50% formamide for 1–2 hrs at 37°C . The oocytes were transferred to 36 µl of hybridization buffer ( 20% dextrane sulfate , 15% formamide in 2× SSCT ) and 100 ng of each fluorescently labelled probe was added . Probe and chromosomal DNA were denatured at 91°C for 2 min and the hybridization reaction was carried out overnight at 37°C . After hybridization , pre-warmed ( 37°C ) 2× SSCT-50% formamide was added to the sample . Oocytes were washed three times with pre-warmed 2× SSCT-50% formamide , once with 2× SSCT-40% formamide , and 2× SSCT-20% formamide for 10 min/wash . Then , the oocytes were washed three times with 2× SSCT for 10 min each , rinsed three times with PBST and treated with Hoechst 33258 ( 1 µg/ml in PBS ) to stain DNA . Finally , the oocytes were washed once with PBS for 5 min and mounted in 70% glycerol , 50 mM Tris-Cl ( pH 9 . 5 ) , 10 mg/ml propyl gallate , 0 . 5 mg/ml p-phenylenediamine in 1× PBS . To enrich for oocytes progressing through meiosis II , approximately 300 females fattened for three days on yeast were put in collection cages and after a pre-collection for 1 h at 25°C , eggs were collected every 20 to 40 minutes for 5 hours . The eggs were immediately dechorionized and fixed with Methanol . Eggs from all collections were pooled and subjected to FISH as described above .
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Meiosis is a specialized form of cell division that ensures production of germ cells with the right number of chromosomes , so that at fertilization the embryo receives complete sets of paternal and maternal chromosomes . The accurate distribution of chromosomes during cell divisions is dependent on a ring-shaped protein complex called cohesin . Cohesin is thought to embrace the chromosomes from the time of their duplication during S-phase until their segregation in the ensuing division . This segregation is facilitated by the controlled proteolytic cleavage of one of the cohesin ring components . Most eukaryotes express specialized variants of this protein: for mitosis the variant Rad21/Scc1/Mcd1 and for meiosis the related protein Rec8 . Because Drosophila lacks a clear Rec8 homolog , we have analyzed in the present study whether the mitotic variant Rad21 may also function during meiosis . We have destroyed Rad21-based cohesin by premature cleavage of an engineered Rad21 variant during oogenesis . While we find no indication for effects on the accuracy of meiotic chromosome segregation , Rad21 cleavage results in a premature disassembly of the synaptonemal complex ( SC ) , a structure required for meiotic recombination in Drosophila oocytes . Our interaction studies provide intriguing hints how Rad21 might contribute to SC maintenance .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"meiosis",
"invertebrates",
"animal",
"genetics",
"cell",
"cycle",
"and",
"cell",
"division",
"cell",
"processes",
"animals",
"animal",
"models",
"mitosis",
"developmental",
"biology",
"drosophila",
"melanogaster",
"model",
"organisms",
"organism",
"development",
"chromatin",
"drosophila",
"research",
"and",
"analysis",
"methods",
"chromosome",
"biology",
"organogenesis",
"insects",
"arthropoda",
"cell",
"biology",
"genetics",
"biology",
"and",
"life",
"sciences",
"molecular",
"cell",
"biology",
"organisms"
] |
2014
|
The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohesion, during Drosophila Female Meiosis
|
Ebolavirus species Zaire ( ZEBOV ) causes highly lethal hemorrhagic fever , resulting in the death of 90% of patients within days . Most information on immune responses to ZEBOV comes from in vitro studies and animal models . The paucity of data on human immune responses to this virus is mainly due to the fact that most outbreaks occur in remote areas . Published studies in this setting , based on small numbers of samples and limited panels of immunological markers , have given somewhat different results . Here , we studied a unique collection of 56 blood samples from 42 nonsurvivors and 14 survivors , obtained during the five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo . Using Luminex technology , we assayed 50 cytokines in all 56 samples and performed phenotypic analyses by flow cytometry . We found that fatal outcome was associated with hypersecretion of numerous proinflammatory cytokines ( IL-1β , IL-1RA , IL-6 , IL-8 , IL-15 and IL-16 ) , chemokines and growth factors ( MIP-1α , MIP-1β , MCP-1 , M-CSF , MIF , IP-10 , GRO-α and eotaxin ) . Interestingly , no increase of IFNα2 was detected in patients . Furthermore , nonsurvivors were also characterized by very low levels of circulating cytokines produced by T lymphocytes ( IL-2 , IL-3 , IL-4 , IL-5 , IL-9 , IL-13 ) and by a significant drop of CD3+CD4+ and CD3+CD8+ peripheral cells as well as a high increase in CD95 expression on T lymphocytes . This work , the largest study to be conducted to date in humans , showed that fatal outcome is associated with aberrant innate immune responses and with global suppression of adaptive immunity . The innate immune reaction was characterized by a “cytokine storm , ” with hypersecretion of numerous proinflammatory cytokines , chemokines and growth factors , and by the noteworthy absence of antiviral IFNα2 . Immunosuppression was characterized by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of peripheral CD4 and CD8 lymphocytes , probably through Fas/FasL-mediated apoptosis .
Ebolavirus ( EBOV ) and Marburgvirus ( MARV ) are among the most deadly human pathogens , causing a severe hemorrhagic fever syndrome in both humans and non human primates [1]–[2] . EBOV is subdivided into five species with different pathogenicities [3] . Zaire ebolavirus ( ZEBOV ) , the most lethal species ( case-fatality rate up to 90% ) , caused numerous human outbreaks between 1976 and 2008 in Democratic Republic of Congo , Republic of Congo ( RC ) and Gabon [4]–[9] . Sudan ebolavirus ( SEBOV , case-fatality rate about 50% ) has caused three documented outbreaks in Sudan and one in Uganda [10]–[14] . Côte d'Ivoire ebolavirus ( CIEBOV ) has been linked to a single , non fatal human case [15]–[16] , while the newly discovered Bundibugyo ebolavirus ( BEBOV ) caused an outbreak with a 25% case-fatality rate in 2007 in Uganda [17] . Finally , Reston ebolavirus ( REBOV ) , which has caused outbreaks in non human primates and swine in the Philippines , appears to be non pathogenic for humans [18]–[20] . EBOV and MARV initially replicate massively in macrophages and dendritic cells ( DC ) , then spread rapidly to all vital organs , infecting endothelial cells , epithelial cells , hepatocytes , and other cell types [21]–[25] . Infection by MARV and the most lethal EBOV species , described virtually exclusively in vitro and with experimental animal models , is associated with high-level viremia , abundant proinflammatory cytokine and chemokine production , massive bystander lymphocyte apoptosis , and widespread focal tissue destruction , resulting in increased endothelial cell permeability , multiorgan failure , and severe clotting disorders , and culminating in a final septic shock-like syndrome [26]–[29] . Fatal outcome in experimentally infected mice and non human primates is associated with impairment of innate immune responses , including rapid and important secretion of certain inflammatory mediators and the absence of type I interferon ( IFN ) production , and also with profound suppression of adaptive immune responses , including impaired humoral responses and B and T lymphocyte apoptosis [30]–[35] . Most information on filovirus pathogenesis comes from in vitro studies and experimental models . However , experimentally infected animal models fail to reproduce certain features of human ZEBOV infection . For instance , rodent models do not exhibit hemorrhagic manifestations and often fail to develop disseminated intravascular coagulation . Lymphocyte apoptosis is consistently observed in mice infected with an adapted variant of ZEBOV but has not been reported in ZEBOV-infected guinea pigs despite histological evidence in the spleen or lymphoid organs of fatally infected animals [26] , [36]–[38] . Non human primate models best mimic fatal human infection , but they do not reproduce the survival of a small percentage of patients [39]–[43] . The paucity of data on human immune responses to ZEBOV is largely due to the fact that most outbreaks occur in remote areas where the facilities required to handle and process clinical samples are lacking . Only four studies of human filovirus infection have been reported so far , only two of which focused on ZEBOV . These two studies were small , involved few immunological markers , and gave conflicting results . The first study involved 9 patients infected during the 1995 Kikwit outbreak ( 7 fatal cases and 2 survivors ) , and showed slightly higher serum levels of IFN-γ , IFN-α , TNF-α , IL-2 and IL-6 in the non survivors than in the survivors , suggesting that stronger immune activation was associated with fatal outcome [44] . The second study , involving 12 patients infected during the 1996 Gabon outbreaks ( 8 fatal cases and 4 survivors ) , failed to confirm the link between elevated IFN-α , TNF-α or IL-2 secretion and fatal outcome [45]–[48] . This latter study suggested that fatal outcome was associated with generalized immune suppression , including intravascular apoptosis , a lack of ZEBOV-specific IgG production , and defective early inflammatory responses when compared to non fatal and asymptomatic infection . However , evidence of lymphocyte apoptosis was based on DNA fragmentation in peripheral blood mononuclear cells ( PBMC ) and reduced CD3 , CD8 , IFN-γ , IL-2 and IL-4 mRNA levels , which cannot distinguish apoptosis from necrosis or anergy , or identify the different target cell subsets . Innate immunity has only been investigated in 8 fatal cases and 4 survivors . Because of the known variability of human immune responses to a given pathogen , and differences in immune status at the time of infection , due for example to concomitant infections by other pathogens , larger studies are needed to observe statistically meaningful trends . To further characterize human immune responses during the acute phase of ZEBOV infection , we analyzed a unique collection of 56 blood samples collected during the five outbreaks that occurred between 1996 and 2003 in Gabon and RC .
This study was implemented as part of an Ebola outbreak control operation coordinated by Ministries of Health ( MoH ) of Gabon and RC , and therefore no ethics committee approval was deemed necessary , as it is usually the case in this kind of emergencies . The patients described here are anonymous . Blood samples were collected by a team from CIRMF ( Centre International de Recherches Médicales de Franceville ) , Gabon , participating in the international response to the different outbreaks along with healthcare workers from MoH of Gabon and RC , the World Health Organization ( WHO ) , Médecins sans Frontières , the Centers for Disease Control and Prevention ( CDC ) , Atlanta , USA , and the National Microbiology Laboratory , Winnipeg , Canada . Blood samples were collected at the patient's home or in hospital isolation wards , with WHO and MoH authorizations ( File S1 and File S2 ) , and with verbal consent from the patient . The two study protocols were reviewed and approved together by the scientific committee of CIRMF . All suspected cases , identified by the international medical teams , were isolated , sampled and received symptomatic treatment and palliative care . Blood samples were collected during the acute phase from patients with laboratory-confirmed ZEBOV infection , during all the documented ZEBOV outbreaks that occurred in Gabon and RC between 1996 and 2005 . The first outbreak hit the villages of Mayibout I and II , located in north-eastern Gabon , from January to February 1996 , causing 10 non fatal clinical cases and 21 deaths . The second outbreak caused 45 deaths among 60 cases between October 1996 and March 1997 in the Booué area , ∼150 km southwest of Mayibout . The infection spread to several villages around Booué , then to Libreville , where 15 cases were recorded . The third outbreak occurred between October 2001 and May 2002 in the Mekambo area of Gabon and the Mbomo area of RC , ∼150 km east of Mayibout . This outbreak in fact consisted of several independent epidemic chains of human transmission that arose from infected animal carcasses ( mainly chimpanzees and gorillas ) . A total of 207 human cases ( 58 survivors and 149 deaths ) were recorded . There were 15 survivors and 128 deaths recorded during the third outbreak which again affected the region of Mbomo in RC , between December 2002 and May 2003 . This outbreak had two independent sources , both due to handling of animal carcasses , one in Yembelengoye village and one in Mvoula , a gold-digger camp located further east , and mainly affected Mbomo and Kelle . Finally , the last outbreak affected the region of Mbomo , causing 6 deaths among 35 cases between October and December 2003 . Initial cases occurred in Mbanza , a village located about 30 km north of Mbomo . Upon collection , blood samples were immediately transported to CIRMF . Plasma and sera were separated by centrifugation and stored at −80°C until use . When enough blood was available , PBMC were separated from whole blood by density gradient centrifugation on lymphocyte separation medium ( Eurobio ) at 2300 rpm for 20 min at room temperature , then washed with phosphate buffered saline ( PBS ) /2% fetal calf serum ( FCS ) , and cryopreserved in FCS containing 10% DMSO in liquid nitrogen in CIRMF secure facilities . Thirty control plasma samples were randomly selected among 4 , 349 samples collected from healthy individuals throughout Gabon during a previous study [49] . These individuals were themselves randomly selected among the Gabonese rural population excluding children and elderly persons ( more than 65 years ) . All controls were native Gabonese . ZEBOV infection was confirmed by detection of circulating antigens with reagents kindly provided by the CDC , Atlanta . Levels of 50 cytokines , chemokines and growth factors were measured in plasma samples by using Luminex technology ( Bio-Rad ) . Two kits , the Bio-plex human cytokine 27-plex assay and the Bio-plex human cytokine 23-plex assay ( Bio-Rad ) , were used as recommended by the manufacturer . The target cytokines were interleukin-1β ( IL-1β ) , IL-1 receptor antagonist ( IL-1RA ) , IL-2 , IL-4 , IL-5 , IL-6 , IL-7 , IL-8 , IL-9 , IL-10 , IL-12p70 , IL-13 , IL-15 , IL-17 , eotaxin , basic fibroblast growth factor ( FGF-basic ) , granulocyte colony-stimulating factor ( G-CSF ) , granulocyte macrophage colony-stimulating factor ( GM-CSF ) , IFN-γ , IFN-inducible protein 10 ( IP-10 ) , monocyte chemoattractant protein-1 ( MCP-1 ) , macrophage inflammatory protein-1α ( MIP-1α ) , MIP-1β , platelet-derived growth factor-ββ ( PDGF-ββ ) , regulated-on-activation normal T-cell expressed and secreted ( RANTES ) , tumor necrosis factor-α ( TNF-α ) , and vascular endothelial growth factor ( VEGF ) for the 27-plex assay; and Il-1α , IL-2Rα , Il-3 , Il-12p40 , IL-16 , IL-18 , cutaneous T cell attracting chemokine ( CTACK or CCL27 ) , growth regulated oncogene alpha ( GRO-alpha or CXCL1 ) , hepatocyte growth factor ( HGF ) , intracellular adhesion molecule 1 ( ICAM-1 ) , IFN-α2 , leukemia inhibitory factor ( LIF ) , MCP-3 ( or CCL7 ) , macrophage colony-stimulating factor ( M-CSF ) , monokine induced by interferon-gamma ( MIG or CXCL9 ) , nerve growth factor-β ( NGF-β ) , stem cell factor ( SCF ) , stem cell growth factor-β ( SCGF-β ) , SDF-1α ( or CXCL12 ) , tumor necrosis factor-β ( TNF-β ) , TNF-related-apoptosis-induced-ligand ( TRAIL ) and vascular cell adhesion molecule 1 ( VCAM-1 ) for the 23-plex assay . Briefly , 25 µL of plasma was diluted 1∶4 and incubated with anti-cytokine antibody-coupled beads for 1 h . All incubations were performed at room temperature . Between each step , the complexes were washed three times in wash buffer ( Bio-Rad ) using a vacuum manifold . The beads were then incubated with a biotinylated detector antibody for 1 hour , before incubation with streptavidin-phycoerythrin for 30 min . Finally , the complexes were resuspended in 125 µL of detection buffer and 200 beads were counted during acquisition in the Luminex 200 device ( Bio-Rad ) . Mean fluorescence intensity was used to calculate final concentrations in pg/mL . Cryopreserved PBMC were rapidly thawed in a 37°C water bath , washed three times and incubated overnight at 37°C in RPMI 1640 culture medium ( Life Technologies , UK ) with 10% heat-inactivated FCS ( full RPMI-10% FCS ) , 1% penicillin-streptomycin , 1% non essential amino acids , and 1 M HEPES . The cells were then washed in RPMI medium , adjusted to a density of 1×106 cells/mL , and cultured for 18 hours before harvesting and a further wash in RPMI . Approximately 1×106 cells were labeled for 20 min at room temperature with anti CD3-FITC , CD4-PE , CD8-PC7 and CD95-PC5 antibodies ( Beckman-Coulter , Geneva , Switzerland ) . The cells were washed and resuspended in PBS 2% FCS , then 100 , 000 events were analyzed with an FC500 four-color flow cytometer ( Beckman Coulter ) . Results were analyzed with CXP software ( Beckman Coulter ) . PBMC from three healthy individuals living in rural areas of Gabon who were sampled at the time of the outbreak served as controls . Student's t test or the Mann-Whitney-Wilcoxon test was used to compare values in patient groups and controls . STATA software version 9 . 0 ( Stata Corporation , College Station , USA ) was used , and statistical significance was assumed when p<0 . 05 .
The patients were subdivided according to clinical outcome ( survivors , S , and deceased , D ) and the number of days between symptom onset and sampling ( early , S1 and D1; late , S2 and D2 ) . S1 and D1 samples were collected 1–4 days after symptom onset; S2 and D2 were collected ≥5 days after symptom onset ( Table S1 ) . Given that disease course in all fatal cases lasted 6–7 days , D2 group contained patients in their last 2–3 days before death . Levels of the following soluble mediators did not differ significantly between the patient population ( S1 , S2 , D1 , D2 ) and the controls: IFN-α2 , IFN-γ , IL-7 , IL-12p40 , IL-12p70 , IL-17 , IL-18 , TNF-α , TNF-β , TRAIL , FGF-basic , LIF , MIG , MIP-1α , MCP-3 , SDF-1α , IL-2rα , G-CSF , GM-CSF , VEGF , PDGF-ββ , SCGF-β , ICAM1 , VCAM1 , RANTES , IL-1α , HGF , β-NGF , SCF and CTACK ( Figure S1 ) . The only significant differences between S1/D1 and controls were higher levels of IL-6 , IL-8 , MCP-1 , M-CSF , MIF ( only D1 ) and IP-10 in the patients ( p<0 . 05 ) ( Figures 1 and 2 ) , while the only significant differences between S1 and D1 were higher IL-8 , MCP-1 , and MIF levels in non survivors than in survivors ( p<0 . 05 ) ( Figures 1 , 2 and 3 ) . Somewhat surprisingly , the only significant differences between S1 and S2 were higher values of IL-8 , MIF and GRO-α in the later samples ( p<0 . 05 ) . By contrast , marked differences in the levels of several cytokines , chemokines and growth factors were observed between D1 and D2 . We also found significant differences between the control samples and D2 , and between D2 and S2 . Circulating levels of the inflammatory cytokines IL-1β , IL-1RA , IL-6 and IL-16 were significantly ( p<0 . 05 ) higher in D2 samples than in controls , S1 , S2 and D1 samples ( Figure 1 ) . Levels in D2 were higher than S1 or controls for IL-8 and IL-15 . Levels of all these cytokines were between 5 and 1 , 000 times higher in D2 samples than in controls . D2 samples contained very high levels of IL-1RA ( mean 4 . 8 ng/mL , SD 5 . 5 ng/mL; 10 times the control value ) , IL-6 ( mean 1 . 2 ng/mL , SD 1 . 6 ng/mL; 100 times the control value ) , and IL-8 ( mean 2 . 4 ng/mL , SD 4 . 2 ng/mL; 1000 times the control value ) ( Figure 1 ) . Similarly , levels of the chemokines MIP-1α , MIP-1β , MIF , IP-10 , GRO-α and eotaxin were significantly ( p<0 . 05 ) higher in D2 samples than in controls , S1 , S2 and D1 samples ( Figure 2 ) . Levels in D2 were higher than S2 or controls for MCP-1 and M-CSF . Again , levels of these chemokines were between 5 and 1 , 000 times higher in D2 samples than in controls . The following two chemokines were found at very high levels in D2 samples: MCP-1 ( mean 1 . 3 ng/mL , SD 1 . 4 ng/mL; 500 times the control value ) and IP-10 ( mean 7 . 9 ng/mL , SD 5 . 9 ng/mL; 1000 times the control value ) . Levels of circulating cytokines mainly produced by T lymphocytes ( IL-2 , IL-3 , IL-4 , IL-5 , IL-9 , IL-13 ) were either similar or significantly lower ( p<0 . 05 ) in surviving and in non surviving patients than in the controls , especially in early samples ( Figure 3 ) . Levels of cytokines did not differ significantly between different outbreaks , ruling out any temporal bias ( Data not shown ) . The samples used in this part of the study , including those from healthy individuals , were all obtained during the 2001 ZEBOV outbreak in Gabon . Frozen PBMC from two healthy controls , three patients sampled 0–1 days before death , one survivor sampled 5 days after symptom onset , and three survivors sampled two weeks after recovery were analyzed by flow cytometry . Positive gating for lymphocytes based on forward and side scatter was followed by CD3+CD4+ and CD3+CD8+ gating , and specific populations were further defined by using antibodies specific for CD95 . The results are reported as the percentage of PBMC found in each gate . As expected , the percentages of CD3+CD4+ and CD3+CD8+ cells in the two healthy controls were normal ( respectively 43 . 6% and 22 . 4% ) and similar to those both in the survivor sampled during the acute phase ( 46 . 2% and 24 . 1% respectively ) and the three survivors sampled after recovery ( mean: 36 . 6% and 17 . 4% , respectively , Figure 4 ) . By contrast , the percentages of these two lymphocyte populations in the three fatally infected patients were drastically lower than in the controls and survivors: 9 . 4% CD3+CD4+ cells and 6% CD3+CD8+ cells ( Figure 4 ) . These data were compatible with the massive lymphocyte death observed elsewhere in experimentally infected animals and in vitro . In order to identify the underlying mechanisms during human ZEBOV infection , we determined the percentages of CD3+CD4+ and CD3+CD8+ cells also expressing CD95 ( Fas ) , a specific surface marker of apoptosis . CD3+CD4+CD95+ and CD3+CD8+CD95+ cells represented respectively 54 . 1% and 75 . 8% of PBMC in the three ZEBOV fatalities , compared to 5 . 6% and 6 . 8% in the two healthy individuals ( Figure 5 ) . The percentage of CD3+CD4+CD95+ cells in the survivor sampled during the acute phase of ZEBOV infection was 11% , while the mean percentages of CD3+CD4+CD95+ and CD3+CD8+CD95+ cells in the three post-recovery samples were 20 . 8% and 18 . 5% , respectively ( Figure 5 ) .
This study , the largest to date , shows that human fatal ZEBOV infection is associated with a markedly impaired innate immune reaction , characterized by strong proinflammatory cytokine production , undetectable antiviral IFNα , and profound immunosuppression resulting from massive peripheral T lymphocyte apoptosis mediated probably in great part by Fas/FasL interactions . Non survivors had extremely high circulating levels of numerous proinflammatory cytokines ( IL-1β , IL-1RA , IL-6 , IL-8 , IL-15 and IL-16 ) , as well as chemokines and growth factors ( MIP-1α , MIP-1β , MCP-1 , M-CSF , MIF , IP-10 , GRO-α and eotaxin ) . Levels of these mediators rose rapidly after symptom onset in non survivors , reaching very high levels in the two days before death and creating a ‘cytokine storm’: shortly before death , average levels were between 5 and 1 , 000 times higher ( even more in some individuals ) than those observed in both healthy individuals and survivors . Proinflammatory cytokines , chemokines and growth factors are mainly synthesized by monocytes and dendritic cells and represent the cornerstone of the innate immune reaction to pathogens . At moderately elevated concentrations , these soluble mediators act at various points in the first line of defense , recruiting circulating mononuclear cells to the site of infection , increasing endothelial permeability , activating macrophage and DC cytotoxic functions , and inducing adaptive immune responses by providing co-stimulatory signals for naïve T cells . By contrast , we never detected a raise in IFN-α2 in either survivors or non survivors , suggesting that direct antiviral activity is lacking in ZEBOV-infected patients . Some of these results are consistent with those of in vitro studies and animal models . Indeed , macrophages challenged with EBOV and MARV in vitro release large quantities of several proinflammatory cytokines and chemokines , while production of type I IFNs is inhibited . Dendritic cells ( DCs ) , on the other hand , fail to produce cytokines when infected [23] , [25] , [50]–[52] . Similarly , fatal outcome in experimentally infected mice and non human primates is associated with impairment of innate immune responses , including rapid and important secretion of inflammatory mediators , contrasting with the absence of type I interferon production [30]–[35] . Numerous studies have shown that VP35 and VP24 play an essential role in the ZEBOV suppression of IFN-α/β production and/or response by infected DCs and macrophages [53]–[57] . VP24 interrupts nuclear accumulation of tyrosine-phosphorylated STAT1 and STAT2 in infected cells , making them insensitive to IFN-α/β [58]–[59] . VP35 inhibits phosphorylation , activation and nuclear localization of the interferon regulatory factors 3 and 7 ( IRF-3 and IRF-7 ) , transcription factors crucial for IFN-α/β synthesis [60]–[65] . VP35 is also reported to inhibit activation of dsRNA-binding protein kinase ( PKR ) and the RNAi pathway , again antagonizing the interferon response [66]–[67] . The second remarkable finding of this study is that human fatal ZEBOV infection is associated with a lack of adaptive immunity , reflected by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of CD4 and CD8 lymphocytes . Using Luminex technology , we found that levels of numerous circulating T cell cytokines ( IL-2 , IL-3 , IL-4 , IL-5 , IL-9 , IL-13 ) were much lower in non survivors than in healthy individuals . Furthermore , using cytometry analysis , we found that CD4 and CD8 lymphocytes represented only 9 . 2% and 6% , respectively , of PBMC in ZEBOV fatalities , compared to more than 40% and 20% in healthy individuals and survivors . Respectively 54 . 1% and 75 . 8% of these cells expressed CD95 , values about 10 times higher than those observed in the healthy individuals . These findings , although they are based on a small sample size , confirm and extend the results of the only previous study in natural human ZEBOV infection , which showed marked DNA laddering of PBMC and release of the apoptotic 41/7 NMP protein in ZEBOV fatalities [45] , [68] . This latter study did not specify which PBMC subsets underwent apoptosis , or provide information on the underlying mechanism . We found that T CD4 and CD8 lymphocytes underwent massive apoptosis in ZEBOV fatalities , largely through Fas/FasL interaction , whereas the level of lymphocyte apoptosis seen in the survivors was close to that found in the healthy controls . These findings are consistent with the marked bystander lymphocyte apoptosis associated with fatal ZEBOV infection in experimental animals . Studies using flow cytometry , electron microscopy and TUNEL staining have shown that NK , CD4 and CD8 T cells are markedly depleted both through classical apoptosis and through apoptosis-like programmed cell death in the blood and spleen of ZEBOV-challenged BALB/c mice [69]–[70] . Similarly , using the same in situ techniques as those mentioned above , recent studies have shown that lymphocytes undergo massive apoptosis in the spleen and lymph nodes of experimentally infected non human primates [24] , [30] , [33] . In addition , ZEBOV infection of human PBMC in vitro has been shown to induce apoptosis of CD4 and CD8 T lymphocytes [71] . Our findings , together with those of in vitro studies and animal models , indicate that lymphocyte apoptosis may account for the lymphopenia and lymphoid depletion associated with ZEBOV infection . This lymphocyte apoptosis is likely to involve several pathways , but we show that apoptosis via Fas/FasL interaction is largely responsible for the massive lymphocyte death observed in human fatal ZEBOV infection . This is consistent with in vitro studies and experimental ZEBOV infection of macaque monkeys , showing that Fas/FasL and , to a lesser extent TNF-TRAIL mechanisms , may largely account for lymphocyte apoptosis in this setting . Infection of naïve PBMC with ZEBOV in vitro strongly upregulates Fas/FasL expression on CD4 and CD8 T lymphocytes and also TNF-related apoptosis-inducing ligand ( TRAIL ) mRNA expression in the same cells [71] . Similarly , TRAIL and Fas transcript levels were shown to be transiently increased in ZEBOV-infected cynomolgus monkeys [24] , [32] . Alternatively , DC dysfunction may lead to bystander lymphocyte apoptosis . Dendritic cells , and to a lesser extent macrophages , play a pivotal role in both innate and adaptive immunity to many viruses . First , these cells secrete antiviral type I IFNs ( mainly IFN-α/β ) and also proinflammatory cytokines and chemokines that upregulate and guide the adaptive immune response to express specific functions . Second , DCs initiate adaptive immune responses by presenting antigens to T lymphocytes and by stimulating T and B cell differentiation . Thus , early productive replication of EBOV and MARV in macrophages and DCs is likely to impair both innate and adaptive immune responses . The soluble apoptotic factor nitric oxide ( NO ) , synthesized by infected macrophages , as well as the apoptosis-inducing ligands FasL and TRAIL , and immunosuppressive sequences in the viral glycoprotein , have also been implicated in lymphocyte apoptosis in this setting [32] , [71]–[73] . Another possibility is that marked DC functional impairment may induce an overall immunosuppressive state . Indeed , several in vitro studies have shown that EBOV and MARV infection of DCs fails to activate these cells , thereby inducing altered cytokine expression and interfering with the ability of DCs to express co-stimulatory molecules [24]–[25] , [33] , [52] . Such DC functional impairment is thought to reduce T cell stimulatory activity and to abrogate functional adaptive immune responses . This work shows that fatal outcome is associated with aberrant innate immune responses and global suppression of adaptive immunity . The innate response in non survivors leads to a “cytokine storm” which probably rapidly triggers disseminated intravascular coagulation , vascular dysfunction and hypotension and , together with massive lymphocyte apoptosis , likely contributes to vascular collapse , multiple organ failure and the shock-like syndrome associated with human fatal ZEBOV infection .
|
Ebolavirus , especially the species Zaïre ( ZEBOV ) , causes a fulminating hemorrhagic fever syndrome resulting in the death of most patients within a few days . In vitro studies and animal models have brought some insight as to the immune responses to ZEBOV infection . However , human immune responses have as yet been poorly investigated , mainly due to the fact that most outbreaks occur in remote areas of central Africa . Published studies , based on small numbers of biological samples have given conflicting results . We studied a unique collection of 50 blood samples obtained during five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo . We measured the plasma levels of 50 soluble factors known to be involved in immune responses to viral diseases . For the first time , using a cell staining technique , we analyzed circulating lymphocytes from ZEBOV-infected patients . We found that fatal outcome in humans is associated with aberrant innate immunity characterized by a “cytokine storm , ” with hypersecretion of numerous proinflammatory mediators and by the noteworthy absence of antiviral interferon . The adaptive response is globally suppressed , showing a massive loss of CD4 and CD8 lymphocytes and the immune mediators they produce . These findings may have important pathological and therapeutic implications .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"immunology/immune",
"response",
"infectious",
"diseases/viral",
"infections",
"immunology/immunity",
"to",
"infections"
] |
2010
|
Human Fatal Zaire Ebola Virus Infection Is Associated with an Aberrant Innate Immunity and with Massive Lymphocyte Apoptosis
|
PML/TRIM19 , the organizer of nuclear bodies ( NBs ) , has been implicated in the antiviral response to diverse RNA and DNA viruses . Several PML isoforms generated from a single PML gene by alternative splicing , share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences . Recent studies of all the PML isoforms reveal the specific functions of each . The knockout of PML renders mice more sensitive to vesicular stomatitis virus ( VSV ) . Here we report that among PML isoforms ( PMLI to PMLVIIb ) , only PMLIII and PMLIV confer resistance to VSV . Unlike PMLIII , whose anti-VSV activity is IFN-independent , PMLIV can act at two stages: it confers viral resistance directly in an IFN-independent manner and also specifically enhances IFN-β production via a higher activation of IRF3 , thus protecting yet uninfected cells from oncoming infection . PMLIV SUMOylation is required for both activities . This demonstrates for the first time that PMLIV is implicated in innate immune response through enhanced IFN-β synthesis . Depletion of IRF3 further demonstrates the dual activity of PMLIV , since it abrogated PMLIV-induced IFN synthesis but not PMLIV-induced inhibition of viral proteins . Mechanistically , PMLIV enhances IFN-β synthesis by regulating the cellular distribution of Pin1 ( peptidyl-prolyl cis/trans isomerase ) , inducing its recruitment to PML NBs where both proteins colocalize . The interaction of SUMOylated PMLIV with endogenous Pin1 and its recruitment within PML NBs prevents the degradation of activated IRF3 , and thus potentiates IRF3-dependent production of IFN-β . Whereas the intrinsic antiviral activity of PMLIV is specific to VSV , its effect on IFN-β synthesis is much broader , since it affects a key actor of innate immune pathways . Our results show that , in addition to its intrinsic anti-VSV activity , PMLIV positively regulates IFN-β synthesis in response to different inducers , thus adding PML/TRIM19 to the growing list of TRIM proteins implicated in both intrinsic and innate immunity .
The establishment of an antiviral state in cells is the defining property of interferons ( IFNs ) , as well as the activity that led to their discovery . IFNs are the first line of defense against viral infections . IFN-regulatory factor 3 ( IRF3 ) , a ubiquitously expressed transcription factor , is responsible for the primary induction of IFN and is a crucial player in the establishment of innate immunity in response to viral infection [1] . IFNs bind to their receptors and activate the canonical JAK/STAT pathway , leading to the induction of IFN-stimulated genes ( ISGs ) , whose products mediate their biological effects [2] , [3] . Vesicular Stomatitis Virus ( VSV ) belongs to the Rhabdoviridae family . Its single stranded negative sense RNA genome ( about 12 kb ) , that encodes 5 viral proteins , is encapsidated by the nucleoprotein N to form the nucleocapsid that is associated with the RNA-dependent RNA polymerase L and its cofactor the phosphoprotein P . Inside the viral particle , this nucleocapsid is associated with the matrix protein M and surrounded by a membrane containing a unique glycoprotein G . The virus enters the host cell through the endosomal transport pathway via a low-pH-induced membrane fusion process catalyzed by the glycoprotein G [4] . The nucleocapsid released into the cytoplasm serves as a template for transcription and replication processes that are catalyzed by the L-P polymerase complex [5] , [6] . During transcription , a positive–stranded leader RNA and five capped and polyadenylated mRNAs are synthesized . These viral mRNA are translated by the cellular machinery to give the viral proteins N , P , M , G and L , then the replication process yields full-length antigenome-sense RNA , which in turn serve as templates for the synthesis of genome-sense RNA . During their synthesis , both the nascent antigenome and the genome are encapsidated by N proteins . The neo-synthesized genome either serves as a template for secondary transcription or is assembled with M proteins to allow budding of the neosynthesized virion at a cellular membrane . VSV replication is highly sensitive to the inhibitory action of IFN and is routinely used to assay the antiviral activity of IFN in vitro [7] . Although IFN treatment induces the expression of hundreds of ISGs , only a few of them have been demonstrated to be responsible for the inhibition of VSV replication . Indeed , ISG products such as double-stranded RNA-activated protein kinase ( PKR ) [8] , myxovirus resistance protein ( Mx ) [9] , p53 [10] , ISG20 [11] , Ifit2/ISG54 [12] and ProMyelocytic Leukemia ( PML ) [13] , [14] have been reported to confer resistance to VSV infection . In addition , 34 ISG products including PML have been shown to elicit an antiviral effect on VSV replication [15] . PML ( also named TRIM19 for TRIpartite Motif protein 19 ) is the organizer of small nuclear-matrix structures named nuclear bodies ( NBs ) [16] . In response to diverse stimuli , PML NBs recruit a growing number of proteins implicated in different cellular processes such as DNA damage response , apoptosis , senescence , protein degradation and antiviral defense [17]–[25] . PML is covalently conjugated to small ubiquitin modifier ( SUMO ) on three major lysine residues ( K65 , K160 , K490 ) [26] . This modification , which affects PML localization , stability and ability to interact with other partners , is critical for NB functions [16] , [27] . Several PML isoforms generated by alternative splicing from a single gene are designated PMLI to PMLVIIb [28] , [29] . They share the same N-terminal region , which encodes the RBCC/TRIM ( RING finger , B-box , and Coiled-Coil ) motif , but differ in their C-terminal region due to alternative splicing . The variability of the C-terminal part of PML isoforms is important for the recruitment of specific interacting partners and for the specific function of each [29] . The implication of PML in antiviral defense against RNA and DNA viruses from different families has been demonstrated in cells stably expressing individual PML isoform or in cells depleted for PML by RNA interference ( reviewed in [17] , [21] ) . We have previously shown that PMLIII confers resistance to VSV [13] . The antiviral effect of PML has been observed in vivo , as PML deficiency renders mice more susceptible to VSV infection [14] . The role of the other PML isoforms in VSV-infected cells is so far unknown . Therefore , we studied the implication of all PML isoforms during VSV infection . We show that only stable expression of PMLIII or PMLIV conferred resistance to VSV infection . Whereas the activity of PMLIII was saturated at a high multiplicity of infection ( MOI ) , PMLIV conferred a higher protective effect towards VSV infection , even at high MOIs . Finally , unlike PMLIII whose anti-VSV activity is strictly IFN-independent , we show that PMLIV confers a strong resistance to VSV infection via two independent mechanisms . Indeed , PMLIV is able to block viral replication in an IFN-independent manner , and also to trigger innate immunity pathways , leading to higher activation of IRF3 and specific enhancement of IFN-β production . Both activities of PMLIV required its SUMOylation . The peptidyl-prolyl isomerase ( Pin1 ) that is known to interact with phosphorylated IRF3 and to promote its degradation via the ubiquitin-proteasome pathway [30] , was recruited within PML NBs in human cells expressing PMLIV . Therefore , the interaction of endogenous Pin1 with SUMOylated PMLIV and its recruitment within NBs resulted in an enhanced IRF3-dependent production of IFN-β in response to VSV infection and also to other inducers such as Sendai virus ( SeV ) , encephalomyocarditis virus ( EMCV ) , human T-lymphotropic virus type 1 ( HTLV-1 ) , influenza virus , vaccinia virus or poly ( I:C ) . Remarkably , specific depletion of PMLIV in human cells reduced both SeV-induced IRF3 activation and IFN-β production . Our results demonstrate for the first time PMLIV's involvement in both intrinsic and innate immunity .
As PML knockout mice are more sensitive to VSV infection than parental mice [14] , we studied viral protein expression and viral production in MEFs derived from these mice . The five structural proteins of VSV are the nucleocapsid , N , the matrix protein , M , the glycoprotein , G , and two minor proteins , the phosphoprotein , P , and the polymerase protein , L , but only the G , N , and M proteins were revealed with our rabbit anti-VSV antibodies . VSV proteins and VSV production were lower in MEFs WT compared to MEFs PML-/- ( Figure 1A ) . In addition , down-regulation of PML expression by RNA interference in human U373MG cells boosted VSV protein expression ( Figure 1B ) . To determine which PML isoforms were capable of inhibiting VSV replication , we infected at an MOI of 0 . 1 U373MG cells transfected with an empty vector ( EV ) or stably expressing each PML isoform ( PMLI , PMLII , PMLIII , PMLIV , PMLV , PMLVI , or PMLVIIb ) . The expression level of the different PML isoforms is shown in Figure S1A . Double immunofluorescence staining for PML and VSV antigens revealed that only PMLIII and PMLIV inhibited expression of viral proteins ( Figure 2 ) . Western blot of extracts from all these cells infected at different MOIs confirmed these results ( Figure S1B ) . As previously shown [13] , the capacity of PMLIII to inhibit viral protein expression was observed at low MOIs but decreased at higher MOI , whereas PMLIV inhibited viral protein synthesis even at an MOI of 2 ( Figure S1B ) . To further confirm this result , extracts from U373MG-EV , U373MG-PMLIII and U373MG-PMLIV cells infected at MOIs of 0 . 2 or 1 were analyzed in the same Western blot and their supernatants were used for the determination of virus yield by standard plaque assay . As seen in Figure 3 , compared to PMLIII , PMLIV had a higher capacity to inhibit viral protein synthesis and VSV multiplication . Compared to control cells , PMLIII had a slight effect on VSV production ( 2-fold inhibition ) when cells were infected at an MOI of 1 for 8 h , whereas PMLIV exhibited a 500-fold inhibition ( Figure 3B ) . Furthermore , compared to control EV cells , U373MG-PMLIV cells were protected against cell lysis 12 h post-infection at different MOIs ( Figure 3C ) , whereas U373MG-PMLIII cells were protected only at a low MOI of 0 . 2 ( data not shown ) . To further confirm the effect of PMLIV on VSV infection , we tested another cell clone stably expressing PMLIVa , a PMLIV variant , lacking exon 5 but having the same C-terminal region . This PML isoform was also able to inhibit VSV protein expression , as observed with PMLIV ( data not shown ) . Taken together , these results demonstrate that among all PML isoforms , only PMLIII , PMLIV and PMLIVa conferred resistance to VSV . However , PMLIV and PMLIVa mediated a much higher protection , suggesting the crucial role of their specific C-terminal portion . To investigate which viral step is targeted by PMLIV , we first investigated whether VSV entry was affected . Thus , we used a MLV virus encoding GFP pseudotyped with the receptor-binding G protein ( VSV-G ) [31] . This pseudovirus can undergo VSV-G-mediated entry but cannot produce its own VSV-G envelope , and hence is only capable of a single-round of infection . MLV-G-GFP was used to transduce U373MG-EV and U373MG-PMLIV cells and GFP expression was readily detected 48 h later by immunofluorescence ( Figure 4A , left panel ) and flow cytometry ( Figure 4A , right panel ) . These analyses revealed that U373MG-EV and U373MG-PMLIV cells had similar GFP staining reaching 80 . 1% and 84 . 4% GFP+ cells , respectively . These results demonstrate that VSV entry was not affected in PMLIV expressing cells . Next , to determine whether viral transcription was altered by PMLIV expression , U373MG-EV and U373MG-PMLIV cells were left uninfected or infected with VSV at an MOI of 3 for 4 h . Total RNA from cell extracts was analyzed by Northern blot for VSV-N mRNA ( Figure 4B , left panel ) and VSV-L mRNA quantified by RT-qPCR ( Figure 4B , right panel ) . Compared to U373MG-EV cells , the amount of N and L mRNAs was highly reduced in U373MG-PMLIV cells ( Figure 4B ) . It is known that treatment of cells with the protein synthesis inhibitor cycloheximide ( CHX ) results exclusively in primary mRNA synthesis , as viral genome replication requires the ongoing synthesis of N protein [32] . Interestingly , the N mRNA level was comparable to the level synthesized in the presence of CHX , indicating that secondary transcription was inhibited by the PMLIV expression ( Figure 4B , left panel ) whereas primary transcription was not . These data suggest that PMLIV had no effect on steps preceding transcription but restricts a post-transcriptional step involving protein synthesis and replication . It is therefore possible that PMLIV induces IFN synthesis during VSV infection , which may in turn inhibit viral mRNA and protein expression . To determine whether PMLIV affects the expression of IFNs and/or pro-inflammatory cytokines , we analyzed IFN-α , IFN-β , TNF-α and IL8 mRNAs by RT-qPCR in extracts from U373MG-EV and U373MG-PMLIV cells infected with VSV at an MOI of 0 . 2 for various lengths of time . PMLIV did not significantly alter the mRNA expression of IFN-α , TNF-α or IL8 following VSV infection ( Figure 5A ) . Thus , PMLIV had no effect on the induction of TNF-α and IL-8 mRNAs that are regulated by NF-κB , or on the induction of IFN-α mRNA that is regulated by IRF7 [33] . In contrast , as early as 8 h post-infection , IFN-β mRNA expression that is known to occur through activated IRF3 , was enhanced by PMLIV leading to as high as a 2-log increase 12 h post-infection . Like PMLIV , PMLIVa increased IFN-β mRNA synthesis upon VSV infection ( data not shown ) . Thus , both PMLIV and its variant PMLIVa sharing the same C-terminal region unique to this isoform , boosted IFN-β induction upon VSV infection . Next , we asked whether PMLIV-dependent boost of IFN-β transcription was specific to VSV infection or whether it was a broader mechanism triggered by pattern recognition receptor ( PRR ) activation . To do this , U373MG-EV or U373MG-PMLIV cells were either infected with SeV ( 40HAU/ml ) or transfected with poly ( I:C ) ( 1 µg/ml ) . After 8 h , mRNAs were extracted and IFN-βtranscripts quantified by RT-qPCR . As shown in Figure 5B , we found that PMLIV also enhanced IFN-β mRNA synthesis in U373MG cells infected with SeV , EMCV or transfected with poly ( I:C ) ( Figure 5B ) . Also , HeLa cells transduced with PMLIV-expressing or with noncoding parental ( EV ) lentiviral vector and infected with HTLV-1 , influenza or vaccinia virus revealed that PMLIV positively regulated IFN-β mRNA production ( Figure 5B ) . This demonstrates that the ability of PMLIV to potentiate IFN-β synthesis is not a specific feature of VSV infection but a more general mechanism following RNA or DNA detection by PRRs . We investigated the role of SUMOylation in the antiviral property of PMLIV by using PMLIV-3KR mutant in which the three major SUMO-target lysines were substituted with arginines . Double immunofluorescence analysis of PML and viral proteins revealed that PMLIV inhibited viral protein synthesis , whereas PMLIV-3KR did not ( Figure S2A , left panel ) . PML and its SUMOylated forms were indeed produced in cells stably expressing PMLIV but , as expected , only the unmodified form was detected in cells stably expressing the SUMO-deficient PML mutant , PMLIV-3KR ( Figure S2A , right panel ) . Western blot analysis of extracts from infected U373MG-EV , U373MG-PMLIV and U373MG-PMLIV-3KR cells confirmed that VSV protein synthesis was inhibited by PMLIV but was not affected by PMLIV-3KR ( Figure S2B , left panel ) . In addition , VSV growth was inhibited in cells expressing PMLIV , but not in cells expressing PMLIV-3KR ( Figure S2B , right panel ) . To determine the role of PMLIV SUMOylation on IFN-β synthesis , we quantified by RT-qPCR IFN-β mRNA in extracts from U373MG-EV , U373MG-PMLIV and U373MG-PMLIV-3KR cells infected with VSV ( Figure 5C ) . As PMLIII also inhibits VSV multiplication , we also quantified IFN-β mRNA in extracts from U373MG-PMLIII and U373MG-PMLIII-3KR cells . Again , we observed a nearly 2 log increase of IFN-β mRNA expression in VSV infected cells expressing PMLIV . Strikingly , the induction was completely lost when the PMLIV-3KR was expressed , demonstrating that SUMOylation of PMLIV is also required for the enhancement of IFN-β mRNA expression . In contrast , following VSV infection , PMLIII did not increase the mRNA synthesis of IFN-β , IFN-α , TNF-α or IL8 ( Figure 5C and data not shown ) . Taken together , these results demonstrate that SUMO modification of PMLIV is required to confer resistance towards VSV infection and also to increase IFN-β synthesis . To determine the effect of other PML isoforms on IFN-β synthesis , U373MG-EV cells and cells stably expressing each PML isoform ( PMLI , PMLII , PMLIII , PMLIV , PMLV , PMLVI or PMLVIIb ) were infected with VSV at an MOI of 0 . 2 and IFN-β mRNA was quantified in their extracts by RT-qPCR ( Figure 6A ) . Among all PML isoforms , only PMLIV expression resulted to a dramatic increase of IFN-β mRNA synthesis following VSV infection , reaching a nearly 2 log rise . To rule out the possibility that this increase could be due to the stabilization of IFN-β encoding mRNA by PMLIV , we performed a transcriptional assay . To do this , we transfected control or PMLIV-expressing cells with a reporter plasmid containing the firefly luciferase gene under the control of the human IFN-β promoter and infected the cells with VSV . Using this reporter assay , we were able to confirm that PMLIV greatly enhances the transcription driven by the IFN-β promoter ( data not shown ) , confirming that this increase is at the transcriptional level . Among members of the IFN regulatory factor family , IRF3 plays an essential role in virus-induced IFN-β gene expression [34] . Interestingly , IFN produced by PMLIV-expressing cells upon VSV infection was due to activated IRF3 , since a higher amount of phosphorylated IRF3 ( P-IRF3 ) was detected 6 h post-infection of cells expressing PMLIV compared to control cells ( Figure 6B ) . Interestingly , PMLIV also enhanced IRF3 phosphorylation in cells transfected with poly ( I:C ) ( Figure 6B ) . In contrast , the level of IRF3 was not altered following VSV infection or poly ( I:C ) transfection . To further determine the role of IRF3 in anti-VSV activity and enhanced IFN synthesis , U373MG-EV and U373MG-PMLIV cells depleted for IRF3 were infected at an MOI of 1 for 8 h and their extracts were analyzed by RT-qPCR for IFN-β mRNA and by Western blot for IRF3 and viral protein expression . As seen in Figure 6C , in PMLIV expressing cells infected for 8 h , depletion of IRF3 abrogated IFN-β mRNA synthesis ( Figure 6C , left panel ) without affecting the inhibition of VSV protein synthesis ( Figure 6C , right panel ) and viral production ( data not shown ) . This demonstrates that the intrinsic anti-VSV activity of PMLIV is independent of IRF3 . Thus , taken together , our results demonstrate that PMLIV is the only PML isoform able to inhibit VSV at a high MOI independently of IRF3 and also to stimulate IFN-β synthesis via an increase of IRF3 activation . PMLIV conferred viral resistance in cells 8 h or 12 h post-infection ( Figure 3 ) . This resistance was correlated with an induction of IFN-β mRNA synthesis observed as early as 8 h and increased as high as 2 log at 12 h post-infection ( Figure 5 ) . To determine whether or not the observed antiviral effect of PML at these times of infection was a secondary response to IFN synthesis , we tested the capacity of PMLIV to inhibit VSV in cells treated with an anti-IFNAR1 mAb targeting the extracellular domain of the IFNAR1 chain of the human IFN-α/β receptor . The anti-IFNAR1 mAb inhibits the binding and biological activity of type I IFN [35] as well as the IFN-β-induced STAT1 expression and anti-VSV activity ( Figure 7A , left panel ) . In contrast , this antibody did not alter the inhibition of VSV protein synthesis by PMLIV in cells infected for either 8 h or 12 h ( Figure 7A , right panel ) . Since STAT1 is the central transcription factor required for the biological responses of all types of IFN , we determined the effect of its downregulation on PMLIV-induced VSV resistance ( Figure S2C ) . The capacity of PMLIV to inhibit VSV protein synthesis was still maintained in cells depleted for STAT1 ( Figure S2C ) , further demonstrating the intrinsic anti-VSV effect of PMLIV at 12 h post-infection . Collectively , these results demonstrate that PMLIV exerts an early intrinsic anti-VSV activity that is independent of IFN . To determine whether the IFN produced and secreted after a longer period of infection was active , culture supernatants from U373MG-EV cells ( SEV ) and U373MG-PMLIV cells ( SPMLIV ) infected with VSV for 20 h were tested , in comparison with IFN-β , for their capacities to induce ISG products and to inhibit viral protein synthesis . Therefore , HeLa cells treated with medium ( Ctrl ) , IFN-β , SEV or SPMLIV supernatant for 24 h were uninfected ( Figure 7B , left panel ) or infected with VSV for 8 h ( Figure 7B , right panel ) . As seen in Figure 7B ( left panel ) , STAT1 and PKR expression was increased only in cells treated with IFN-β and SPMLIV supernatant . In addition , VSV proteins were inhibited only in extracts from cells pretreated with IFN-β or SPMLIV supernatant ( Figure 7B , right panel ) . It should be noted that the capacity of SPMLIV supernatant to induce STAT1 and PKR expression as well as to inhibit VSV was higher than that observed with 100 units/ml of IFN-β . In addition , SPMLIV but not SEV supernatant was able to induce STAT1 phosphorylation in HeLa cells ( Figure 7C , left panel ) . Taken together , these results show that the IFN produced and secreted by U373MG-PMLIV cells 20 h post-VSV infection , activates STAT1 , induces ISG products and protects HeLa cells from viral infection . Next , we quantified the amount of type I IFN in supernatants from VSV-infected U373MG-EV and U373MG-PMLIV cells using the human HL116 cell line carrying the luciferase gene under control of the IFN-inducible 6–16 promoter . This experiment showed that PMLIV boosted the amount of type I IFN synthesized in infected cells by up to 200 to 300 international units/ml ( IU/ml ) , depending on the experiment . A typical experiment is presented in Figure 7C ( right panel ) . In addition , SEV and SPMLIV supernatants were also titrated on HeLa cells . The IFN titer of SEV was below the detection limit ( less than 2 IU/ml ) and that of SPMLIV was 250 IU/ml . Taken together , these results demonstrate that PMLIV has a dual effect on viral infection: ( i ) an early intrinsic anti-VSV activity that was not eradicated by treatment with anti-IFNAR1 mAb , knockdown of STAT1 or IRF3 and ( ii ) an activation of innate immune signaling that occurs later and leads to the production and the secretion of type I IFN , which can protect other cells from viral infection . Pin1 is known to interact with and to promote phosphorylated IRF3 degradation [30] . Since PMLIV increased IRF3 activation upon VSV infection , we asked whether PMLIV can specifically recruit endogenous Pin1 within PML NBs . Double immunofluorescence studies were performed on endogenous Pin1 and PML in cells expressing PMLIII , PMLIV or PMLIV-3KR . In both uninfected and infected cells endogenous Pin1 was found both in the cytoplasm and the nucleus ( Figure 8A and data not shown ) . Importantly , Pin1 was found colocalizing with PMLIV within the NBs in uninfected or VSV-infected cells ( Figure 8A and data not shown ) . Indeed , Pin1 was found diffuse in the nucleus of EV , PMLIII and PMLIV3KR cells , whereas in PMLIV-expressing cells , it formed speckles colocalizing with PML NBs . Interestingly , such colocalization was not observed in cells expressing PMLIII or PMLIV-3KR ( Figure 8A , left panel ) . Fluorescence intensities were quantified using Image-J software and revealed that the portion of Pin1 associated to PML NBs highly increased in PMLIV-expressing cells ( Figure 8A , right panel ) . Thus , PMLIV induced a relocalization of Pin1 from the nucleoplasm to the NBs . The recruitment of Pin1 to PML NBs by PMLIV was further demonstrated by Western blot analysis of the RIPA soluble and insoluble fractions ( Figure 8B ) . In EV cells , most of the Pin1 was found in the RIPA-soluble fraction that included both the cytoplasm and the nucleoplasm , whereas a small fraction was associated to the nuclear matrix ( RIPA-insoluble fraction ) . As observed by immunofluorescence , the expression of PMLIV resulted in a shift of Pin1 to the nuclear matrix , resulting in an enrichment of Pin1 in the RIPA insoluble fraction ( Figure 8B ) . Co-immunoprecipitation assays revealed that PMLIV and PMLIV-3KR interacted with endogenous Pin1 whereas a very slight interaction was detected with PMLIII ( Figure 8C ) . The recruitment of Pin1 within PML NBs was observed in various human cell lines including HeLa cells transduced with a lentiviral vector expressing PMLIV ( Figure 9A , and data not shown ) . The recruitment of Pin1 within PML NBs by PMLIV in HeLa cells was also associated with a positive regulation of both IRF3 activation ( Figure 9B and data not shown ) and IFN-β synthesis upon VSV or SeV infection ( Figure 9C ) . Thus , PMLIV expression in different infected human cells resulted in enhanced IRF3 phosphorylation and IFN-β mRNA production ( Figures 5 , 6 and 9 ) . At the opposite , the expression of PMLIV in MEF cells neither induced the recruitment of endogenous mouse Pin1 within NBs ( Figure 9A ) nor enhanced IRF3 activation or IFN-β synthesis upon viral infection ( Figure 9C and data not shown ) . Our results suggest that the recruitment of Pin1 by PMLIV within PML NBs is required for PMLIV-induced enhancement of IFN-β synthesis . Next , we asked whether the intrinsic anti-VSV activity of PMLIV was observed in PML-/-MEF cells . To do this , PML-/- MEFs were transduced with EV- or PMLIV-encoding lentivector before infection with VSV . As seen in Figure 9D , PMLIV was still able to inhibit VSV protein synthesis when expressed in PML-/- cells , thus revealing that the intrinsic anti-VSV activity of PMLIV did not require the expression of endogenous PML . To further confirm the specific positive regulation of PMLIV on IFN-β transcription , U373MG cells were infected with SeV 48 h after transfection with scramble siRNA ( Sc ) , siRNA targeting IRF3 , siRNA common to all PML isoforms ( siRNA PMLc ) , or with the siRNA specific to PMLIII or to PMLIV that we have previously validated [23] . As expected , the siRNA IRF3 completely abolished IFN-β mRNA synthesis upon SeV infection ( Figure 9E ) . SeV-induced IFN-β expression was not altered by PMLIII depletion but was highly reduced following the depletion of all PML isoforms or the specific depletion of PMLIV ( Figure 9E ) . Furthermore , specific suppression of PMLIV expression reduced SeV-induced IRF3 activation ( Figure 9F ) . This demonstrates that endogenous PMLIV is required for the efficient synthesis of IFN-β transcription upon viral infection and validates our data obtained in cells overexpressing PMLIV . Taken together , these results show that PMLIV and PMLIV-3KR interacted with Pin1 but only PMLIV was able to recruit it within PML NBs where both proteins colocalized . Therefore , the interaction of endogenous Pin1 with SUMOylated PMLIV and its recruitment in PML NBs could alter Pin1-induced downregulation of activated IRF3 thus resulting in a higher amount of phosphorylated IRF3 during viral infection . In addition , the intrinsic anti-VSV activity of PMLIV is independent of the expression of other PML isoforms .
Many reports implicate PML and PML NBs in antiviral responses targeting diverse cytoplasmic replicating RNA viruses through different mechanisms [13] , [17] , [19] , [21] , [23]–[25] , [36] . An antiviral effect of PML against rhabdoviridae has been observed in vivo , as PML deficiency renders mice more susceptible to VSV infection [14] . In this report , we show that cells derived from these mice or human cells depleted of PML produced a higher level of viral proteins . Among the various PML isoforms tested , only stable expression of PMLIII and PMLIV conferred resistance to VSV . This inhibitory effect did not alter VSV entry , but was observed at the level of viral mRNA and protein synthesis , resulting in a reduction of VSV yields and in cell lysis protection . The protective property of PMLIV was found to be higher than that of PMLIII , as PMLIV was able to inhibit virus growth up to an MOI of 2 , resulting in a 500-fold reduction of VSV yields . Whereas PMLIII confers viral resistance in an IFN-independent way , PMLIV displays two antiviral activities during VSV infection: an early IFN-independent activity targeting VSV replication followed by the activation of innate immunity pathways , leading to an enhanced type I IFN synthesis , which protects yet uninfected cells from viral infection ( Figure 10 ) . Interestingly , PMLIV-3KR failed to confer resistance to VSV and also to induce IFN-β synthesis , demonstrating that SUMOylation of PMLIV is required for both intrinsic antiviral activity and innate immune property . The induction of IFN-β expression is the key event in the initiation of the innate antiviral response . Central to this process is the activation of IRF3 via its phosphorylation [34] . Here we demonstrate for the first time that one particular isoform of PML ( PMLIV ) is implicated in innate immunity , triggering a dramatic increase of IFN-β synthesis via IRF3 phosphorylation upon VSV infection . Depletion of IRF3 further demonstrates the dual activity of PMLIV , as it abrogated PMLIV-induced IFN synthesis but not PMLIV-induced inhibition of viral replication . Pin1 was shown to interact with phosphorylated IRF3 and to promote its ubiquitin-mediated proteasomal degradation [30] . We report here that endogenous Pin1 interacts with PMLIV and that both proteins colocalize in PML NBs . This results in sustained IRF3 activation and higher IFN-β induction during VSV infection . Thus , the recruitment of endogenous Pin1 in PML NBs might antagonize Pin1-induced P-IRF3 degradation [30] and could be a novel mechanism for inhibiting Pin1 function . Consistently , host antiviral responses are boosted in the presence of PMLIV . Collectively , our report characterizes PMLIV as a positive regulator of antiviral innate immune responses , which maintains stability of IRF3 phosphorylation through the interaction of endogenous Pin1 with SUMOylated PMLIV and its recruitment in PML NBs . Thus , our results suggest that , whereas the direct antiviral activity of PMLIV is specifically targeting VSV , the positive regulation of innate immunity should also be observed using other stimuli than VSV infection since PMLIV also recruited Pin1 within the NBs in uninfected cells . Indeed , we observed that IRF3 activation and IFN-β production were also drastically enhanced in PMLIV-overexpressing cells following transfection with poly ( I:C ) or infection with viruses from different families such as SeV , EMCV , HTLV-1 , influenza virus or vaccinia virus . These observations confirm that PMLIV has a specific anti-VSV activity and a much wider positive effect on innate immunity pathways . Remarkably , specific depletion of endogenous PMLIV in human U373MG or HeLa cells reduced VSV- as well as SeV-induced activation of IRF-3 and consequent production of IFN-β ( this paper and data not shown ) . This is the first demonstration of the implication of PML in the enhancement of IFN-β production upon viral infection . In contrast , the intrinsic antiviral activity of PML has been documented to act on viruses from different families [17] , [21] . In the case of EMCV , PMLIV is the only PML isoform that confers viral resistance , by interacting with the viral polymerase 3D , and sequestering it within PML NBs where both proteins colocalize [23] . Interestingly , PMLIV is also able to sequester the ORF23 capsid protein within PML NBs , leading to the inhibition of varicella-zoster virus ( VZV ) an alphaherpesvirus [37] . PML has also been shown to interfere with retrovirus replication , since PMLIII interacts with Tas , the transcriptional transactivator of human foamy virus , resulting in viral restriction [24] . The mechanism by which the nuclear PMLIV directly inhibits VSV , whose replication takes place entirely in the cytoplasm , remains to be elucidated . We demonstrated that the intrinsic anti-VSV property of PMLIV did not require the expression of other PML isoforms and was independent of IFN since it was maintained in cells treated with antibodies against type I IFN receptors , depleted for IRF3 or depleted for STAT1 . However , further investigations are needed to demonstrate how PMLIV and PMLIII exert their intrinsic anti-VSV activity by interacting with a viral or a cellular protein required for VSV replication . Understanding the intrinsic antiviral activity of PML may introduce new ways for targeted antiviral therapy that would bypass the need for IFN treatment . Thus , PML confers viral resistance in two ways ( Figure 10 ) . It can exert an intrinsic anti-VSV activity independent of IRF3 , STAT1 and IFN . In addition , we show here that PML enhanced both IRF3 activation and IFN-β synthesis upon viral infection . This produced IFN-β is secreted and protects yet uninfected cells from oncoming infection . Our results demonstrate that PML , an ISG product with a broad intrinsic antiviral activity , is also able to trigger IFN-β synthesis upon viral infection . There are an increasing number of ISG products that are also implicated in innate immunity processes . PKR for example , long known to mediate the antiviral activities of IFNs , also plays an important role in the induction of type I IFN , particularly IFN-β during measles virus infection [38] or double-stranded RNA treatment [39] . Indeed , activation of PKR increases IRF3 activation , and knockdown of PKR reduces both activated IRF3 level and IFN-β induction [39] . The role of the endoribonuclease RNase L in the innate antiviral immune response has been demonstrated in vivo . Indeed , injection of 2′5′-linked oligoadenylates leads to IFN-β synthesis in wild-type mice but not in RNase L deficient mice . In addition , EMCV- or Sendai virus-induced IFN is highly reduced in mice lacking RNase L [40] . Interestingly , many members of the TRIM protein family , which PML belongs to , ( i ) are products of ISGs [41] , ( ii ) display a direct antiviral activity [42] and/or have been identified as important players of innate immunity [43] . TRIM5α for instance is induced by type I IFN [44] , [45] , inhibits retroviral infections [46] and is also able to promote innate immune signaling [47] . TRIM25 protein plays a key role in innate immunity , since it is essential for RIG-I-mediated antiviral activity [48] . Strikingly , TRIM21 is also able to enhance IRF3-mediated antiviral response . Indeed , TRIM21 was shown to interact directly with IRF3 upon viral infection and to interfere with its interaction with Pin1 [49] . Recent studies performed on the entire TRIM protein family allowed the identification of several other members of this family as key components of inflammation and innate immunity signaling pathways [50] , [51] . Our results show that TRIM19/PML , another member of the TRIM protein family , acts , through Pin1 recruitment in PML NBs , as a positive regulator of IRF3 phosphorylation , enhancing the strength and duration of IFN-β-induced antiviral response . Thus , PML can therefore be added to the list of TRIM proteins implicated in both intrinsic and innate immunity . Initially considered as two independent arms of the immune system , our results further suggest a closer crosstalk between intrinsic and innate immunity .
Human recombinant IFN-β was purchased from Biogen Inc . Rabbit polyclonal ( sc-5621 ) and mouse anti-PML ( sc-966 ) , rabbit anti-IRF3 ( sc-9082 ) , rabbit anti-STAT1 ( sc-345 ) , rabbit anti-phospho-STAT1 ( Tyr701 , sc-7988 ) and rabbit anti-PKR antibodies ( sc-707 ) were obtained from Santa-Cruz Biotechnology . The rabbit anti-phospho-IRF3 ( Ser 396 ) and rabbit anti-Pin1 antibodies were obtained from Cell Signaling , and HRP-conjugate monoclonal anti-Actin antibody from Sigma . The 64G12 monoclonal antibody against human IFN-α/β receptor ( anti-IFNAR1 mAb ) was a gift from P Eid ( INSERM UMR1014 ) [35] . The rabbit anti-VSV polyclonal antibodies ( home-made ) were obtained by repeated injection of purified virus . Reactivities against the viral proteins N , M or G were different depending on the batch used in western-blot experiments . Human glioblastoma astrocytoma U373MG , epithelial HeLa and fibrosarcoma HL116 cells as well as mouse embryonic fibroblasts ( MEFs ) from wild-type ( WT ) or knockout PML ( PML-/- ) mice [52] , were grown at 37°C in DMEM supplemented with 10% FCS . U373MG cells transfected with empty vector or stably expressing individual PML isoforms ( PMLI to VIIb ) , PMLIII-3KR or PMLIV-3KR were kept in medium supplemented with 0 . 5 mg/ml of neomycin . HL116 cells were grown in medium supplemented with HAT ( Hypoxanthine: 20 µg/ml , Aminopterin: 0 . 2 µg/ml , Thymidine: 20 µg/ml ) . The accession number ( GenBank ) for PML isoforms are AF230401 ( PMLI ) , AF230403 ( PMLII ) , S50913 ( PMLIII ) , AF230406 ( PMLIV ) , AF230411 ( PMLIVa ) , AF230402 ( PMLV ) , AF230405 ( PMLVI ) , AF230408 ( PMLVIIb ) . In PMLIII-3KR and PMLIV-3KR mutants , the three SUMO-target lysines ( at positions 65 , 160 , and 490 ) were replaced with arginines . Stable U373MG cells expressing each of the PML isoforms ( PMLI to VIIb ) , PMLIII-3KR or PMLIV-3KR were obtained via transfection with constructs corresponding to each cloned in pcDNA3 . 1 and subsequent neomycin selection at a final concentration of 0 . 5 mg/ml [25] . Control U373MG cells were generated in the same way using the empty vector ( U373MG-EV ) . For expressing PMLIV in MEFs wild-type , MEFs PML-/- and HeLa cells , we constructed an HIV-derived lentiviral vector ( pTRIP-PMLIV ) , which was used to transduce the cells . The pTRIP plasmid was provided by P Charneau ( Institut Pasteur , Paris , France ) [53] . VSV ( Mudd-Summer strain , Indiana serotype ) was grown in BSR cells . BSR cells were infected at an MOI of 0 . 1 . After 24 h , supernatants were collected and cellular debris removed by low-speed centrifugation . Virus titers ( 109 PFU/ml ) were determined by standard plaque assay onto BSR cells . EMCV was produced as described [20] and has a titer value of 2 . 108 PFU/ml . Vaccinia virus has a titer of 2 . 109 PFU/ml . Sendai virus was kindly provided by E Meurs ( Institut Pasteur , Paris , France ) . It was used at 40 HAU/ml to activate RIG-I , as described [54] . HTLV-1 , and influenza A virus ( strain A/PR 8/34 ) were provided by JP Herbeuval ( CNRS UMR 8601 ) . HTLV-1 and influenza virus were used at 600 ng/ml of p19-equivalent and at 40 HAU/ml , respectively . MLV-derived vectors encoding GFP pseudotyped with VSV-G , provided by FL Cosset ( ENS Lyon ) , had a titer value of 107 IU/ml and were generated as described [31] . Cells were seeded in six-well plates and transfected with siRNA using Lipofectamine RNAiMax transfection reagents ( Invitrogen ) . The mRNA sequence targeted by the siRNA PMLc ( common to all PML isoforms ) is 5′-AUGGCUUCGACGAGUUCAATT-3′ , by siRNA PMLIII is 5′-AGUGCAUGGAGCCCAUGGATT-3′ and by siRNA PMLIV is 5′UGAAAGUGGGUUCUCCUGGTT-3′ . The siRNA scramble sequence is the following: 5′-GCAUGAACCGGAGGCCCAUUU-3 . STAT1 or IRF3 expression was silenced using ON-TARGETplus SMARTpool siRNAs purchased from Thermo Scientific . Cells were infected with VSV at an MOI of 3 in the absence or the presence of cycloheximide ( 100 µg/ml ) . After adsorption for 1 h , cells were washed and fresh medium with or without cycloheximide ( 100 µg/ml ) was added . After 4 h , total RNA was isolated from cells with the RNA NOW Kit ( Ozyme ) . Total RNA was separated on 1 . 5% agarose gel under denaturing conditions and blotted onto Nylon membranes ( Roche Molecular Biochemicals ) . Hybridizations were performed with digoxigenin ( DIG ) -labeled oligonucleotides recognizing the VSV-N gene sequence and by incubation with anti-DIG antibody conjugated to alkaline phosphatase followed by CDP Star . Total RNA was extracted using RNeasy Mini Kit ( Qiagen ) and cDNAs were prepared using Oligo ( dT ) primer and SuperScript II Reverse Transcriptase ( Invitrogen ) . Real-time PCR reactions were performed in duplicates using Platinum SYBR Green qPCR SuperMix-UDG ( Invitrogen ) following manufacturer's instructions . GAPDH , IFN-α1 , IFN-β , TNF-α , IL8 and VSV-L encoding cDNAs were amplified on a Mastercycler ep realplex ( Eppendorf ) with a denaturation step of 5 min at 95°C followed by thirty-five cycles of 10 s at 95°C , 10 s at 60°C and 20 s at 72°C . Threshold cycle ( Ct ) values were converted to 2−Ct in order to be proportional to the amount of transcripts in the samples . To compare samples , 2−ΔCt were calculated by normalizing the data by the expression of GAPDH: 2−ΔCt = 2−Ct ( sample ) /2−Ct ( GAPDH ) . Primers used for quantification of transcripts by real time quantitative PCR are the following: VSV L ( Forward: TGATACAGTACAATTATTTTGGGAC and Reverse: GAGACTTTCTGTTACGGGATCTGG ) , GAPDH ( Forward: ACTTCAACAGCGACACCCACT and Reverse: GTGGTCCAGGGGTCTTACTCC ) , IFN-α1 ( Forward: CCAGTTCCAGAAGGCTCCAG and Reverse: TCCTCCTGCATCACACAGGC ) , IFN-β ( Forward: TGCATTACCTGAAGGCCAAGG and Reverse: AGCAATTGTCCAGTCCCAGTG ) , TNF-α ( Forward: GGCGTGGAGCTGAGAGATAAC , Reverse: GGTGTGGGTGAGGAGCACAT ) and IL-8 ( Forward: AAGGGCCAAGAGAATATCCGAA and Reverse: ACTAGGGTTGCCAGATTTAACA ) . Immunofluorescence analyses were performed as described [23] . PML was detected with mouse anti-PML antibody and the corresponding anti-IgG antibody conjugated to Alexa 594 . The VSV antigens or Pin1 protein were detected with rabbit anti-VSV or anti-Pin1 antibodies followed by Alexa 488 . The cells were mounted onto glass slides by using Immu-Mount ( Shandon ) containing 4 , 6-diamidino-2-phenylindole ( DAPI ) to stain nuclei . Confocal laser microscopy was performed on a Zeiss LSM 510 microscope . Quantitative analysis of immunofluorescence data was carried out by histogram analysis of the fluorescence intensity at each pixel across the images using Image J software . The results of the analysis of 20 images acquired in each experimental condition were then combined to allow quantitative estimates of changes in Pin1 localization . Cells were washed and re-suspended in PBS , lysed in hot Laemmli sample buffer and boiled for 5 min . For cell fractionation , cells were dissociated and washed twice in PBS . The cytoplasmic and nucleoplasm fractions were extracted by incubating the cell pellet in 50 µl of RIPA buffer for 20 min on ice followed by centrifugation at 15000 g for 15 min to separate the RIPA soluble fraction ( R ) from the pellet ( P ) . This RIPA insoluble fraction ( P ) was suspended in 50 µl of PBS . Fifty µl of 2X Laemmli buffer were added to each fraction , and the samples were boiled for 5 min before Western blot analysis . Protein extracts were analyzed on a 10% SDS-PAGE gel , and transferred onto a nitrocellulose membrane . The proteins were blocked on the membranes with 5% skimmed milk in PBS for 2 h and incubated overnight at 4°C with rabbit polyclonal anti-PML ( clone H-238 ) , anti-VSV , anti-IRF3 , anti-phospho-IRF3 or anti-Actin antibodies . The blots were then washed extensively in PBS-Tween and incubated for 1 h with the appropriate peroxidase-coupled secondary antibodies ( Cell Signaling Technology ) . All of the blots were revealed by chemiluminescence ( ECL , Bio-Rad ) . Cells ( 107 ) were incubated for 30 min at 4°C in 0 . 5 ml of buffer containing 20 mM Tris-HCl pH 7 . 4 , 1 M NaCl , 5 mM MgCl2 , 1% triton , and 1 mM phenylmethylsulfonyl fluoride ( PMSF ) . After cell lysis , 1 . 25 ml of immuno-precipitation buffer ( IB ) ( 20 mM Tris-HCl pH 7 . 4 , 150 mM NaCl , 0 . 5% DOC , 1% Triton X-100 , 0 . 1% SDS and 1 mM EDTA ) were added . Rabbit anti-Pin1 antibodies were added and the samples incubated overnight at 4°C . Protein G beads ( Sigma ) in IB were then added and the samples mixed 2 h at room temperature . The beads were collected , washed four times with IB buffer and bound proteins were subjected to Western blotting . To inactivate the virus , culture supernatants from infected cells were brought to pH 2 for 24 h and neutralized before titration . IFN secretion was quantified using the reporter cell line HL116 that carries the luciferase gene under the control of the IFN-inducible 6–16 promoter [55] . HL116 cells ( 2×104 ) were plated in 96-well plate and incubated for 8 h with the desired culture supernatants or a standard of human IFN-β reference ( Gb-23-902-531 ) . Cells were then lysed ( Luciferase Cell Culture Lysis Reagent , Promega ) and luciferase activity measured using Perkin Elmer Wallac 1420 . IFN titers were also quantified on HeLa cells challenged with VSV . IFN titers , defined as the amount of IFN leading to 50% inhibition of the cytopathic effect , were expressed in international unit/ml relative to the human IFN-β reference ( Gb-23-902-531 ) of the NIH . PMLI ( AF230401 ) , PMLII ( AF230403 ) , PMLIII ( S50913 ) , PMLIV ( AF230406 ) , PMLIVa ( AF230411 ) , PMLV ( AF230402 ) , PMLVI ( AF230405 ) , PMLVIIb ( AF230408 ) , STAT1
|
PML is expressed as seven isoforms , designated PMLI to PMLVIIb , each with specific functions conferred by their C-terminal regions . PML isoforms are implicated in several cell processes , including antiviral defense . Very few studies have been performed with all PML isoforms to investigate their individual antiviral properties . Our comparative study with all PML isoforms on VSV replication revealed that only PMLIII and PMLIV are able to inhibit this virus in an IFN-independent way . Importantly , PMLIV is also able to enhance IRF3 phosphorylation resulting in a dramatic IFN-β production in response to viral infection , thus protecting yet uninfected cells . At the opposite , the specific suppression of PMLIV expression in human cells reduced virus-induced IRF3 activation and subsequent IFN-β production . We found that PMLIV affects the endogenous distribution of Pin1 , by recruiting this protein in PML NBs where both proteins colocalize . This prevents activated IRF3 degradation , thus enhancing the production of IFN-β . Here we show for the first time that , in addition to its intrinsic anti-VSV activity , PMLIV is implicated in antiviral innate immune response . Thus , PML is an IFN stimulated gene whose products have a broad intrinsic antiviral activity and one of them , PMLIV , is also a potent regulator of innate immune pathways .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"mechanisms",
"of",
"resistance",
"and",
"susceptibility",
"immunity",
"virology",
"innate",
"immunity",
"host-pathogen",
"interaction",
"biology",
"microbiology",
"viral",
"replication"
] |
2014
|
Implication of PMLIV in Both Intrinsic and Innate Immunity
|
HIV latency is a major obstacle to curing infection . Current strategies to eradicate HIV aim at increasing transcription of the latent provirus . In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA ( vorinostat ) , despite effective inhibition of histone deacetylases . To identify steps that were not susceptible to the action of SAHA or other latency reverting agents , we used a primary CD4+ T cell model , joint host and viral RNA sequencing , and a viral-encoded reporter . This model served to investigate the characteristics of latently infected cells , the dynamics of HIV latency , and the process of reactivation induced by various stimuli . During latency , we observed persistence of viral transcripts but only limited viral translation . Similarly , the reactivating agents SAHA and disulfiram successfully increased viral transcription , but failed to effectively enhance viral translation , mirroring the ex vivo data . This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir .
Successful antiretroviral therapy reduces HIV plasma viremia to undetectable levels; however , the virus is not eradicated [1] . Current and non-mutually exclusive hypotheses to explain this phenomenon include ( i ) the presence of anatomic viral reservoirs that are inaccessible to drugs , ( ii ) ongoing viral replication and cell-to-cell spread , and ( iii ) the existence of a long-lived cellular reservoir that is infected but does not produce viral particles and can be reactivated . With the last hypothesis , it is proposed that reactivating viral transcription in latently infected cells may help purging the HIV reservoir and contribute to HIV eradication [2] . Therefore , a better understanding of the mechanisms involved in viral transcriptional silencing and in the reactivation from latency is essential . Less attention has been paid to post-transcriptional blocks which prevent completion of the viral replication cycle , thus limiting expression of viral proteins , de novo production of viral particles and cytopathogenesis . Currently , there are two competing models on how HIV establishes latency in resting CD4+ T cells [3] , [4] . The first model suggests that activated cells are infected by HIV , and that most of them are productively infected and die within a few days . However , a minority of cells revert to a resting memory state , following the natural biology of CD4+ T cells . The second model proposes that HIV is able to directly infect resting CD4+ T cells , even if this process is poorly efficient . To study HIV latency experimentally , cell lines have been used extensively to investigate the molecular mechanisms underlying impairment of viral gene expression . Cell lines highlighted viral transcriptional silencing as a major determinant of HIV latency ( reviewed in [5]–[8] ) . There is however increasing interest for work in more representative primary cell settings . In recent years , multiple systems using primary cells from various CD4+ T cell subpopulations have been developed . These systems yield sufficient numbers of cells allowing investigations that may recapitulate more closely in vivo processes ( reviewed in [4] , [9] . Primary cell latency models have been used for the screening and assessment of molecules that promote viral transcription [10] . Multiple compounds [11] reactivate viral transcription from latently infected cells in vitro , reflecting the various mechanisms involved in viral transcriptional control , including epigenetic regulation ( such as histone modifications and DNA methylation ) and immune modulation ( such as T cell receptor engagement and protein kinase C signaling ) [8] , [11] , [12] . The efficiency of these agents varies according to the HIV latency model used [11] , [13] , underscoring that the mechanisms leading to repression and reactivation of viral transcription may be cell-specific . Some of the agents are moving forward to clinical trials [1] , [11] , [14]–[16] . We have previously completed a detailed analysis of the cellular response to HIV infection in a T cell line and derived a model of cell reprogramming upon viral invasion [17] . Here , we use an extension of this experimental approach to investigate viral latency and reactivation in a model of primary CD4+ T cells . We hypothesized that a comprehensive assessment of the paired transcriptomes of the host and the virus by RNA sequencing , coupled to the analysis of viral protein expression , would allow for addressing the extent of viral transcriptional and post-transcriptional silencing , the structure of the viral transcriptome , and the nature of the cellular transcriptional program induced by pharmacological and immunological reactivating compounds . Our in vitro and ex vivo data reveal defects in viral translation and particle production that reflect a state of post-transcriptional cellular latency which may not be reversed by agents merely enhancing viral transcription .
We compared the efficacy of T-cell receptor ( TCR ) stimulation and SAHA treatment to stimulate viral particle production ex vivo . For this , we tested virus release from latently infected cells isolated from HIV-infected individuals under successful treatment ( Table S1 ) . We isolated resting CD4+ T cells and cultured them for 1 , 2 , and 4 days in the presence of IL-2 supplemented with DMSO ( control ) , SAHA or TCR stimulation . TCR stimulation resulted in successful viral particle production in 7 of 11 samples , with viral RNA copies ranging from 42 to 2940 copies/ml of supernatant . Viral RNA copies in controls ( mock-treated cells ) were at or below the limit of detection of the assay for 6 samples , and values ranging from 27 to 287 copies/ml for 5 samples ( Fig . 1 ) . SAHA was undistinguishable from control , suggesting a failure to stimulate particle production from latently infected cells ex vivo , confirming previous studies [18] . We observed effective inhibition of histone deacetylases confirming that primary cells were exposed to appropriate concentrations of SAHA ( Fig . 1 ) . To further investigate the mechanisms underlying SAHA inefficiency to reactivate latently infected CD4+ T cells ex vivo and generate viral particles , we used the cellular model described by the laboratories of Cloyd [19] and Karn [20] that preferentially produces latently infected CD4+ T cells with a central memory phenotype . These cells constitute the major cellular reservoir of HIV in blood [21] . The viral vector , previously used in latency studies , encodes Tat , Rev , and a destabilized GFP protein with an estimated half-life of 6 hours [22] . Starting with the transduction of two million cells from a healthy donor , we sorted a population of 200 , 000 successfully infected cells and amplified them to obtain 50 million cells . The infected cell population was then co-cultured on a feeder layer of the H80 human brain tumor cell line over 10 weeks ( Fig . S1 ) . We obtained a population of latently infected cells carrying a stable number of viral integration events , measured at 1 . 06 proviruses per cell . This measure of quality control , as well as the quasi-universal reactivation of the cell population upon T cell receptor ( TCR ) stimulation with anti-CD3/anti-CD28 and IL-2 ( see below ) excluded outgrowth by non-infected cells . The expression from the env open reading frame of a destabilized viral-encoded GFP decreased significantly following infection ( entry phase ) , and reached its nadir upon co-culture on feeder cells ( week 0 ) . However , low level GFP expression remained stable at around 3-fold above background ( mock uninfected cells ) through 10 weeks of co-culture ( latency phase ) ( Fig . S2 ) . Upon reactivation by TCR stimulation ( reactivation phase ) , we observed up to 9-fold increase of GFP expression; with 92% of cells expressing the viral-encoded GFP , Figure 2 . The activation marker IL2Rα ( CD25 ) remained undetectable during the latency phase . Thus , the model recapitulates the process of latency , although the basal degree of GFP expression suggests incomplete silencing of HIV . To understand the extent of viral silencing during latency , we assessed three parameters of viral transcriptional activity using RNA sequencing data . First , we estimated the proportion of viral transcripts among the total number of detected transcripts . From week 0 ( W0 ) through week 10 ( W10 ) , this proportion was stable with a mean of 1 . 23% ( 95%CI; 1 . 15 , 1 . 32 ) . Second , we evaluated the distribution of viral reads along the viral vector genome . We identified a conserved pattern of coverage of the viral vector genome through the latency phase ( Fig . 3A ) . Third , we assessed the pattern of viral splicing . All main splice variants were well represented and in conserved proportions [23] ( Fig . 3B , Fig . S3 , Fig . S4 ) . Upon TCR stimulation , there was a proportional shift in viral splice forms . Indeed , cell activation resulted in a profound modification of the transcriptome and doubling of the RNA content of the blasts . The RNA sequencing approach captures polyadenylated viral RNA ( i . e . fully elongated ) . On these RNA species we did not observe major deficits in viral genome coverage , or splicing during latency . Thus , these viral transcripts could contribute to the residual viral-encoded GFP expression . However , these analyses do not discriminate between continuous translation from a stable pool of viral transcripts from the original infection and de novo transcription from leakiness of the latency model . The host RNA profile during the dynamic process of latency was studied in time-course transcriptome analyses by grouping host genes according to their transcriptional response to HIV and to TCR stimulation with anti-CD3/CD28 . Whereas the virus only minimally perturbed the transcriptional state of the cell , TCR stimulation resulted in massive cell reprogramming , impacting 66% of all detected genes . Numbers of genes differentially expressed under the various conditions are detailed in the legend of Figure 4 . Principal component analysis revealed two transcriptionally distinct phases: entry ( W0–W2 ) and latency ( W4–W10 ) ( Fig . 4B , Fig . S5 ) . The tight cluster of samples during the 6 weeks of latency includes both infected and uninfected samples , indicating a stable process that is only minimally perturbed by the virus . To assess the differences between infected and uninfected cells , we compared gene expression in paired samples to pinpoint potential markers of HIV latency , consistently differentially expressed during the 6 weeks of maintenance of latency ( W4–W10 ) : we identified 103 cellular genes as upregulated and 124 as downregulated in HIV-infected samples during this phase . The differentially expressed genes exhibit moderate enrichment for pathways including chemokine and cytokine receptors and immune response ( Fig . S6 ) . All differentially expressed genes and analyses can be queried and downloaded from the open access interactive web resource http://litchi . labtelenti . org . The RNA sequence data also allowed for estimating the diversity of integration sites during the various time points and upon reactivation . Out of these , we identified 15886 integration events with reads at a junction between host and HIV transcripts . Importantly , the data reflects host-virus splicing events that , while globally identifying the general location of an integration event , do not allow precise mapping . From this we identified 12868 integration events in the host genome across conditions; ranging from 3718 at the earliest time point ( i . e . 14 days before W0 ) to 2216 at the latest time point ( TCR 24h ) ; 92 . 5% represented unique integration sites . Overall , these findings indicate minimal clonal bias in the model . In summary , the stability of the cellular transcriptome profile between weeks 4 and 10 is consistent with maintenance of a resting state of primary CD4+ T cells and limited contribution from viral infection . TCR stimulation creates a cellular environment highly supportive of HIV expression . After ten weeks of co-culture on H80 , cells were collected and incubated in the presence of various pharmacological ( vorinostat [SAHA] , disulfiram [DSF] , 5′-azacytidine [AZA] ) , or immunological ( interleukin 7 [IL-7] ) stimuli , as well as TCR stimulation . We collected RNA for sequencing and cells for FACS analysis at 8 and 24 hours after treatment start . The removal of cells from the feeder cell layer induced a 2-fold reduction of the proportion of HIV to all cellular transcripts ( from 1 . 26% to 0 . 64% at 8 hours ) , possibly due to loss of a trophic environment provided by H80 cells ( Fig . 5A ) . In contrast , exposure to SAHA resulted in an increase in the proportion of viral transcripts to 1 . 77% at 8 hours and 2 . 14% at 24 hours , three times that of DMSO control ( Fig . 5A ) . The effect of TCR stimulation on the proportion of HIV transcripts was modest , but cell activation was accompanied by blast morphology and a 2-fold increase in total cellular RNA at 24 hours . Disulfiram had a transient effect , with an increase in the proportion of HIV transcripts to 1 . 63% at 8 hours . IL-7 had a slower effect with 1 . 21% at 24 hours . AZA had no detectable impact on viral transcription . The use of the various agents did not result in profound changes in viral vector genome coverage or splicing . However , the use of SAHA was associated with a relative increase in the proportion of unspliced transcripts ( Fig . S3 ) . Viral transcription induced by activating agents such as SAHA and disulfiram was not proportionally reflected at the protein level as expression of the viral-encoded GFP remained low ( Fig . 5B ) . As previously reported , AZA [24]-[26] alone , or IL-7 [27] had no discernible or minimal effects on viral protein expression . This observation was in contrast to the strong increase in GFP expression induced by TCR stimulation . The divergent response was maximal when estimating GFP translation per transcript ( Fig . 5C ) . Ratios were also assessed by considering the production of GFP relative to the amount of single-spliced HIV forms , which have the capability of being translated into GFP . The general profile of transcription , translation and ratios was not modified in this analysis . To understand the differential effect of reactivating agents on viral transcription , we compared the transcriptional signatures induced by these agents ( Fig . 6 , and http://litchi . labtelenti . org ) . TCR stimulation showed a very strong effect involving 3664 upregulated and 3220 downregulated genes , with a strong enrichment signal for upregulation of the cellular machinery ( signal transduction , generic gene expression , protein synthesis , and metabolism ) , and upregulation of genes previously proposed to play a role in HIV biology [28] . In contrast , the various other agents exerted limited influence on the host transcriptome ( Fig . S7 ) . SAHA induced upregulation of 730 genes and downregulation of 559 genes . Genes involved in immune response and T-cell activation processes were preferentially downregulated by SAHA . Disulfiram showed weak effects on cellular gene expression , with only 132 upregulated genes enriched for HIV cofactors , innate immunity , and apoptosis , and 57 downregulated genes enriched in defense response genes . Very little transcriptomic effects were observed in response to the other reactivating compounds ( Fig . 6 and http://litchi . labtelenti . org ) . To confirm the main findings , we repeated the latency and reactivation experiment in the same and on an independent donor . These analyses also included spike-in internal controls for RNA sequencing and used culture supernatant only from H80 feeder cells ( without direct cell-cell contact ) . The data confirmed the reproducibility of the model , and the inability of SAHA to induce viral protein expression in proportion to the increase in viral transcripts ( Fig . S8 ) . Taken together , these results suggest that , in this model of latency , agents successfully inducing HIV transcription , such as SAHA and disulfiram , do not lead to proportional effects on HIV translation . These agents may not generate a cellular environment that effectively sustains HIV protein expression . In contrast , cell activation upon TCR stimulation creates a permissive environment by systematic activation of the cellular machineries required for viral replication . These results underscore the need to explore strategies that facilitate successful completion of the viral cycle in combination with agents that act primarily on viral transcription .
A number of agents have been identified as latency reverting drugs . SAHA has been extensively studied and brought forward to clinical trials [14] , [28] , [29] . However , data from Blazkova et al . [18] , our data , and recent reports at the time this work was under consideration [30]-[32] indicate that SAHA fails to effectively increase particle production ex vivo . We tested the ability of SAHA to increase viral production from latently infected CD4+ T cells from virologically suppressed HIV-infected individuals . While SAHA effectively inhibited histone deacetylation , it failed to stimulate particle production as compared to TCR stimulation . Data from the Siliciano laboratory also indicates that reactivation induced by SAHA is not sufficient to trigger a cytopathic effect and cell death [33] . We explored this paradox by using the primary CD4+ T cell model described by Cloyd [19] and Karn [20] . Use of a primary cell model allowed the detailed analysis of the dynamic process of HIV latency and persistence . We examined cellular transcriptional dynamics over 10 weeks and jointly defined cellular and viral expression patterns using RNA sequencing and FACS analysis . In this cellular model , latency appears to be a stable process . However , viral transcripts are continuously present accompanied by residual expression of viral-encoded GFP . We reviewed printed material and figures from various primary models and observed comparable levels of residual GFP or p24 expression that are generally interpreted as background [10] , [19] , [20] , [34] , [35] – indeed , the original paper by Tyagi et al . identified up to 28 . 77% of cells expressing low levels of GFP during latency [20] . Upon TCR stimulation , there is near universal induction of GFP expression , generally validating the model . The use of SAHA and disulfiram successfully increased viral transcription; however the reactivated cells failed to produce proportional amounts of viral protein . Overall , these observations underscore the importance of the cellular environment to allow any effect on viral transcription to translate into an efficient purging strategy [1] . We observed a remarkable stability of the cellular transcriptional profile after 4 weeks of co-culture on H80 feeder cells despite the presence of HIV transcripts in the cells . Few cellular genes were differentially expressed between non-infected and infected cells . These genes may represent biomarkers of HIV latency , or markers of the original subset of cells that were successfully transduced . Moreover , the pool of viral transcripts might persist from the original infection or reflect ongoing transcription . Cell-associated viral RNA can also be detected in latently infected resting memory CD4+ T cells in individuals under effective antiretroviral therapy ( [14] , [36]-[38] , and Hu et al . , abstract 405 , and Fromentin et al . , abstract 412 , CROI 2014 ) , and in animal models ( [39] , [40] , and Okoye et al . , abstract 136LB , CROI 2014 ) . The short half-life of the viral vector-encoded destabilized GFP [10] suggests that some residual translation takes place . Leakiness of the system could be induced by the trophic environment of the experimental system , including secreted factors from the feeder cells , or by the small concentration of IL-2 used in the model . Some level of residual activation has been observed in other primary cell models [41] . Here , however , we only detected negligible levels of cellular activation as assessed by expression of the activation marker IL2Rα ( CD25 ) . To better understand the nature of the persisting viral transcripts , we examined the amount and distribution of HIV reads along the viral vector genome , and the pattern of splicing . The proportion of HIV transcripts remained stable at about 1 . 26% of all cellular transcripts during 10 weeks , without apparent defects in structure . However , the RNA sequencing approach captures polyadenylated RNA; therefore it would not represent coverage of other species of viral transcripts , including non-polyadenylated paused viral transcripts that could be relevant and contribute to the process of latency or reactivation . The leaky system bears some similarities with that of infection of cells belonging to the monocyte/macrophage lineage , which , as discussed by Van Lint et al . [41] , may not reach complete silencing but maintain a low level of viral replication . Therefore , the model used here may recapitulate both latency and persistence of viral transcription as already observed in vivo [1] . However , the experimental system used here has shortcomings . In our hands , the amount of materials generated is limiting , and the use of a complete viral clone is too toxic , which led us to use an attenuated viral vector that only expresses GFP ( from env ORF ) , Rev and Tat . The use of such a truncated HIV vector may affect the level of basal HIV transcription and thus the process of latency [42] . Although we are aware of the possibility that , in other types of vectors , specific transcripts and viral proteins other than GFP could be selectively expressed ( e . g . Gag ) [43] , the detailed splice analysis militates against major differences in protein expression across the viral genome . We tested three pharmacological agents to induce viral transcription . SAHA was an efficient inducer , resulting in over 3-fold increase in the proportion of viral transcripts in the cell , consistent with previous reports [14] , [16] , [44] , [45] , reviewed in [5]–[7] . The effect of disulfiram was short-lived , with a 2 . 5-fold induction of viral transcription at 8 hours; also consistent with previous reports [46] . Despite the observed effects on viral transcription , these compounds contributed only minimally to viral protein expression . AZA did not affect viral transcription or translation , consistent with the mode of action of this drug which needs to be incorporated into DNA to be active and prevent methylation . This finding suggests that the cells are not dividing , which again supports the accuracy of our model . In contrast , in cell lines , which are dividing , a longer exposure allows observation of biological effects [24] . In addition , the cellular transcriptional environment was hardly affected by the various drugs , emphasizing that the cells retain a resting state [18] , [46] , [47] . Use of IL-7 contributed minimally to enhanced viral transcription or translation , despite exerting more pronounced effects on cellular transcription , consistent with recent reports on the use of this cytokine in vivo [48] . In contrast , the effect on the transcriptome and the cellular environment created by TCR stimulation strongly favored translation . Our data raise the possibility of post-transcriptional block as one of the mechanisms of HIV latency . This is consistent with studies from the Fauci laboratory that indicated that resting CD4+ T cells from aviremic patients did not produce quantifiable cell-free virions despite the presence of cell-associated HIV-1 RNA [18] , [49] . Proposed mechanisms underlying post-transcriptional blocks include splicing defects [43] , inhibition of nuclear RNA export [50] , inhibition of expression of viral proteins in a codon-usage-dependent manner [51] , and inhibition of HIV translation by microRNAs [41] , [52] . Our current work , and results from Iglesias-Ussel et al . [53] suggest that latently infected cells may have multiple biochemical and metabolic blocks that are not completely released by some of the reactivating agents currently being evaluated . Cell line models and primary CD4+ T cell models have advanced the understanding of basic mechanisms of HIV transcriptional latency and served to screen for latency reversing agents . In particular , cell lines highlighted the role of multiple cis- and trans-acting factors in the regulation of basal viral transcription [5] , [7] . Primary cell models provided a broader view on the complexity of latency and variability in response to reactivating agents [13] , [54] . Our work on the dynamics of latency and reactivation using a primary cell model underscores the existence of barriers beyond transcriptional silencing – thus helping explain clinical trials and ex vivo data that identify persistent viral transcription but impaired viral protein expression and particle production . Overcoming post-transcriptional blocks may thus require additional interventions .
We collected 25 ml of total blood from HIV-infected individuals participating in the Swiss HIV Cohort Study ( http://www . shcs . ch ) with controlled viremia ( Table S1 ) . Resting CD4+ T cells were purified by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell enrichment kit supplemented with anti-HLA-DR , anti-CD25 and anti-CD69 ( Stem Cell Technologies ) . Cells were resuspended in Opti/FCS/IL-2 , split in three wells ( approximately 1 million/ml/well ) containing DMSO , 0 . 5 µM SAHA , or TCR stimulation as described below . After 1 , 2 or 4 days of incubation , cell supernatant was collected and assessed for viral presence in a COBAS AMPLICOR analyzer ( Roche ) , with a limit of detection of 20 unspliced RNA copies/ml . Resting CD4+ T cells from HIV-infected individuals were isolated and treated for 24 h with DMSO , SAHA or TCR stimulation as indicated above . Histone extraction was performed according to Abcam's protocol . Briefly , cells were washed twice with ice-cold PBS , resuspended in Triton Extraction Buffer ( PBS–0 . 5% Triton X100–protease inhibitors–0 . 02% NaN3 ) at 107 cells/ml and incubated 10′ on ice to allow cell lysis . Cells were centrifuged at 380 g for 10′ at 4°C and washed once with Triton Extraction Buffer . After centrifugation , the pellet was resuspended at 4×107 cells/ml in 0 . 2N HCl and incubated over night at 4°C to allow for histone extraction . Samples were centrifuged at 380 g for 10′ at 4°C and the histone-containing supernatant was collected and protein concentration was measured using Qubit Protein Assay ( Life Technologies ) following manufacturer's protocol . Histone extracts ( 1 µg ) were separated on an 8–16% SDS-polyacrylamide gel , transferred to a nitrocellulose membrane and processed for immunoblotting . Briefly the membranes were washed with PBS , blocked for 2 h at RT in PBST-milk ( PBS/0 . 2% Tween-5% non fat dry milk ) , incubated over night with anti-histone antibodies ( 1∶1000 ) at 4°C , washed three times for 10′ in PBST , incubated with swine anti-rabbit-HRP ( Dako P-0217 , 1∶2000 ) for 2 h at RT , washed again and revealed using Luminata Crescendo Western HRP substrate ( Millipore ) . Primary antibodies were rabbit polyclonal from Abcam: anti-H3K27Ac ( ab4729 ) , anti-H3K9Ac ( ab10812 ) , anti-H3 ( ab1791 ) . Primary CD4+ T cells were purified by Ficoll gradient separation followed by negative selection and magnetic separation using the human CD4+ T Cell Isolation kit II ( Miltenyi Biotec ) ( Fig . S1 ) . Cells were activated using CD3/CD28 co-stimulation in presence of IL-2 ( mimicking TCR stimulation ) as described previously [10] . Briefly , anti-CD3 antibodies ( 10 µg ) were plated in 1 ml PBS per well of a 6-well plate and incubated for 1–2 h at 37°C . Wells were then washed once with 3 ml of PBS and filled with 106 cells/ml of primary CD4+ T cells supplemented with 1 µg/ml anti-CD28 antibodies in Optimizer CTS T-Cell expansion SFM culture medium containing 5% heat-inactivated fetal calf serum ( FCS ) and 100 U/ml human recombinant IL-2 ( Opti/FCS/IL-2 culture medium ) . Three days post-stimulation , cells were collected , washed and resuspended at 106 cells/ml in Opti/FCS/IL-2 for infection with an HIV-based vector ( NL4-3-Δ6-drEGFP/CXCR4; a kind gift from R . F . Siliciano ) . This HIV vector uses a CXCR4 tropic HIV envelope for entry , contains functional tat and rev genes , as well as a gfp reporter gene containing a PEST sequence ( yielding a protein half time of ∼6 hours ) ; this vector contains mutations ( stop codons , frameshift or deletion ) in gag , vif , vpr , vpu , env and nef and is thus less cytotoxic for the infected cell , thereby promoting latency in a higher proportion of infected cells [10] . HIV particles were produced by transfection of HEK293T cells as previously described [22] . Infection was carried out by spinoculation for 3 hours at 25°C and at 1500 g using 50 µg p24 equivalent HIV vector/106 cells in presence of 5 µg/ml polybrene . After spinoculation , cells were washed and resuspended at 106 cells/ml in Opti/FCS/IL-2 . Forty-eight hours post-infection , cells were resuspended in PBS/FCS ( to eliminate the interference of phenol red ) and sorted by FACS according to GFP expression , yielding a typical purity of >98% . FACS sorting was performed with a Mo-Flo Astrios instrument . Upon sorting , successfully infected GFP+ cells were further processed to investigate HIV latency . Establishment and maintenance of HIV latency was mostly carried out as previously described [19] , [20] ( Fig . S1 ) . Infected GFP+ cells stimulated using anti-CD3 and anti-CD8 antibodies in presence of IL-2 as described above were expanded in Opti/FCS/IL-2 for ∼3 weeks , typically allowing ∼2 log of cell multiplication . At this time ( week 0 ) , cells were washed , resuspended in R-10/IL-2 ( RPMI-1640 culture medium supplemented with 10% heat-inactivated FCS and 40 U/ml IL-2 ) and added to an adherent H80 feeder cell layer . The H80 human brain tumor cell line , known to promote ex vivo survival of primary CD4+ T cells , were prepared by plating 106 cells/25 cm2 flask ( T25 ) or 3×106 cells/75 cm2 flask ( T75 ) the day before starting co-culture with primary CD4+ T cells . CD4+ T cells were resuspended in R-10/IL-2 and added on top of the feeder cell layer , in 16 ml R-10/IL-2 in T75 if 30-100×106 primary CD4+ T cells or in 5 ml R-10/IL-2 if >30×106 cells . Culture medium was changed three times a week by renewing half of it . Every two weeks , CD4+ T cells were collected and transferred onto a new feeder cell layer flask . During this transfer , cell samples were collected for analyses ( see below ) . The co-culture of primary CD4+ T cells with H80 feeder cells was carried over a period of 10 weeks to allow CD4+ T cells to revert to a resting phenotype and to ensure entry into and maintenance of latency . Collected samples were used for: ( i ) assessing the expression of virally-encoded GFP and of the marker of activation IL2Rα ( CD25 ) by FACS analysis , and ( ii ) assessing dynamic changes of the cellular and viral transcriptomes using mRNA-Seq . This analysis allowed for ( a ) following the cellular dynamic process from activated to resting state at the transcriptional level in non-infected as well as infected cells , ( b ) highlighting genes differentially expressed in HIV-infected cells as compared to mock cells , and ( c ) measuring concomitant viral transcriptional activity in resting CD4+ T cells . The latency process was also repeated using H80 supernatant as alternative to cell-to-cell contact conditions . We included pharmacological and immunological agents that affect HIV transcriptional activity by different mechanisms of action and that are currently under clinical consideration . Specifically , we used a histone deacetylase inhibitor ( vorinostat or SAHA , 0 . 5 µM ) , a DNA methylation inhibitor ( 5′-azacytidine , 1 µM ) , an alcohol dehydrogenase inhibitor ( disulfiram , 0 . 5 µM ) . All drugs were suspended in dimethyl sulfoxyde ( DMSO ) , thus DMSO was used as a negative control ( of note , final DMSO concentration was 0 . 0033% corresponding to 1∶30 , 000 dilution ) . We also used immunological stimulation by interleukin-7 cytokine ( 100 ng/ml ) , and a full TCR stimulation using anti-CD3/anti-CD28 antibodies in presence of interleukin-2 . At 8 h and 24 h after treatment , cells were analyzed for GFP expression by FACS and for transcriptome composition as described below . Cell samples were stored in RNALater at the time of collection . Once all samples , from week 0 to week 10 , were collected , total RNA extraction was performed using Illustra RNAspin mini isolation kit ( GE Healthcare ) and further processed for mRNA-Seq library preparation ( TruSeq RNA sample prep kit , Illumina – that starts with capture of polyA-containing transcripts ) , followed by cluster generation ( TruSeq single-end cluster generation kit , Illumina ) and high-throughput sequencing on Illumina HiSeq2000 at the Genomics Technology Facility , University of Lausanne . Addition of RNA spike-ins were also added according to manufacturer's instructions ( ERCC Exfold RNA Spike-in Mixes , Life Technologies and Loven et al . [55] ) . We obtained about 100 mio single end reads of 100 nucleotides for each sample . The sequencing reads were cleaned before alignment in order to improve the accuracy of downstream analyses . The cleaning steps included the removal of ( i ) Illumina's adapter ( if present at the 3' end of the read ) with cutadapt v0 . 9 . 5 [56] , ( ii ) low quality ( PHRED score<6 ) nucleotides at the 3′ or 5′ end of the reads , ( iii ) reads with mean PHRED score lower than 20 and ( iv ) polyA tails with prinseq v0 . 17 [57] . Only reads of 30 nucleotides or longer after trimming were kept for further analyses . The cleaned reads were aligned to the reference genome with TopHat v2 . 0 . 6 [58] using the ensembl gene GRCh37 release 68 annotation file . The reference genome was built by concatenating the human genome ( hg19 ) and the HIV vector sequence . The number of reads per gene was extracted with a modified version of HTSeq-count v0 . 5 . 3 ( http://www-huber . embl . de/users/anders/HTSeq/doc/count . html ) using the same annotation file and considering the whole HIV vector as a single gene . The modifications allowed multiply aligned reads to be weighted accordingly . Ambiguous reads were randomly attributed to one of the genomic regions they aligned to . To identify integration sites we used Tophat fusion with bowtie1 and default parameters . Two-way analysis of variance and principle component analysis ( PCA ) were used for quantitative assessment of the transcriptome data presented in Fig . 4 . In order to fulfill the underlying assumptions of these models , we conservatively discarded lowly expressed genes to minimize heteroscedasticity due to shot noise . Retaining genes expressing at least 100 reads in more than half of the samples yielded 14 , 513 genes . The resulting dataset was smoothed by adding pseudocounts ( 10 extra reads per gene ) , and log-transformed for variance stabilization . Differential expression tests were performed assuming negative-binomially distributed read counts using the Bioconductor package DESeq [29] . Paired-sample differential expression tests were performed using generalized linear models and dispersions were estimated using the Cox-Reid-adjusted maximum likelihood estimator . Lowly expressed genes were discarded after estimating dispersion . All p-values were corrected using a false discovery rate of 5% ( Benjamini-Hochberg procedure [59] ) . Enrichment analyses were performed using Fisher's exact test to detect overrepresentations of functional classes , regulatory motifs ( among miRNA and transcription factor targets ) , physical locations on chromosomes , and HIV-related pathways , proteins and co-factors as described previously [17] . HIV co-factors include genes in HIV related pathways from the Reactome pathways database Ver . 40 , genes identified in previous siRNA studies [60]–[63] , and genes encoding protein interaction partners for each viral protein ( [28] and http://mint . bio . uniroma2 . it/virusmint ) . All p-values were corrected using a false discovery rate of 5% . For visualization in Fig . 6 , we only considered enrichment hits from Reactome pathways and HIV co-factors databases . The major pathway categories were chosen from maximal pathways , ( Pi ) , among all the reported unique enrichment hits pooled over all reactivating agents . In order to choose the most representative pathway categories , we defined the asymmetric coverage score of the ith pathway , Pi , over the jth pathway , Pj , as mi , j = |Pi ∩ Pj|/|Pj| and the total coverage of the ith pathway as Si = ∑j mi , j . The set of representative categories was built by those with the highest total coverage score Si . We used the nine top categories plus a pseudo-pathway called “miscellaneous” for the visualization . Each pathway was assigned to the major category that holds the highest coverage score over it . Subsequently , the pathways that are not covered more than 95% in any of the chosen major categories are assigned to the miscellaneous category . All data , including differentially expressed genes and enrichment analyses , are freely available in interactive mode or for downloading at http://litchi . labtelenti . org . Two splice junction aligners were used to identify HIV splicing events: Tophat v2 . 0 . 6 and STAR v2 . 3 . 0 [64] . All acceptor-donor pairs were retrieved and their proportion calculated . Both splice junction aligners gave similar and consistent results . The HIV transcriptome profile was assessed using the Python package HTSeq ( http://www-huber . embl . de/users/anders/HTSeq/doc/overview . html ) . Coverage was normalized by the sample size factor estimated using DESeq . To calculate the percentage of HIV transcripts among all transcripts , we compared the gene length normalized counts using the length of the longest annotated transcript ( isoforms with intron retention were not considered ) . The main viral RNA forms were estimated as follows . The number of reads overlapping the D1 junction corresponds to unspliced reads ( US , non-spliced in D1 ) . The number of reads aligning to the left of D1 and broken at D1 ( up to a different HIV position ) corresponds to reads spliced in D1 and thus belongs to singly spliced or multiply spliced HIV RNA ( MS+SS ) . The number of reads overlapping the D4 junction corresponds to singly spliced or unspliced HIV transcripts ( US+SS ) . The number of reads aligning to the left of D4 but broken at D4 ( up to different HIV position ) corresponds to reads spliced in D4 and thus belong to multiply spliced transcripts ( MS ) . Finally , the total proportion of reads ( 100% ) is US+MS+SS , and SS can be obtained as SS = 100%–US–MS .
|
HIV-infected individuals must receive lifelong antiviral therapy because treatment discontinuation generally results in rapid viral rebound . The field has identified a state of latency at the level of transcription of the integrated provirus as the major mechanism of persistence . A number of drugs are now tested that aim at inducing viral transcription as a step to purge the reservoir . The assessment of viral production in cells from HIV-infected individuals with optimal viral suppression revealed the failure of SAHA/vorinostat to efficiently generate viral particle production . To further investigate and characterize the process of latency at the transcriptome level , and the response to SAHA as well as various reactivating agents , we use a model of primary CD4+ lymphocytes . The main observation from this study is that viral transcripts persist during latency , and that the accumulation of viral transcripts does not result in efficient viral protein expression upon reactivation with agents such as SAHA . Our data suggest that post-transcriptional blocks also contribute to latency , and that additional strategies need to be explored to efficiently purge the viral reservoir .
|
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2014
|
Dynamics of HIV Latency and Reactivation in a Primary CD4+ T Cell Model
|
Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms . These small molecules can be used as starting points for drug discovery programs that target such mechanisms . A major challenge of the approach is the identification of the cellular targets . Here we report GNF7686 , a small molecule inhibitor of Trypanosoma cruzi , the causative agent of Chagas disease , and identification of cytochrome b as its target . Following discovery of GNF7686 in a parasite growth inhibition high throughput screen , we were able to evolve a GNF7686-resistant culture of T . cruzi epimastigotes . Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b . Cytochrome b is a component of complex III ( cytochrome bc1 ) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites , QN and QP . The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays . Additionally , the mutant cytochrome b confers resistance to antimycin A , another QN site inhibitor , but not to strobilurin or myxothiazol , which target the QP site . GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T . cruzi amastigotes with a half maximal effective concentration ( EC50 ) of 0 . 15 µM , and is highly specific for T . cruzi cytochrome b . No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686 . Our approach , which combines T . cruzi chemical genetics with biochemical target validation , can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease .
Chagas disease , or American trypanosomiasis , is a neglected disease caused by the kinetoplastid protozoan Trypanosoma cruzi ( T . cruzi ) . Endemic to Latin America , Chagas disease is increasingly globalized due to population migration from endemic regions into developed countries , and the U . S . in particular . About eight million people are estimated to harbor the infection with 40 , 000 new cases added annually [1 , 2] . In the 100+ years that have passed since the first description of Chagas disease by Carlos Chagas , only two drugs have been developed to treat this infection: nifurtimox and benznidazole [2 , 3] . While both these drugs can clear T . cruzi from the infected mammalian hosts , they are both inadequate to address the medical need of millions of patients chronically infected today . The drug shortcomings include toxicity , prolonged treatment time , and high rate of treatment failure [2 , 3] . T . cruzi is transmitted to mammalian hosts primarily via hematophagous triatomine bugs [4] . While in the vector insect , T . cruzi cells propagate as flagellated epimastigotes that transform into non-dividing infective trypomastigotes . As the T . cruzi-infected bug takes a blood meal from a host , it deposits motile T . cruzi trypomastigotes near the wound site . Following invasion of host cells in the bite wound or at mucous membranes , intracellular trypomastigotes undergo a morphological transformation into amastigotes and start to replicate [1 , 4 , 5] . After completing several rounds of intracellular division , the amastigotes transform into trypomastigotes that then leave the infected cell and initiate a new cycle of infection . The acute phase of Chagas disease is often asymptomatic , characterized by readily detectable parasitemia , and usually resolves within a few weeks through control of parasite proliferation by the adaptive immune system [4] . In the chronic stage , infected individuals rarely display symptoms or evidence of the disease for decades . However , ~30% of these patients eventually go on to develop a severe cardiomyopathy and ~10% of patients progress with gastrointestinal complications [1 , 4] . New drug discovery for Chagas disease is hampered by very limited number of validated T . cruzi drug targets . Drug discovery efforts have focused on the trypanosome ergosterol biosynthesis pathway and cruzain , a T . cruzi cysteine protease [6] . During the last decade , much attention has been paid to inhibitors of sterol 14 α-demethylase ( CYP51 ) , an essential enzyme in the ergosterol biosynthesis pathway [6 , 7] . To a large degree , this interest has been fueled by the availability of drugs targeting fungal sterol 14 α-demethylase , such as posaconazole or ravuconazole [8 , 9] . Both these drugs are exceptionally potent on T . cruzi in vitro and have been shown to effect radical parasitological cure in mouse models of Chagas disease [10] . Also , treatment with posaconazole cured T . cruzi infection in an immunosuppressed patient following benznidazole treatment failure [11] . However , a 60-day treatment with posaconazole , while transiently clearing the parasite from Chagas patients , did not prevent recrudescence of infection in a majority of patients ( 81% ) as determined by PCR . A similar trial testing the efficacy of ravuconazole prodrug E1224 has recently reported failure to cure infection in a majority ( ~70% ) of the treated Chagas patients [12 , 13] . These failures in clinical phase 2 trials have been attributed to insufficient drug exposure or dosing duration [14] . In addition to inhibitors of the parasite ergosterol biosynthesis , several inhibitors of cruzain were reported as promising candidates for treating Chagas disease . Of these , the most advanced is K777 , a vinyl sulfone peptidomimetic inhibitor [15 , 16] . K777 has been shown to be safe and efficacious in animal models of acute and chronic Chagas disease [17 , 18] and is currently undergoing preclinical development evaluation . Identification of T . cruzi growth inhibitors by phenotypic screening represents a viable alternative to target-based Chagas disease drug discovery . The approach allows efficient discovery of small molecules that perturb new molecular targets . Limitations of this approach stem from ignorance of the molecular mechanism , which precludes the use of structure-assisted drug design and prevents early predictions of toxicity through inhibition of homologous host enzymes . Chemical optimization of hit molecules in the absence of target-based activity measurements can be confounded by complex structure-activity-relationships , as biochemical activity and cellular permeability cannot be distinguished [19 , 20] . To overcome these limitations , we have established a chemical genetics approach to the determination of the mechanism of action of small molecule growth inhibitors in T . cruzi . Starting with a novel T . cruzi inhibitor GNF7686 , we evolved resistant T . cruzi mutants in vitro , and then identified the resistance-conferring mutation by whole genome sequencing . Finally , we demonstrated inhibition of the putative target in a biochemical assay . An expansion of this approach to other T . cruzi growth inhibitors could lead to identification of many additional drug targets and associated lead inhibitors for Chagas disease , and is already underway in our laboratory . As with the case of the T . cruzi cytochrome b target reported in this study , such an approach could point to drugs and drug targets from other fields , and substantially accelerate the introduction of novel Chagas disease treatments into the clinic .
With the exception of decylubiquinone ( MP Biomedicals ) , all chemicals were purchased from Sigma-Aldrich Corporation . T . cruzi CL strain was propagated in NIH/3T3 fibroblast cells . NIH/3T3 cells were grown in RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum ( FBS , Hyclone ) and 100 IU penicillin / 100 μg streptomycin ( Hyclone ) per mL at 37°C / 5% CO2 , and passaged every three to four days . To establish infection , 6 . 25 × 105 of 3T3 cells were seeded in a T-175 flask . After attachment , cells were infected with 20–40 × 106 T . cruzi trypomastigotes . Following cell infection , parasites cycled between the trypomastigote and the intracellular proliferative amastigote forms and medium was changed biweekly . T . cruzi epimastigotes were maintained in liver infusion tryptose ( LIT ) medium ( 9 g / L liver infusion broth , 5 g / L tryptose , 1 g / L NaCl , 8 g / L Na2HPO4 , 0 . 4 g / L KCl , and 1 g / L glucose , pH = 7 . 2 ) supplemented with 10% heat-inactivated FBS and 15 μM hemin , and passaged every five days during middle to late logarithmic growth phase ( maintained at 26°C / 0% CO2 ) . For differentiation of epimastigotes into trypomastigotes , saturated cultures of T . cruzi CL epimastigotes were harvested by centrifugation ( 1 , 000 × g for 10 min at 10°C ) , resuspended in artificial triatomine urine medium ( TAU; 190 mM NaCl , 17 mM KCl , 2 mM MgCl2 , 2 mM CaCl2 , 0 . 035% NaHCO3 , 8 mM phosphate buffer , pH 6 . 9 ) at a density of 5 x 108 cells / mL , and incubated at 26°C . Two hours later , the parasites were transferred to TAU 3AAG medium ( TAU supplemented with 10 mM L-proline , 50 mM sodium L-glutamate , 2 mM sodium L-aspartate and 10 mM D-glucose ) in T-25 culture flasks with a layer of culture medium approximately 1 cm in depth . This cell density was previously shown to be the optimal density for epimastigote differentiation [21 , 22] . After 72 hour incubation , the mixture of epimastigotes and newly differentiated trypomastigotes was used for infection of mammalian host cells ( NIH/3T3 mouse embryonic fibroblast line ) . Briefly , NIH/3T3 cells were plated at a density of 0 . 025 million cells / mL into T-175 flasks and infected with 5 mL of pelleted epimastigote / trypomastigote mixture ( collected from 25 mL of transformed culture ) resuspended in RPMI-1640 medium supplemented with 10% FBS and 100 IU penicillin / 100 μg streptomycin per mL . After 24 hour incubation , non-internalized extracellular epimastigotes and trypomastigotes were removed , and infected NIH/3T3 cells were cultured for additional seven days . By then , newly formed trypomastigotes released from infected NIH/3T3 host cells were present in the medium and further used as described in the “T . cruzi in vitro efficacy assays” section . To determine compound efficacy on T . cruzi intracellular amastigotes , NIH/3T3 cells were seeded ( 1 , 000 cells / well , 40 μL ) into microscopy-grade , clear bottom , 384-well plates ( Greiner ) in RPMI-1640 medium containing 5% heat-inactivated fetal bovine serum and 100 IU penicillin / 100 μg streptomycin per mL . Plates were incubated overnight at 37°C / 5% CO2 . Cells were infected with T . cruzi trypomastigotes at a multiplicity of infection ( MOI ) of 10 for the wild-type strain , and 20 for the GNF7686-resistant mutant strain . After six hours of infection ( at 37°C / 5% CO2 ) , the plates were washed by aspirating the medium and replacing with fresh screening medium to remove remaining extracellular trypomastigotes . The plates with infected cells were incubated overnight ( 37°C / 5% CO2 ) and compounds dissolved in DMSO were added to plate wells on the following day ( 0 . 2% DMSO final concentration ) . After 48-hour compound treatment , infected cells were fixed ( 4% paraformaldehyde in phosphate-buffered saline containing 0 . 5 mM CaCl2 and 0 . 5 mM MgCl2 ) , permeabilized ( 0 . 1% Triton X-100 in PBS ) , and then stained using a 1:125 , 000 dilution ( prepared in PBS ) of SYBR green ( Life Technologies ) . The plates were then scanned using the Evotec Opera High Content Screening System ( Perkin Elmer ) and amastigote proliferation was assessed by counting parasites within the 3T3 cells using CellProfiler version 2 . 1 . 0 cell image analysis software [23] . In some experiments , an alternative protocol for measurement of compound activity on intracellular T . cruzi was used [24] . To determine compound activity on the T . cruzi epimastigote form , epimastigotes ( 20 μL; 2 . 5 × 105 parasites / mL ) were added to 384-well assay plates containing 20 μL of LIT media with pre-dispensed compounds ( 0 . 2% DMSO final concentration ) and incubated for seven days at 26°C / 0% CO2 . Parasite viability was assessed at the end of this incubation period using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . Luminescence as a measure of parasite viability was measured on the EnVision plate reader . To assess compound efficacy on trypomastigotes , parasites ( 30 μL; 2 × 106 trypomastigotes / mL ) were added to 384-well plates containing 10 μL of RPMI-1640 without phenol red ( Invitrogen ) and supplemented with 10% FBS and 100 IU penicillin / 100 µg streptomycin per mL , and then treated with compounds ( 0 . 2% DMSO final concentration ) . Following a 48-hour incubation period , viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . Leishmania donovani ( L . donovani ) axenic amastigotes ( strain MHOM/SD/62/1S-CL2D ) were maintained at 37°C / 5% CO2 in RPMI-1640 medium containing 4 mM L-glutamine , 20% heat inactivated FBS , 100 IU penicillin / 100 μg streptomycin per mL , 23 μM folic acid , 100 M adenosine , 22 mM D-glucose , and 25 mM 2- ( N-morpholino ) ethanesulfonic acid ( pH 5 . 5 adjusted at 37°C using 1M HCl ) . For compound screening , axenic amastigotes were seeded into 384-well plates containing axenic amastigote medium with pre-dispensed compounds ( 0 . 25% final DMSO concentration ) at a density of 9 , 600 cells / well . Plates were incubated for 48 hours at 37°C / 5% CO2 . Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . Trypanosoma brucei brucei ( T . b . brucei ) bloodstream form ( strain Lister 427 ) was maintained in HMI-9 medium: IMDM ( Iscove's Modified Dulbecco's Media ) , 10% heat-inactivated FBS , 10% Serum Plus medium supplement ( SAFC biosciences ) , 1 mM hypoxanthine , 50 μM bathocuproine disulfonate . Na2 , 1 . 5 mM cysteine , 1 mM pyruvate , 39 μg/mL thymidine , and 0 . 2 mM 2-mercapthoethanol ) at 37°C / 5% CO2 . For compound screening , parasites were seeded into 384-well assay plates containing HMI-9 with pre-dispensed compounds ( 0 . 25% final DMSO concentration ) at a density of 6 , 000 cells / well , and plates were then incubated for 48 hours at 37°C / 5% CO2 . Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . For conversion from the bloodstream form to the procyclic form , bloodstream form parasites were transferred from HMI-9 medium to Differentiating Trypanosome Medium ( DTM , pH = 7 . 2 ) , consisting of: 6 . 8 g / L NaCl , 400 mg / L KCl , 200 mg / L CaCl2 , 140 mg / L NaH2PO4 . H2O , 200 mg / L MgSO4 . 7H2O , 7 . 94 g / L HEPES , 2 . 2 g / L NaHCO3 , 110 mg / L sodium pyruvate , 10 mg / L phenol red , 14 mg / L hypoxanthine , 1 mg / L biotin , 760 mg / L glycerol , 640 mg / L proline , 236 mg / L glutamic acid , 1 . 34 g / L glutamine , 7 . 5 mg / L hemin ( in 50 mM sodium hydroxide ) , 1X MEM amino acid solution ( Invitrogen ) , 1X MEM non-essential amino acids solution ( Invitrogen ) , 28 . 2 mg / L bathocuproine disulfonate . Na2 , 182 mg/L cysteine , 0 . 2 mM 2-mercaptoethanol , 15% heat-inactivated FBS , and 5 mM sodium citrate and cis-aconitate [21 , 22] . Following medium exchange , parasites were incubated at a lower temperature ( 27°C / 5% CO2 ) , monitored for change in morphology to procyclic parasites , and sub-cultured for long-term cultivation . For compound screening , 20 μliters ( 5 , 000 parasites ) of T . b . brucei procyclic culture were added to 384-well plate wells filled with 20 μliters of DTM medium and pre-dispensed compounds ( 0 . 25% final DMSO concentration ) . Plates were then incubated for 72 hours at 27°C / 5% CO2 . Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . GNF7686 and cytochrome b inhibitors were assayed for activity on two Plasmodium falciparum ( P . falciparum ) lines: D10attB and yDHODH-D10attB . The D10attB line is reliant on the coenzyme Q-dependent malarial dihydroorotate dehydrogenase ( PfDHODH ) , whereas the yDHODH-D10attB line expresses also the fumarate-utilizing Saccharomyces cerevisiae ( S . cerevisiae ) DHODH , which circumvents reliance on PfDHODH and renders this line fully resistant to cytochrome b inhibitors [25] . The use of these two lines allows for distinction of selection of potent cytochrome b inhibitors as described in detail in the Results section [26 , 27] . Growth and viability of P . falciparum cell lines ( in the presence or absence of drug ) in infected erythrocytes were assessed using a SYBR Green-based proliferation assay exactly as described previously [28] . NIH/3T3 fibroblast cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 100 IU penicillin / 100 μg streptomycin per mL at 37°C / 5% CO2 . For compound screening , cells were diluted to 4 × 104 cells / mL in assay medium ( RPMI-1640 , 5% FBS , and 100 IU penicillin / 100 μg streptomycin per mL ) and seeded at 50 μL / well into 384-well plates . Following overnight incubation , compounds were added to each well ( 0 . 2% DMSO final concentration ) and plates were further incubated for 96 hours . Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay ( Promega ) . Measured luminescence values were normalized to the value obtained for 0 . 2% DMSO , and plotted against the corresponding compound concentration for half maximal cytotoxic concentration ( CC50 ) value determination . The minimal inhibitory concentrations ( MIC ) of the compounds for inhibition of S . cerevisiae drug pump knock-out strain NF7061 ( MATa his3Δ 1; leu2Δ 0; met15Δ 0; ura3Δ 0; snq2::KanMX; pdr5::KanMX; pdr1::NAT1; pdr3::KanMX; yap1::NAT1; pdr2::LEU2; yrm1::MET; yor1::LYS2 ) were determined by a modification of the microdilution technique described elsewhere [29] . Briefly , two-fold dilutions of the compound solution in DMSO were made . They were added to each well of 96-well assay plate containing 200 μL per well of either YPD ( 1% Difco Yeast Extract , 2% Difco Bacto Peptone and 2% Dextrose ) or YPG ( 3% of glycerol in replacement of 2% Dextrose in YP ) medium . Early stationary yeast NF7061 cells grown in either medium were collected and resuspended to 2 × 105 cells / mL . Ten microliters of the yeast cell suspension were inoculated to each well of the plates containing medium and compound to achieve a final inoculum of approximately 104 CFU / mL . The plates were incubated at 30°C for 24 hours ( containing YPD ) or 48 hours ( containing YPG ) . The MIC end point was defined as the lowest compound concentration exhibiting no visual growth . T . cruzi epimastigotes were initially treated with GNF7686 at EC20 value ( 0 . 01 μM , 0 . 2% DMSO ) and continually passaged at the same concentration until the culture growth rate matched that of epimastigotes growing in the medium containing 0 . 2% DMSO . Parasites were subsequently passaged in a similar manner in the presence of increasing concentration of GNF7686 until significant resistance was achieved ( ~5-fold increase in the EC50 value ) . The time to generate resistance was approximately eleven months . Resistant epimastigotes were cloned by the limiting dilution technique . Following expansion of GNF7686-resistant T . cruzi clones in LIT media , T . cruzi total DNA was isolated using Qiagen DNeasy Blood and Tissue Kit from 108 parasites per sample . Whole genome sequencing was performed using Ilumina HiSeq1000 next-generation sequencing platform . Sequencing reads were aligned by Burrows-Wheeler Aligner ( BWA , version 5 . 9 . 0 ) to the T . cruzi JR cl . 4 genome ( version 1 . 0 ) . Simple single nucleotide variants ( SNVs ) were called ( using Samtools 1 . 19 ) looking for SNVs or small indels with an overall quality > 100 where the control was the drug-sensitive parental CL clone . Approximately 600900 reads called a ‘T’ and 520 reads called a “G” at L197F position resulting in L197F mutation . Heterozygous calls were determined by Samtools , and verified in the Integrated Genomics Viewer . Putative SNVs were manually checked in IGV [30 , 31] . To further confirm the presence of L197F mutation , the T . cruzi cytochrome b gene was amplified by PCR ( forward primer 5’-AGCTACTGTTCCTGTATTCGGC-3’ and reverse primer 5’-ACAAAAACAAAGTCGCTCACAA-3’ ) and cloned into the pCR2 . 1 vector . The insert DNA was sequenced using M13R and M13F ( -21 ) primers ( Genewiz ) . GNF7686 and cytochrome b inhibitors ( 0 . 2% DMS0 final concentration ) were added to 384-well assay plates containing 20 μL of assay buffer ( 250 mM sucrose , 15 mM KCl , 5 mM MgCl2 , 1 mM EGTA , 30 mM K2HPO4 , pH 7 . 4 ) and allowed to dissolve for two hours . Meanwhile , T . cruzi epimastigotes were harvested ( 800 × g for 5 min at 4°C ) , washed twice with buffer A ( 10 mM Tris-HCl , pH = 7 . 4 , 0 . 23 M mannitol , 0 . 07 M sucrose , 0 . 2 mM EDTA , 0 . 2% bovine serum albumin , 0 . 5 mM phenylmethanesulfonyl fluoride ) , and finally resuspended in buffer A at a final concentration of 150 × 106 epimastigotes / mL . Next , 20 μL of T . cruzi epimastigote suspension ( 3 x 106 parasites ) was added to each sample well , and then 15 μL of MitoXpress-Xtra probe ( Cayman Chemicals ) was added ( final assay volume of 60 μL ) . Probe phosphorescence is quenched in the presence of oxygen and is inversely proportional to the amount of oxygen present in the solution . HS mineral oil ( 20 μL , Cayman Chemicals ) was added to wells to prevent oxygen exchange between the assay buffer and air . Blank wells containing assay buffer without parasites , corresponding compound , and mineral oil were prepared in parallel and values were subtracted from sample values to specifically measure changes in oxygen concentration caused by parasite respiration . All sample and blank wells were prepared in duplicate . The assay plate was transferred to a Gemini XPS fluorescence plate reader ( Molecular Devices ) and read at 3 minute intervals at excitation and emission wavelengths of 380 nm and 650 nm , respectively , for 30 minutes at 37°C . The slope for the linear portion of time course of fluorescence increase ( rate of oxygen consumption ) was calculated after subtraction of blank well values . Obtained slope values were normalized to the slope obtained for 0 . 2% DMSO , and plotted against the corresponding compound concentration for half maximal inhibitory concentration ( IC50 ) value determination . T . cruzi epimastigotes ( 57 day old culture , density of 5 × 107 parasites / mL ) were harvested by centrifugation ( 800 × g for 5 minutes at 4°C ) and resuspended at 10 mg / mL of protein in buffer A containing 0 . 1 mg digitonin / mg protein . The parasites were then incubated with the detergent for 10 minutes at 26°C . The pellet fraction was collected by centrifugation ( 13 , 000 × g for 5 min at 4°C ) and immediately used in the complex III assay . Complex III activity was monitored using a coupled decylubiquinol / cytochrome c reaction [32 , 33] . Decylubiquinone was first reduced to decylubiquinol as described [33] . The freshly reduced decylubiquinol ( 80 μM final concentration in the reaction ) was added to a reaction buffer ( 25 mM potassium phosphate , 5 mM MgCl2 , 2 . 5 mg / mL BSA , pH = 7 . 2 ) containing 1 mM KCN , 0 . 1 mM yeast cytochrome c , 0 . 6 mM n-D-B-maltoside , and 12 μg of digitonin-solubilized T . cruzi epimastigotes . The reduction of cytochrome c was monitored at 550 nm using the SpectraMax Plus384 absorbance microplate reader . Blank samples containing all components excluding decylubiquinol were processed in parallel and absorbance values were subtracted from sample absorbance values to specifically measure decylubiquinone-dependent reduction of cytochrome c . All sample and blank wells were prepared in triplicate . For IC50 determination , the slope of the linear portion of the corrected respiration trace was determined , and normalized to the slope obtained for 0 . 2% DMSO condition . Sprague Dawley rats were euthanized and skeletal muscle ( from hind legs ) was removed and stored in CP1 buffer ( 0 . 1 M KCl , 0 . 05 M Tris , 2 mM EGTA , pH 7 . 4 at 4°C ) on ice . Using the ‘Herb Mincer‘ , tissue was minced 34 times and then transferred into CP1 buffer ( on ice ) to wash away fatty and connective tissues from muscle . Following two rounds of wash and decantation with CP1 buffer , the rinsed tissue was transferred to CP2 buffer ( CP1 + 0 . 5% BSA , 5 mM MgCl2 , 1 mM ATP , 250 units / 100 mL Protease Type VIII ( Sigma P5380 ) , pH 7 . 4 at 4°C ) and incubated on ice for 5 minutes prior to homogenization using the Polytron PT3100 . Following homogenization and centrifugation ( 500 × g for 10 min at 4°C ) , the supernatant was decanted using cheesecloth and further centrifuged ( 10 , 000 × g for 11 min at 4°C ) . The crude mitochondrial pellet was resuspended in 10 mL of CP1 buffer ( carefully avoiding red blood cell pellet ) and subjected to an additional centrifugation step ( 10 , 000 × g for 10 min at 4°C ) . The resulting pellet was again separated from the red blood cell pellet and centrifuged ( 600 x g for 6 min at 4°C ) . The supernatant containing resuspended mitochondria was subjected to one final round of centrifugation ( 5 , 000 × g for 11 min at 4°C ) and the pelleted mitochondria were resuspended in CP1 buffer and stored at -80°C . Respiration in isolated rat mitochondria was measured using the same protocol as described for T . cruzi epimastigotes using 150 μg protein / sample .
GNF7686 ( Fig 1A ) was identified in a high throughput screen designed to find new small molecules with growth inhibition activity on L . donovani axenic amastigotes . A library of 700 , 000 compounds was assembled with a particular focus on drug-like properties and structural diversity and these compounds were tested for inhibitory activity on L . donovani at 4 μM concentration . The library has been previously profiled in more than 60 other high throughput screens , including biochemical and cell-based assays against human and pathogen targets . The screen history allowed rapid identification and elimination of compounds with a ‘frequent hitter’ property . The screen yielded 2 , 306 primary hits ( 0 . 3% hit rate ) that inhibited growth by > 50% . Data from more than 95% of the assay plates had Z′ > 0 . 7 , using DMSO as the negative control and 5 μM pentamidine as the positive control . Primary hits from the screen were further characterized using a dose−response assay format to determine the EC50 values . In parallel , cytotoxicity of these compounds was determined against a proliferating mouse fibroblast cell line ( NIH/3T3 ) . The final set of condirmed hits consisted of compounds that had EC50 < 4 μM against L . donovani , as well as low or no 3T3 cytotoxicity ( CC50 > 10 μM or SI > 10; SI = CC50/EC50 ) . The final set of confirmed L . donovani hits consisted of 1003 inhibitors . The confirmed hits were further assayed for activity on other two medically important kinetoplastid parasites T . cruzi and T . brucei . GNF7686 was selected for further investigation because of potent in vitro activity on all three T . cruzi morphological forms ( intracellular amastigote EC50 = 0 . 15 μM , trypomastigote EC50 = 0 . 71 μM , epimastigote EC50 = 0 . 16 μM; Table 1 and Fig 1C ) . GNF7686 also inhibited the growth of L . donovani axenic amastigotes ( EC50 = 0 . 46 μM ) and promastigotes ( EC50 = 0 . 46 μM ) , but not the growth of T . b . brucei bloodstream form trypomastigotes ( EC50 > 25 μM ) . Curiously , GNF7686 was active on T . b . brucei procyclics ( EC50 = 0 . 59 μM ) , the parasite form found in the tsetse fly vector that mediates T . b . brucei transmission [34] . GNF7686 did not inhibit growth of 3T3 cell line ( CC50 > 20 μM ) . To investigate the mechanism of action of GNF7686 , we selected a population of drug-resistant T . cruzi epimastigotes through a long-term parasite culture in the presence of this compound . As tolerance for GNF7686 gradually increased over time , we periodically escalated the selection pressure by raising the inhibitor concentration . In the course of eleven months , the EC50 of the T . cruzi culture shifted ~ 4-fold from 0 . 16 µM to 0 . 73 μM ( Table 1 ) . Populations of evolving microbes often comprise cells harboring alternative mutations that are derived from different mutation events [35 , 36] . To simplify analysis of genomic changes accumulated during the selection by characterizing homogenous culture populations , we isolated three clones from the GNF7686-resistant culture . All three clones exhibited the same extent of GNF7686 resistance ( EC50 ~ 1 μM ) as the parent culture . Importantly , the sensitivity of clones to benznidazole remained at the same level as observed for the wild-type T . cruzi strain ( Table 1 ) , demonstrating that resistance to GNF7686 did not arise through a broadly pleiotropic mechanism . When cultured in medium lacking GNF7686 , all three clones grew at a rate similar to the wild-type strain ( mutant epimastigote doubling time = 5355 hours vs 60 hours for the wild-type strain ) , but reached stationary phase at a lower cell density ( ~60% of the wild-type strain , Fig 1B ) . Whole genome sequencing identified the same set of five mutations in all three clones that included L197F in cytochrome b , L283M in the ATPase subunit of HsIVU protease , R75C in TCSYLVIO008926 hypothetical protein , and two mutations in non-coding regions . The observation that the three clones were identical at the genome sequence level suggests that they were siblings derived from one founding cell that expanded in the passaged culture during the selection . During whole genome sequencing , multiple sequence reads ( up to 100 in total ) from many independent DNA molecules are obtained for each nucleotide position in the genome . We uncovered that the L197F mutation in the cytochrome b gene and one of two mutations mapped to non-coding regions fully replaced the corresponding wild-type alleles , while the other three mutations remained heterozygous during the selection . Interestingly , the two mutations map both to the kinetoplast maxicircle DNA , which is present in 2050 copies per cell [37 , 38] . This indicates that these two mutations , presumably appearing on one maxicircle copy at first , replaced the corresponding wild-type alleles during the selection to the point of achieving homoplasmy . In addition to selection of T . cruzi mutants resistant to GNF7686 , we subjected the inhibitor also to chemogenomic profiling in S . cerevisiae . The genome-wide deletion collections available for this eukaryotic model system provide powerful genetic tools for investigation of bioactive molecules [39 , 40] , and the approach was successfully applied to mechanism of action studies of various growth inhibitors in the past [41 , 42] . In the haploinsufficiency profiling assay ( HIP ) , complete collection of heterozygous yeast deletion strains , in which each strain has only one copy of a particular gene , is profiled for hypersensitivity to a compound . Gene deletions associated with increased compound sensitivity indicate pathways directly affected by the compound [43] . We observed that growth of S . cerevisiae is inhibited when the yeast was cultured in media containing glycerol but not glucose as the carbon source ( see also below ) . We therefore conducted a HIP profiling experiment in medium containing glycerol . Testing of GNF7686 at its EC30 concentration in two independent HIP experiments resulted in a reproducible profile ( Fig 2A ) . Identified hypersensitive heterozygous strains included those with deletions in genes involved in mitochondrial metabolism , such as CYT1 ( cytochrome c1 ) , HAP4 ( a transcription factor involved in regulation of the respiratory chain including CYT1 ) , CBP1 ( a regulator of cytochrome b mRNA stability ) , and QCR6 ( a subunit of the cytochrome c reductase complex ) [44] . As all these hits pointed at inhibition of mitochondrial respiration by GNF7686 , we performed additional HIP experiments with strobilurin , an inhibitor of cytochrome bc1 complex , and venturicidin , an inhibitor of F-type ATPase [45 , 46] . In a control experiment , we also collected the HIP profile for benomyl , a microtubule binding inhibitor , which does not interfere with ATP production during oxidative phosphorylation . While both venturicidin and benomyl yielded HIP profiles distinctly different from that of GNF7686 ( Fig 2C and 2D ) , treatment of gene deletion strain collection with the cytochrome bc1 inhibitor strobilurin identified essentially the same set of sensitive , heterozygous mutants as GNF7686 and included CYT1 , HAP4 , CBP1 and QCR6 ( Fig 2B ) . It is important to note that the gene coding for cytochrome b , which is the proposed direct target of strobilurin [47] , is encoded by the mitochondrial genome in S . cerevisiae and not amenable to standard gene targeting protocols . Thus , the cytochrome b gene deletion strain is not present in the yeast heterozygous deletion pool and could not be directly identified by the HIP method . The observed HIP compound profiles strongly suggested that GNF7686 directly interferes with function of the S . cerevisiae respiratory chain , possibly at the level of complex III . Genomic analyses of GNF7686 resistance/sensitivity pointed to involvement of the T . cruzi cytochrome b in resistance to growth inhibition by GNF7686 . Cytochrome b is a component of the multisubunit cytochrome bc1 complex , an asymmetric homodimer with two spatially separated catalytic sites QN and QP ( Fig 3A ) . In concert , QN and QP catalyze oxidation of ubiquinol formed by preceding steps of the respiratory chain [48 , 49] . Inhibitors of cytochrome b are already of interest as anti-parasitic drugs . Atovaquone , a hydroxy-naphthoquinone inhibitor of the QP site , is used in the treatment of malaria and fungal pneumonia [50] . Another hydroxy-naphthoquinone , buparvaquone , is used to treat cattle theileriosis , and potently inhibits growth of L . donovani [19 , 51] . Surprisingly , the potential of cytochrome b inhibitors for treatment of Chagas disease has not been explored , even though cytochrome b inhibitors including antimycin A were shown to affect T . cruzi mitochondrial respiration , bioenergetics , and calcium homeostasis [52–55] . To assess the effect of this class of compounds on T . cruzi growth and survival , we tested prototypical QN and QP site inhibitors on intracellular amastigotes , trypomastigotes , and epimastigotes ( inhibitor structures shown in Fig 1A ) [46 , 49] . The QN site inhibitor antimycin A potently inhibited the growth of epimastigotes and rapidly reduced viability of trypomastigotes . We also observed T . cruzi inhibition by compounds targeting the cytochrome b QP site . Two well-characterized QP site inhibitors , myxothiazol and strobilurin , blocked growth of epimastigotes , and reduced viability of trypomastigotes ( Table 1 ) . The effect of antimycin A , myxothiazol , and strobilurin on intracellular amastigotes could not be accurately determined because of the inhibitor toxicity on the host 3T3 cells . T . b . brucei is a kinetoplastid parasite closely related to T . cruzi at the genomic level [56] . While the parasite bloodstream ( mammalian ) form of T . b . brucei relies primarily on the glycolytic pathway for ATP production , growth of the procyclic ( insect ) form requires activity of the conventional respiratory pathway , including cytochrome b [48] . In line with the hypothesis that GNF7686 is a cytochrome b inhibitor , GNF7686 inhibited the growth of the procyclic but not bloodstream T . b . brucei parasites ( EC50 = 0 . 59 μM vs > 25 μM ) . We also observed similar differential activity on the two T . brucei forms with the other tested cytochrome b inhibitors such as antimycin A ( EC50 = 0 . 03 μM vs > 25 μM; Table 2 ) . The inhibition of various morphological forms of T . cruzi by prototypical cytochrome b inhibitors is consistent with the hypothesis that the cytochrome b fulfills an essential function in parasite physiology and that GNF7686 inhibits its function . However , with the exception of antimycin A , the anti-parasitic potency of the other tested inhibitors is too weak to be of therapeutic significance . Through a literature search , we identified a previously published report that described L198F mutation in S . cerevisiae cytochrome b , which is equivalent to the L197F mutation in T . cruzi cytochrome b ( Fig 3B ) . The yeast L198F mutation confers resistance to ilicicolin H , a cytochrome b inhibitor with potent anti-fungal activity [46 , 57] . Inspection of high resolution crystal structure of the yeast cytochrome bc1 complex further revealed that the Leu198 side chain is positioned in close proximity ( < 5 Å ) to ubiquinol bound inside the QN pocket and next to His197 , which coordinates the iron atom in the bH heme . In accordance with the structure , L198F mutation also conferred resistance in yeast to other tested QN site inhibitors such as funiculosin and antimycin A [57 , 58] . Additional characterization of the GNF7686-resistant T . cruzi mutants revealed that they were selectively resistant to antimycin A , a QN site inhibitor [46] . While antimycin A displayed very potent activity on wild type epimastigotes , a sharp decrease in potency ( 40-fold ) was observed with GNF7686-resistant epimastigotes ( EC50 = 1 . 8 μM , Table 1 ) . Similarly , a steep shift in potency ( 20-fold ) was observed between the wild-type and mutant trypomastigotes ( Table 1 ) . In contrast , QP inhibitors myxothiazol and strobilurin showed comparable activity on both wild-type and resistant strains ( Table 1 ) . In summary , the L197F mutation in the T . cruzi cytochrome b is likely located within the QN site and can interfere with binding of QN site inhibitors in a similar way as was previously described for the L198F mutation in the S . cerevisiae cytochrome b . During aerobic respiration , the electron transport chain ( ETC ) conducts electrons derived from reduced carbon substrates through a series of redox reactions to the terminal electron acceptor , molecular oxygen , which is then reduced to water [33 , 48] . Inhibition of electron flow through the ETC at any step , including cytochrome b , results in a block of oxygen consumption . To evaluate whether GNF7686 disrupts the function of the T . cruzi ETC , oxygen consumption by intact T . cruzi epimastigotes was monitored in the presence of GNF7686 and prototypic cytochrome b inhibitors ( Fig 4A and 4B ) . Antimycin A potently blocked respiration by wild-type epimastigotes ( IC50 = 0 . 04 μM ) , but was ~7-fold less potent on GNF7686-resistant parasites ( IC50 = 0 . 27 μM ) . Two QP site inhibitors employed in this report , myxothiazol and strobilurin , both inhibited epimastigote respiration , but , in contrast to antimycin A , the respiratory IC50 values of the QP inhibitors were comparable between wild-type and GNF7686-resistant T . cruzi . GNF7686 inhibited respiration by wild-type parasites with an IC50 = 0 . 21 μM . A significant drop in inhibitor potency was observed with the GNF7686-resistant T . cruzi epimastigotes ( oxygen consumption IC50 = 5 . 2 μM ) . As seen for growth inhibition , the results on respiration inhibition distinguish GNF7686 and antimycin A from the QP site inhibitors ( Fig 4B ) . We then asked whether GNF7686 inhibits T . cruzi cytochrome bc1 ( complex III ) directly ( Fig 4C ) . Epimastigotes were permeabilized with digitonin , and KCN ( complex IV inhibitor ) was added to the permeabilized cells to block electron conductance by the parasite ETC . The reaction was then initiated by adding stoichiometric quantities of decylubiquinol ( an electron donor for complex III ) and oxidized yeast cytochrome c ( an electron acceptor ) , and the catalytic activity of complex III was monitored through accumulation of reduced yeast cytochrome c . A control reaction with antimycin A confirmed earlier observations with intact epimastigotes ( Table 1 ) . Antimycin A potently blocked reduction of cytochrome c in the reaction with wild-type permeabilized epimastigotes , but a dramatic loss of inhibitor potency was observed ( ~100-fold ) when GNF7686-resistant permeabilized parasites were used ( Fig 4C ) . In a similar fashion , GNF7686 inhibited wild-type complex III activity with an IC50 of 0 . 40 μM , but a 20-fold loss of potency was observed with the mutant complex III ( Fig 4C ) . These observations validate GNF7686 as a complex III inhibitor that likely targets the QN site of the T . cruzi cytochrome b . The inhibitory effect of GNF7686 on mammalian cytochrome b function was assessed through monitoring oxygen consumption by mitochondria isolated from rat skeletal muscle cells . In the control reaction , antimycin A showed a potent inhibition ( IC50 = 0 . 81 μM ) of mitochondrial respiration ( Fig 4D ) . In a parallel experiment , GNF7686 did not show any effect on oxygen consumption up to 25 μM concentration ( Fig 4D ) . This observation validates GNF7686 as a highly selective inhibitor of the T . cruzi cytochrome b and a promising starting point for Chagas disease drug discovery . We also examined effect of GNF7686 on cytochrome b in the malaria parasite , P . falciparum , and in yeast S . cerevisiae , the latter being used as a surrogate for pathogenic Pneumocystis jirovecii , a causative agent of a pneumocystis pneumonia [59] . Cytochrome b is a validated drug target in both organisms and atovaquone , an inhibitor of cytochrome b , is a clinical treatment for these diseases . For the P . falciparum studies , all inhibitors were tested on two parasite lines—D10attB and yDHODH-D10attB ( Table 2 ) . In the wild-type D10attB line , the parasite de novo pyrimidine biosynthesis is dependent on a type 2 dihydroorotate dehydrogenase ( PfDHODH ) and requires a functional P . falciparum ETC , including cytochrome b , downstream from PfDHODH [26 , 27] . In contrast , the yDHODH-D10attB cell line is modified with a type 1A dihydroorotate dehydrogenase from S . cerevisiae ( yDHODH ) , which is cytosolic and utilizes fumarate as the terminal electron acceptor [25–27] . The assay was validated with antimycin A , which blocked growth of the D10attB line ( EC50 = 0 . 7 μM ) , but was inactive on the yDHODH-D10attB parasite line ( EC50 > 12 . 5 μM ) . Myxothiazol and strobilurin also showed a similar preferential activity on the D10attB line , whereas GNF7686 did not inhibit growth of either P . falciparum cell line . This result suggests that GNF7686 is not active on P . falciparum cytochrome b . A similar , growth inhibition-based assessment of GNF7686 effect on the ETC function was also performed on S . cerevisiae ( Table 2 ) . Growth inhibition of a wild-type S . cerevisiae strain by compounds in two different media was monitored . The first medium contained glucose as the sole carbon source , which allows growth of yeast cells that lack a functional ETC [60] . In the second medium , glucose was replaced with glycerol , a non-fermentable carbon source . Under the latter condition , yeast growth is dependent on cellular respiration and functional cytochrome b [60] . All prototypic cytochrome b inhibitors used in this report potently inhibited yeast growth on medium with glycerol , but were inactive when yeast grew in medium with glucose . Following a similar pattern , GNF7686 weakly inhibited growth of yeast in glycerol medium ( EC50 = 5 . 0 μM ) , but did not affect yeast growing in the medium with glucose . In summary , GNF7686 selectively inhibits T . cruzi cytochrome b and does not affect respiration of mammalian mitochondria nor does it significantly inhibit respiration-dependent growth of P . falciparum and S . cerevisiae .
We have shown that cytochrome b is a possible target for new drug discovery efforts aimed at treating kinetoplastid diseases . The importance of this finding is underlined by the paucity of drug targets for these diseases . For Chagas disease , only sterol 14α-demethylase ( CYP51 ) and cruzain have been explored in depth as possible targets . However , low parasitological cure rates that were observed ( 20–30% ) during clinical testing of anti-fungal drugs targeting sterol 14α-demethylase ( posaconazole and ravuconazole prodrug E1224 ) in Chagas disease patients have lessened enthusiasm for further work on repurposed CYP51 inhibitors as single agents [61 , 62] . Additional clinical evaluation of this class of drugs partnered with benznidazole for combination treatments is still planned . It is also important to note that the failure of posaconazole and E1224 in phase 2 trials have been attributed to insufficient drug exposure or dosing duration [14] , and work on T . cruzi-specific CYP51 inhibitors that could enter clinical development in the future is also ongoing [63 , 64] . Finally , the anti-cancer drug BEZ235 has activity across kinetoplastid parasites , but it requires additional optimization ( improvement of therapeutic index ) before becoming a preclinical candidate [65] . Given this sparse landscape , new chemical starts and new drug targets are urgently needed to anchor drug discovery efforts for the kinetoplastid diseases . Many groups have resorted to a ‘pre-genomic’ approach to drug discovery , in which compounds are screened to identify inhibitors of pathogen growth , without regard to mechanism of action . While this approach typically provides large number of chemical starting points and broad hit diversity , a lack of information on mechanism of action creates additional risk in chemical optimization , and in predicting possible toxicity liabilities . Next generation sequencing of evolved resistant pathogens has been used successfully to identify resistance mechanisms and , in many cases , target mechanisms , for several pathogens . In our own program , we have identified targets for malaria and tuberculosis [66 , 67] . However , the approach has not been reduced to practice in kinetoplastid drug discovery until this study . Several features of the results in this study bode well for future application of the approach . First , the number of mutations associated with the emergence of drug resistance was relatively low ( five point mutations ) , which simplified subsequent target prediction and validation . Second , we were able to generate resistance in T . cruzi epimastigotes despite a relatively long doubling time and stable genome . Selection process required almost a year of drug pressure in this study but was ultimately successful . Finally , we found a mutation in a gene that is a ‘plausible’ drug target , where plausibility is supported by essentiality of the mutated gene in other cellular systems , or precedence from drugs targeting homologous targets in other organisms . Drugs targeting cytochrome b are in clinical use for treatment of malaria and fungal pneumonia , and cytochrome b was also reported as a promising target for treatment of tuberculosis . The current study extends utility of cytochrome b as a drug target also to Chagas disease , and possibly leishmaniasis . Various structurally different cytochrome b inhibitors showed patterns of growth and biochemical inhibition that consistently confirmed that functional cytochrome b is essential for T . cruzi propagation . Based on the presented validation data , T . cruzi growth can be inhibited through targeting either QN or QP cytochrome b site . GNF7686 represents a new cytochrome b inhibitor , likely targeting the QN site , which has high selectivity for T . cruzi and does not show any effect on respiration of mammalian mitochondria . Crystal structures of cytochrome b from several sources ( bovine , chicken , yeast , Rhodobacter , Paracoccus ) were previously published [68 , 69] . This opens opportunities for rational drug design based on homology modeling of T . cruzi cytochrome b structure . Finally , a counter-screen assay to measure inhibitory activity on the cognate human enzyme ( as described here ) can be used to guide chemical optimization away from host toxicity . While GNF7686 appears to be a promising starting point for kinetoplastid drug discovery , it does not inhibit the growth of P . falciparum or S . cerevisiae , and thus may not be a suitable starting point for anti-malaria or anti-fungal drug discovery . Further in vivo characterization of GNF7686 revealed that it has poor pharmacokinetic properties , including low oral bioavailability ( F = 6% ) and high in vivo mouse clearance ( 53 mL * min-1 * kg-1 ) ; thus , extension of the current studies to an in vivo model of Chagas disease will require identification of a GNF7686 analogue that has improved pharmacokinetic profile . In summary , we have established an approach for identification of molecular targets of T . cruzi growth inhibitors that enables transition to target-based drug discovery for compounds with previously unknown mechanism of action . The first application of this approach resulted in identification of a highly selective inhibitor of T . cruzi cytochrome b , GNF7686 , which can serve as an excellent starting point for discovery of new drugs for Chagas disease and leishmaniasis .
|
Chagas Disease , or American trypanosomiasis , is caused by the kinetoplastid protozoan Trypanosoma cruzi and is primarily transmitted to a mammalian host via a triatomine insect vector ( the “kissing bug” ) infected with T . cruzi parasites . Although discovered in 1909 by the physician Dr . Carlos Chagas , the disease gained recognition by the public health community only following a major outbreak in Brazil during the 1960s . Approximately eight million people ( primarily in Central and South America ) are infected with T . cruzi and cases are becoming more widespread due to migration out of the endemic regions . Current treatment options have severe problems with toxicity , limited efficacy , and long administration . Hence , discovery of new drugs for treatment of Chagas disease has become of prime interest to the biomedical research community . In this study , we report identification of a potent inhibitor of T . cruzi growth and use a chemical genetics-based approach to elucidate the associated mechanism of action . We found that this compound , GNF7686 , targets cytochrome b , a component of the mitochondrial electron transport chain crucial for ATP generation . Our study provides new insights into the use of phenotypic screening to identify novel targets for kinetoplastid drug discovery .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease
|
Rabies is an important but preventable cause of death in Ethiopia . We assessed the knowledge , attitudes and practices of animal bite victims attending an anti-rabies health center in Jimma Town , Ethiopia . Between July 2012 and March 2013 a cross-sectional questionnaire was administered to 384 bite victims or their guardians in the case of minors ( aged <15 years ) . Factors associated with knowledge , attitudes and practices were evaluated using generalized linear models . Almost all participants ( 99% ) were aware that rabies was transmitted by the bite or lick of a rabid dog , however only 20 . 1% identified “germs” as the cause of disease . A majority of participants stated rabies could be prevented by avoiding dog bites ( 64 . 6% ) and confining dogs ( 53 . 9% ) ; fewer ( 41 . 7% ) recognized vaccination of dogs/cats as an important preventive strategy . Regarding attitudes , most ( 91 . 1% ) agreed that medical evaluation should be sought as soon as possible . However , most ( 75 . 0% ) also believed that traditional healers could cure rabies . Rural residence ( adjusted odds ratio [OR] = 2 . 1 , p = 0 . 015 ) and Protestant religion ( OR = 2 . 4 , p = 0 . 041 ) were independently associated with this belief . Among 186 participants who owned dogs , only 9 ( 4 . 8% ) had ever vaccinated their dog and more than 90% of respondents indicated that their dog was free-roaming or cohabitated with the family . Only 7 . 0% of participants applied correct first aid following exposure , and the majority ( 47 . 7% ) reported that the animal was killed by the community following the incident . Female sex and Muslim religion were independently associated with higher and lower practices scores , respectively , due largely to differences in animal management practices following the incident . Although respondents demonstrated reasonably sound knowledge of rabies and its transmission , attitudes and practices were inconsistent with rabies prevention . Culturally- and gender-sensitive activities that promote proper first aid and healthcare seeking behavior as well as appropriate animal management , particularly in rural areas , are needed to prevent deaths associated with rabies in this setting .
As a disease that mostly impacts poor communities , rabies is a classic example of a neglected tropical disease [1] . A vaccine-preventable disease , most deaths from rabies arise due to lack of awareness and poor access to proper health services [2] . It is estimated that around half of the global human population lives in canine rabies-endemic countries and is at risk of exposure [3] . Because cases often go unreported , it is agreed that official records vastly under-estimate the true burden of rabies [3–6] . Modelling studies estimate the annual human death toll from rabies to be around 50 , 000–60 , 000 , with 99% of these fatalities occurring in tropical developing countries , overwhelmingly in Africa and Asia [3 , 6–8] . An estimated 21 , 000–24 , 000 these deaths occur annually in Africa alone [6 , 8] . More recently the global burden of rabies ( reflecting the number of human deaths as well as lost productivity due to premature death , adverse events related to the nerve-tissue vaccine and psychological effects of the disease ) was estimated at 3 . 7 million disability-adjusted life years ( DALYs ) , while global economic losses associated with the disease were estimated at 8 . 6 billion USD annually [8] . Rabies is spread principally by domestic dogs [9 , 10] and these animals constitute the major source of infection to humans [6] . In Ethiopia , for example , dogs were the source of infection in 95% of fatal human cases between 1990 and 2000 [11] . Effective vaccines are available to control disease in dogs and large-scale elimination of canine rabies is considered epidemiologically and logistically feasible as well as cost-effective [12 , 13] . Despite effective tools and sound economic rationale for control , rabies remains a neglected disease in many parts of Africa [12 , 14] . Rabies has long been recognized as an important disease in Ethiopia [15] . Several studies have investigated rabies awareness among urban [16 , 17] and rural [18–21] communities in Ethiopia , however none have investigated knowledge , attitudes and practices among bite victims , specifically . One study , conducted in North Gondar Zone ( 95% Ethiopian Orthodox religion [22] ) in northern Ethiopia , found high awareness of the disease but a dependence on traditional healers for treatment [18] . Ethiopia has a diverse population and thus knowledge , attitudes and practices might be expected to differ by geographic region . We assessed the knowledge , attitudes and practices of animal bite victims visiting an anti-rabies health center in Jimma Zone ( 86% Muslim religion [22] ) , which differs substantially from North Gondor in terms of religious composition .
Jimma Town Anti-rabies Health Center ( JTAHC ) is located in Jimma Town , the capital of Jimma Zone in Oromia National Region , Ethiopia . Jimma Town ( 7°13’ to 8°56’ N , 35°52’ to 37°37’E ) is 352 km southwest of Addis Ababa . The Zone lies at an elevation of 880 to 3360 meters above sea level , and experiences hot and humid weather conditions . JTAHC provides a range of health services to the population of Jimma Zone ( approximately 2 . 5 million people in 2007 [22] ) and is the only center providing post-exposure prophylaxis ( PEP ) for humans bitten by animals suspected of rabies within this zone and surrounding areas . The target population for this cross-sectional survey was animal bite victims or their guardians presenting for the first time to JTAHC between July 20 , 2012 and March 12 , 2013 . This research was approved by the research and ethical review committee of the College of Agriculture and Veterinary Medicine of Jimma University . After explaining the study , written consent was obtained from all patients or their guardians ( in case of minors aged <15 years , per cultural norms in Ethiopia ) . A closed and open-ended questionnaire was developed and included questions to explore participant knowledge of rabies ( including means of transmission to humans and its treatment ) ; their attitudes towards the public health risk of rabies; and their practices for prevention and control of the disease . Practices related to seeking PEP were not assessed because the study specifically targeted bite victims presenting to the health center to receive PEP . The questionnaire also collected basic demographic information , including gender , age , educational status ( no formal education , 1–8 ( primary ) , 9–12 ( secondary ) and >12 ( tertiary ) ) , and religion ( Orthodox , Muslim and Protestant ) , residence ( urban and rural ) and occupation ( farmers , students and working professionals ) . The questionnaire was translated into local languages ( Afan Oromo and Amharic ) and then back-translated to English and pre-tested to ensure validity . The statistical approach was adapted from Davlin and colleagues [23] . Briefly , for each of the question types ( knowledge , attitudes , practices ) responses were designated as “favorable” or “unfavorable” and compiled into a score based on the number of “favorable” responses . A favorable responses was defined as a response that is consistent with rabies prevention . For example , knowledge that rabies is transmitted by dog bites was considered “favorable” since people with this knowledge can prevent rabies through avoiding dog bites and/or seeking medical care following dog bite . To reduce the influence of the large number of clinical signs relative to other parameters on the knowledge score , we assigned +1 , +2 or +3 to respondents who identified below the 25th percentile , between the 25th and 75th percentile and above the 75th percentile , respectively , for this question . Potential factors associated with knowledge , attitudes and practice scores were identified in univariate analysis using t-tests and ANOVA , or the non-parametric equivalent , as appropriate . Comparisons yielding a p-value of <0 . 1 were included in subsequent multivariate analysis . Multivariate , Poisson regression was employed to model scores based on potential factors identified in the univariate analysis . In addition , chi square analysis was employed to examine factors specifically associated with a belief that traditional healers and herbal medicines can cure rabies . Factors associated with this belief were included in subsequent multivariate , logistic regression . All models were fitted using the glm function in SPSS , version 21 ( IBM Corporation , Armonk , NY ) .
Table 1 shows the demographic characteristics of 384 respondents who were interviewed as part of the study . Of these , 254 ( 66 . 1% ) respondents presented to the health center because they themselves had been potentially exposed to rabies . Remaining respondents were parents ( 25 . 3% ) or older siblings ( 8 . 6% ) who brought a minor ( <15 years ) to the health center for treatment following potential exposure . Cases presented to the health center within 3 days ( 50 . 5% ) , 4–7 days ( 39 . 8% ) or more than 8 days ( 9 . 7% ) following potential exposure . Eighty-three per cent of potential exposures were inflicted by dogs; other sources included cats ( 6 . 8% ) , bovines ( 3 . 4% ) , equines ( 2 . 6% ) , wildlife ( specifically , foxes and hyenas; 0 . 8% ) and humans ( 3 . 4% ) . In the 319 cases where a dog was the source of potential exposure , respondents indicated the animal was owned by the family ( 25 . 1% ) , owned by a neighbor ( 45 . 8% ) or free-roaming ( 29 . 2% ) . Bites were the main type of exposure ( 82 . 6% ) . Remaining cases sought treatment following contact with broken skin ( 12 . 5% ) , scratches without bleeding ( 1 . 3% ) or non-specific contact with an animal or human suspected of having rabies ( 3 . 6% ) . Sites of exposure were the head/face ( 4 . 2% ) , chest ( 7 . 6% ) , arm/hand ( 26 . 6% ) and leg ( 61 . 7% ) . Table 2 shows the knowledge of participants in relation to rabies transmission and prevention . The vast majority ( 91 . 7% ) of participants stated that they had heard of rabies before they had been exposed . More than 60% of respondents indicated they heard about the disease from family; friends ( 21 . 1% ) , teachers ( 10 . 7% ) and mass media ( 5 . 5% ) were also sources of information . Despite the high awareness , most respondents ( 52 . 3% ) did not know what caused the disease and more than 90% stated that the maximum incubation period was less than 40 days in humans . Most participants were able to identify the main clinical signs of rabies in animals and humans , although hydrophobia was less commonly identified ( 56 . 8% and 63 . 8% respondents recognized this as a sign in animals and humans , respectively ) . Almost all participants ( 99 . 0% ) identified a bite from a rabid dog as the main way that people acquire rabies , with fewer participants appreciating the role of rabid cats ( 76 . 3% ) , farm animals ( 71 . 1% ) and wildlife ( 47 . 7% ) in transmission . More than a third of participants stated that meat and milk of rabid animals could transmit the disease to humans . Participant attitudes towards rabies are shown in Table 3 . Around 75% of respondents believed that traditional healers and herbal medicines cure rabies . In open-ended questions , bite victims from remote rural areas mentioned that they came to the health center due to their past experience of observing clinical disease and deaths among a neighbor , family member or close relative who was taken to a traditional healer . Others mentioned that they came to the health center because they could not comply with instructions provided by a traditional healer . For example , farmers that owned lands spanning two sides of a river could not comply with advice that they not cross a river within 40 days of receiving a herbal remedy . Among 309 respondents who had some experience with PEP by the time of the interview , 134 ( 43 . 3% ) indicated that multiple injections were tolerable , 175 ( 56 . 6% ) indicated that it was difficult to finish all the doses , and 24 ( 7 . 8% ) stated that they prefer traditional medicines . Table 4 shows the practices adopted following potential exposure to rabies . In 62 cases the animal was deemed to have died of the disease while under observation . Among 186 participants who owned dogs , only 9 ( 4 . 8% ) reported that they vaccinated their dog . More than 90% of respondents indicated that their dog was free-roaming or cohabitated with the family; only 16 ( 8 . 6% ) reported that the pet was confined . If a ruminant was suspected of having rabies , 133 ( 34 . 6% ) respondents indicated that they would slaughter and eat the animal , while 251 ( 65 . 4% ) stated that the animal would be killed and burned . Factors associated with knowledge , attitudes and practices scores are shown in Table 5 . Rural residence was the only significant factor associated with attitudes score in univariate analysis , while age , sex and religion were significantly associated with practices score . Female sex ( crude β = 0 . 358 , p = 0 . 035; adjusted β = 0 . 330 , p = 0 . 039 ) and Muslim religion ( crude β = -0 . 442 , p = 0 . 011; adjusted β = -0 . 413 , p = 0 . 018 ) remained significant in multivariate analysis and were associated with higher and lower practices scores , respectively . There was no significant interaction between gender and religion . The proportion of women reporting that the animal was killed was 38 . 3% ( vs . 45 . 8% of men , p = 0 . 015 ) while 59 . 1% of Muslims reported that the animal was killed ( vs . 39 . 1% of other religions , p<0 . 001 ) . Table 6 shows the factors associated with a belief that traditional healers and herbal medicines cures rabies . Rural residence ( odds ratio [OR] = 2 . 1 , p = 0 . 015 ) and Protestant religion ( OR = 2 . 4 , p = 0 . 041 ) were independently associated with this belief . There was no significant interaction between place of residence and religion .
This study is the first to assess the knowledge , attitudes and practices among attendees at an anti-rabies health center in Jimma Town , Ethiopia . Similar to other studies conducted elsewhere in Ethiopia [16 , 18 , 21] , we found moderately high levels of knowledge regarding the role of dogs in transmission as well as clinical signs in both animals and humans . However , unfavorable attitudes and practices were found in terms of the need for appropriate first aid , medical care and animal management . Of the 384 respondents , 91 . 7% had heard of rabies before exposure . This suggests that victims are aware of the presence of rabies in the area and likely reflects the endemicity of the disease in this location . The majority ( 83 . 9% ) of the victims had heard about rabies from their family or friends . This finding is similar to the work of Sambo and colleagues who reported the most common source of information on rabies in Tanzania was from personal contacts ( neighbors , parents and friends ) [24] . Only 16 . 2% of respondents in this study had heard about rabies from sources such as media and teachers . This suggests that dissemination of information from the government is poor , and could be due to the fact that rabies is neglected and not considered a major public health importance in Jimma Zone . Most of the respondents were able to identify the typical clinical signs of rabies for both animals and humans and were aware that rabies is transmitted via bites or licks from rabid dogs , cats and other domestic animals . This is consistent with previous reports from Ethiopia [16 , 17] and other countries [25 , 26] and again suggests that the disease has long been recognized in Jimma Town and surrounding areas . While awareness of rabies was very high , some important gaps were noted . For instance , more than 90% of the respondents estimated that the maximum incubation period of rabies in humans is less than 40 days . This is consistent with the finding of Agarwal and colleagues [27] who reported that the majority of the communities knew that dog bites could cause death but were not aware of the incubation period of rabies . The incubation period of rabies in humans varies depending on the site of the bite , severity of the wound and amount of virus introduced into the wound and ranges from a few days to several years; in most cases signs develop after one to three months [3 , 28 , 29] . That is , the incubation period is not limited to 40 days . Misconceptions about the incubation period in Ethiopia may emanate from misleading information provided by traditional healers . For example , participants stated that they were advised not to cross rivers and travel to health facilities in search of medical care earlier than 40 days post-exposure . According to the healers , if a victim does not develop clinical signs of the disease during this period , the dog initiating the bite was free of rabies . In this study , the majority ( 95 . 6% ) of the respondents believed that a person potentially exposed to rabies should seek medical evaluation promptly . This favorable attitude is consistent with recommendations that anyone who has been bitten by an animal , or who otherwise may have been exposed to rabies , should seek medical attention immediately [30] . However , the fact that 75% of the respondents concurrently believed that traditional healers and herbal medicine could cure rabies may suggest that inappropriate assistance may be sought following bite . Indeed , some bite victims in this study stated that they sought medical care because they could not comply with advice provided by a traditional healer . Ayalew indicated that the widespread use of traditional , ( supposedly ) anti-rabies herbal remedies is common in Ethiopia and noted that some exposed individuals discontinue the vaccination regime to start these herbal remedies [31] . More recently , Deressa and colleagues stated that most fatal human rabies cases recorded at the Ethiopian Health and Nutrition Research Institute were associated with herbal remedies where the majority of human rabies cases were treated by traditional healers [32] . We found that Protestant religion and rural residence were independently associated with a belief that traditional healers could cure rabies , after adjusting for other factors ( Table 6 ) . Individuals who use herbal remedies and do not develop disease may believe that they did not contract rabies due the use of the remedy after exposure . However , this may be a false association because not all animal bites will cause rabies . In this study , only 7% of the respondents indicated that they washed the bite wound with water and soap as first aid , while 43% performed no first aid after being bitten by a suspected rabid animal . Similar low rates of first aid have been reported elsewhere in the Philippines [33] and India [25] . The WHO recommends that wounds be thoroughly flushed with soap and water immediately after a bite injury; povidone iodine or other antiseptic should be applied when available [3] . Early treatment of all bites and scratches is important because the virus can remain within the area of the injury for an indefinite duration of time . It is therefore recommended that first aid be applied even if the person presents long after exposure [30] . In terms of animal management following a bite , 47 . 7% of respondents replied that they immediately kill suspected rabid dogs that bite humans to prevent further attacks . This is similar to reports from Tanzania where most study participants state they would kill the animal without informing livestock health officers in the areas [24] . Such practices are contrary to recommendations that these animals be quarantined for 10 days to determine whether the exposed human should be vaccinated [34] . In most cases , communities kill dogs as soon as they bite humans but do not submit the animal’s head for rabies diagnosis . In this case , all exposed persons require anti-rabies treatment . In reality , not all bites cause rabies; dogs may bite people due to aggressiveness to strangers , behavioral change during breeding seasons , and to protect newly born puppies . While we do not condone withholding PEP following potential exposure to rabies , administration of PEP in people who were not exposed results in unnecessary costs related to post-exposure treatment and transport to the health center , as well as pain associated with multiple injections with a large needle ( the nerve-tissue vaccine ( Fermi type ) remains the most widely available vaccine in Ethiopia [35] ) . In addition , it makes it difficult to appreciate the scale of the problem and take appropriate steps to prevent further transmission because dogs are not quarantined and observed for development of clinical signs . In this study , female sex was independently associated with a higher practices score , while Muslim faith was associated with a lower score . Further investigation revealed that this was due to differences in the way suspect rabid animals were managed , perhaps reflecting dissimilar participation or attitudes towards dogs among these groups . Variation by gender in reported animal management practices after exposure is unlikely to be due to differential recall since males and females were equally likely to report that they did not know the status of the animal ( 16 . 7% vs 15 . 8% , p = 0 . 883 ) . One study in Ethiopia found that management of dogs was more often the responsibility of women and children and so it is possible that females develop different attitudes towards dogs [16] . Muslim households in this study were more likely to report that the animal was killed . Dogs are considered impure in Islamic tradition [36] and so it is conceivable that people of this faith may be less concerned with dog welfare . Muslim households were less likely to own dogs than other households in this study ( 39 . 6% vs 55 . 0% , p = 0 . 003 ) . Dog ownership rates among Muslim households were considerably higher in this study compared to a similar study in Addis Ababa ( 2 . 6% ) [16] . This difference likely reflects the rural focus of the present study; according to Islam , dogs may be used for hunting and guarding crops and livestock [37] . Indeed in Tanzania , where Muslim households were also less likely to own dogs , the effect of religion was not observed when only livestock-owning households were considered [38] . The majority of respondents identified that rabies could be prevented by avoiding dog bites and confining dogs . Despite a good level of knowledge of these preventive measures , these were poorly practiced among dog owners . Only 9% of pet owners tied up their dogs during the whole day . The remaining 91% of dog owners reported that their dogs were untied and free to cohabit with family and forage widely . This is similar to findings from Addis Ababa [16] . Likewise , only 5% of pet owners in the present study vaccinated their dog . Again this is considerably lower than that reported for Addis Ababa , where vaccine is more easily available [16] . Participants also identified eliminating strays or free roaming dogs as a possible control measure . Dog culling is not recommended as a rabies control strategy on its own [39] . However , along with dog vaccination , removal of strays or free roaming dogs may form part of an initial rabies control program in this particular setting where the number of unwanted dogs is considered problematic . Thus , we looked at elimination of free roaming and stray dogs as a short term solution , with awareness creation on responsible dog ownership ( confining dogs , vaccination ) as a longer term goal . About 35% of the respondents reported slaughtering food animals suspected of rabies and eating their meat , considering that the meat from such animals has medicinal value . In contrast , 65 . 4% of the respondents reported killing and avoiding ( not eating ) the body of animals bitten by suspected rabid animals fearing that rabies is transmitted through an animal’s carcass . Rabies virus is inactivated by heating and therefore eating cooked meat or pasteurized milk is not considered an exposure [34] . However , drinking unpasteurized milk or unprotected cutaneous contact with a carcass ( for example , during butchering and dressing ) are considered exposures [34] . In another study , about one quarter of respondents in Tanzania claimed they would throw away the carcass of a rabid animal [24] . This habit poses a risk for scavengers , which may feed on dead infected animals . In South Africa , an outbreak of rabies in endangered wild dogs in 2000 was speculated to be associated with feeding on the carcass of a rabid jackal [40] . This suggests the need to create awareness that the carcasses of all animals that have died of rabies should be burned or buried to stop the transmission of rabies to scavengers and other animals . This study has some limitations . Due to ethical considerations we did not interview minors ( defined in this cultural setting as <15 years ) . Children are a major risk group for rabies [6] and constituted around 40% of the individuals who presented to the health center in this study . We did not evaluate the knowledge , attitudes and practices of these individuals directly but rather interviewed the parent or older sibling who brought them to the health center . In addition , our study focused on people presenting to the health center for treatment following potential exposure . Thus , our findings reflect the knowledge , attitudes and practices of people who have some awareness of medical intervention and may not be generalizable to the entire community . Even so , we found that the view that herbal remedies can cure rabies existed concurrently in the study population . This may suggest that access and availability of medical versus traditional medicines , as well as willingness to comply with the treatment , may be a key determinant of what treatment is sought . In sum , most of the respondents presenting to JTAHC were familiar with the clinical signs of rabies in animals and humans and with the routes of infection . However , there was a lower level of knowledge of the need to vaccinate and confine dogs . Most of the respondents had good attitude towards the public health risk of rabies but their actual practices , particularly with regard to wound washing with soap and water ( first aid ) and dog management following possible exposure , was inadequate and does not favor the control of the disease . Gender and religion were found to influence these practices . The fact that the majority of the respondents concurrently believed that herbal remedies cure rabies contributes to unnecessary deaths and underestimation of the burden of rabies , since these cases remain unreported to the health centers . This belief was significantly more common among rural residents and points to the need for specific measures to counter this attitude in these areas . Campaigns aimed at raising public awareness around responsible pet ownership including regular vaccination and confinement , as well as appropriate first aid and on the use of traditional remedies are needed . Based on the findings of this study , we suggest religious leaders and gender offices should be involved in rabies prevention efforts so that critical gaps can be addressed in culturally- and gender-sensitive ways . For instance , religious leaders can serve as conduits of information about rabies prevention at church and mosque services . Anthropologists and others with expertise in ethnomedicine should also be recruited to rabies prevention efforts , to advise on strategies for communication around the ineffectiveness of herbal remedies . The findings of this study will be shared with the Jimma University Research and Community Based Education Office so that critical gaps identified by this study can be addressed in collaboration with the Jimma Zonal Health Office and other stakeholders .
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Rabies is an important but preventable cause of death in Ethiopia . We assessed the knowledge , attitudes and practices of animal bite victims attending an anti-rabies health center in Jimma Town , Ethiopia . We found generally high levels of knowledge about rabies . Participants recognized domestic dogs as the source and identified a range of appropriate preventive measures , including avoidance of bites and the need for dog confinement . Despite this reasonable level of knowledge , attitudes and practices that were not consistent with rabies prevention were identified . In particular , a belief that rabies could be cured by traditional healers ( herbal medicine ) was common among the participants , especially among those who lived in rural areas or who were of Protestant faith . Further , few of the dog owners actually confined their dogs and practiced vaccination . First aid following a suspect rabid bite was inadequate in the majority of participants and , contrary to established guidelines , most animals were killed rather than placed under quarantine following a bite incident . Female bite victims were more likely to report improved animal management practices while those of Muslim faith tended to report unfavorable animal management practices . These findings highlight the need for culturally- and gender-sensitive awareness raising programs that improve first aid and healthcare seeking behavior as well as appropriate animal management in order to prevent rabies-related deaths in Jimma Zone , Ethiopia .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Knowledge, Attitudes and Practices of Animal Bite Victims Attending an Anti-rabies Health Center in Jimma Town, Ethiopia
|
Amoebiasis ( a human intestinal infection affecting 50 million people every year ) is caused by the protozoan parasite Entamoeba histolytica . To study the molecular mechanisms underlying human colon invasion by E . histolytica , we have set up an ex vivo human colon model to study the early steps in amoebiasis . Using scanning electron microscopy and histological analyses , we have established that E . histolytica caused the removal of the protective mucus coat during the first two hours of incubation , detached the enterocytes , and then penetrated into the lamina propria by following the crypts of Lieberkühn . Significant cell lysis ( determined by the release of lactodehydrogenase ) and inflammation ( marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta , interferon gamma , interleukin 6 , interleukin 8 and tumour necrosis factor ) were detected after four hours of incubation . Entamoeba dispar ( a closely related non-pathogenic amoeba that also colonizes the human colon ) was unable to invade colonic mucosa , lyse cells or induce an inflammatory response . We also examined the behaviour of trophozoites in which genes coding for known virulent factors ( such as amoebapores , the Gal/GalNAc lectin and the cysteine protease 5 ( CP-A5 ) , which have major roles in cell death , adhesion ( to target cells or mucus ) and mucus degradation , respectively ) were silenced , together with the corresponding tissue responses . Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa . In addition , our study demonstrates that E . histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion .
The protozoan intestinal parasite Entamoeba histolytica is the causative agent of human amoebiasis . This disease is primarily a problem in the developing world , where it leads to 50 million clinical cases and 100 , 000 deaths per year [1] . One of the most puzzling clinical aspects of E . histolytica infection is that 90% of individuals are asymptomatic , whereas the remaining 10% develop colitis , diarrhoea , dysentery and ( in a few cases ) extra-intestinal amoebic lesions , such as liver abscess . The factors that protect the host against invasive diseases and which trigger the invasive process in humans are still poorly understood . However , a link between malnutrition and E . histolytica dysentery has been established in infected children in Bangladesh [2]–[4] . Furthermore , genetic reorganization in the parasite [5] and the gender of the host [6]–[8] could play a role in the outcome of the infection . Invasion of the intestinal wall involves several main steps: ( i ) contact with and degradation of the mucus layer by the trophozoites allowing the amoeba to access the epithelial surface , ( ii ) intimate adhesion of the amoeba to the mucosal cells enabling expression of its cytolytic activity , and ( iii ) induction of a host inflammatory response . E . histolytica motility is essential for the invasive processes and the demonstrated in vitro chemotaxis towards cytokines such as tumour necrosis factor ( TNF ) and interleukin 8 ( IL8 ) could have a role in directing the migration [9] , [10] . A number of animal models for intestinal colitis have been investigated but none reproduces the typical colonic lesions that have been observed in intestinal amoebiasis in humans ( for a review , see [11] ) . The early steps in amoebiasis , such as parasite adhesion to the mucosa , have been investigated in the C3H/HeJ mouse in which chronic infection can be obtained after mechanical injury of the caecal epithelium [12] . Experiments in animal models have shown that once inflammation begins , the epithelial cells release cytokines and chemokines ( IL1β , IL8 , TNF-alpha , IFN-γ ) . In the susceptible C3H/HeJ mouse strain , IL-4 secretion was found to regulate the inflammatory response even in the absence of IL-10 and TGF-beta [13] . Furthermore , E . histolytica infections were also produced in C57BL/6 IL-10 deficient mice [14] . It has been reported that tissue damage in amoebic colitis in the severe combined immunodeficient mouse-human intestinal xenograft ( SCID-HU-INT ) arises from both the direct effects of E . histolytica on colonic tissue and the resulting gut inflammatory response [15] , [16] . Zhang et collaborators have also showed that TNF blockade reduces inflammation and intestinal damage , whereas inhibition of IL-1β reduced cytokine production but had less marked effects on inflammation and disease [16] . Although the inflammatory response produced in the above-mentioned SCID-HU-INT mouse model partially reproduces the early steps of human intestinal parasite invasion , this model lacks lymphocyte responses [17] and it does not take into account the role of the human colonic mucus . Among E . histolytica components Gal/GalNAc lectin is a major factor for adherence to mucus [18] and epithelial cells . It has been reported Gal-GalNAc lectin-mediated contact between trophozoites and human epithelial cells and leukocytes induces cell apoptosis [19] . The Gal/GalNAc lectin is a protein complex with a 170 kDa heavy chain and a 35 kDa light chain . The blockage of cell signalling through the lectin in parasites which over-express the carboxyl terminal end of the heavy chain ( the substrain HGL-2 ) significantly reduced amoebic adhesion to epithelial cells and modify abscess formation in animal model [20] . Previous studies have also shown that purified Gal/GalNAc-lectin stimulates the in vitro production of TNF by macrophages [16] and induces Toll like receptor-2 expression [21] and dendritic cell activation in Balb/c mice [22] , [23] , provoking a Th1 immune response . The second factors important for pathogenesis are the amoebapores that kill host cells and are highly homologous to the perforin produced by mammalian NK cells [24] , [25] . Investigations in the SCID-HU-INT mouse model with a substrain of E . histolytica in which expression of the amoebapore A gene was abrogated , have shown that invasion of the intestinal wall was diminished; however , amoebapore A had no impact on the development of colitis or inflammation [26] . To accomplish pathogenesis amoeba also needs proteases . The E . histolytica genome contains at least 40 genes encoding cysteine proteases . Only a few of these are transcribed and even fewer of the proteases are secreted [27] , [28] . Nevertheless , studies with an E . histolytica substrain in which the expression of several cysteine proteinases was down-regulated by an anti-sense transcript have shown that these proteins play a major role in the development of amoebic colitis in the SCID-HU-INT mouse [29] . One of these cysteine peptidases , EhCP-A5 , which is not expressed in E . dispar [30] has been shown to degrade the cysteine-rich domains of the MUC2 mucin , the major structural component of the colonic mucus gel in the human digestive tract [31] , Using the colonic explants model [32] , [33] . We studied both sides of the host-parasite interaction by determining the kinetics of parasite penetration into the mucus and the mucosa , structural changes in the mucosa , cell lysis and the development of an inflammatory response to virulent wild-type ( WT ) E . histolytica strain ( HM1: IMSS ) and compared them with those observed for the non-pathogenic parasite E . dispar . We also experimented with a number of substrains of the HM:1:IMSS strain which had been genetically manipulated to produce trophozoites that lacked one or more of the above-mentioned virulence factors . We used HGL-2 trophozoites lacking Gal-lectin activity [20] and strains epigenetically silenced for expression of the amoebapore A gene ( AP-A ) ( strain G3 ) , the light subunit of Gal/GalNAc lectin ( Lgl1 ) , ( strain RBV ) and the cysteine proteinase-5 ( EhCP-A5 ) ( strain RB8 ) [34] , [35] . Here , we report that the Gal/GalNAc lectin and amoebapores are not required for invasion of human colon explants and suggest that EhCP-A5 is not required for crossing the mucus but contribute directly or indirectly for penetrating the lamina propria and inducing inflammation .
The pathogenic E . histolytica WT strain used was HM1: IMSS . This virulent strain was also used to produce trophozoites silenced for the Ehap-a gene ( strain G3 ) [35] and double-silenced for the Ehap-a and EhLgl1 genes ( strain RBV ) [34] or Ehap-a and EhCP-A5 ( strain RB8 ) [34] . Amoebae were grown axenically in TYI-S-33 medium at 37°C [36] . The non-pathogenic E . dispar ( strain SAW 1734 ) was cultured xenically with Crithidia fasciculata in TYI-S-33 at 37°C . [36] . Ttrophozoites transfected with the plasmid pEhExNeo ( Neo ) [37] or the plasmid containing a truncated hgl2 gene encoding the transmembrane and cytoplasmic domains of HGL2 ( XM_651089 . 1; HGL2 parasites; [20] ) were cultured in the same way , plus supplementation with 10 µg/ml geneticin ( G418 ) ( Gibco-BRL ) . Prior to each experiment , the geneticin concentration was raised to 30 µg/ml for 48 h . All trophozoites were harvested during the logarithmic growth phase ( 48 h ) and collected by centrifugation at 300 g for 5 minutes . Segments of human colon ( ascending , descending and sigmoid colon ) were obtained from 32 fully informed patients undergoing surgery for colon carcinoma ( 23 men and 9 women; range age , 47–81 years ) and they were analyzed anonymously . Patient written consent was obtained , according to the French bioethics law [38] None of the patients had undergone radiotherapy or chemotherapy . According to the pathologist's examination rules for the longitudinally bisected colon , a healthy segment of tissue which was distant from the tumour region and devoid of metastatic cells was removed . Tissues were processed according to the French Government guidelines for research on human tissues and the French Bioethics Act , with the authorization from the “Institut Pasteur Recherche Biomédicale” investigational review board ( RBM . /2004 . 032 ) . The resected tissues were placed in a 50 ml tube containing KREBS medium ( 117 mM NaCl , 4 . 7 mM KCl , 1 . 2 mM MgCl2 . 6H2O , 1 . 2 mM NaH2PO4 , 25 mM NaHCO3 , 2 . 5 mM CaCl2 . 2H2O and 11 mM glucose ) at room temperature and transported immediately to our laboratory by an authorized courier . The tissues were dissected under a stereomicroscope in order to remove fat and muscle and to retain the mucus , the mucosa and the submucosa . The explants were cut into segments measuring 3 cm by 1 . 5–2 cm ( i . e . 5 . 5 to 6 cm2 ) and pinned ( with the submucosa facing down ) onto a 4% agarose layer in tissue culture Petri dishes ( 60×20 mm ) ( Schott Duran , Germany ) . Trophozoites ( 8×105 in 1 ml of KREBS medium ) from the various amoeba strains mentioned above were added to the luminal face of the colon and incubated in KREBS medium at 37°C for different times ( from 1 to 7 hours ) . Amoeba-free segments served as controls for each experiments and time point . The tissue control ( without amoeba ) and the tissue incubated with amoeba together with the supernatant of each experiments were analyzed at the same time . Lactate dehydrogenase ( LDH ) is a well established marker of tissue breakdown and/or cell viability . Thus , for the LDH assay , an aliquot ( 1 ml ) was taken from each explant incubated with 8×105 WT , E . dispar , HGL2 , NEO , RB8 , RBV , G3 or HM1 trophozoites ( or in the absence of amoeba ) at different time intervals ( 1 to 7 hours ) and was then centrifuged at 2000 g for 5 minutes and stored at −20°C until analysis . The supernatant concentration of LDH was quantified using an enzyme assay ( with 1 ml of supernatant ) on an automated analyzer , which expressed the results in IU/L ( the Dimension® clinical chemistry system from Dade Behring , Schwalbach , Germany , 1971 ) , as described in the manufacturer's instructions . Cytokine levels were analyzed in the supernatants of explants incubated with 8×105 WT , E . dispar , HGL2 , NEO , RB8 , RBV , G3 or HM1 trophozoites ( or in the absence of amoeba ) at different time intervals ( from 1 to 7 hours ) with the Bioplex Protein Array system ( Bio-Rad Laboratories ) using beads specific for IL-1β , IL-2 , IL-4 , IL-6 , IL-8 , IL-10 , GM-CSF , IFN-γ , and TNF on the Luminex 100 instrument ( Applied Cytometry System , Sheffield , UK ) , as previously described ) [39] . Each sample was tested in duplicate in all experiments . To identify the mucus , Human colonic fragments were incubated without Entamoeba and with E . histolytica for seven hours , then fixed with Carnoy fixative , stained with Alcian blue and counterstained with haematoxylin . As these conditions were not optimal to preserve and visualise the amoeba after immunostaining , we decided to use an alternative protocol to study the interaction between the amoeba and the mucosa . The tissue architecture was monitored after incubation with ( 8×105 ) WT , HGL2 , NEO or E . dispar trophozoites ( or in the absence of amoeba as a control ) at 1-hour time intervals up to 7 hours . Eight individual experiments comparing the control tissue , E . histolytica wild type and E . dispar , as well as three distinct experiments comparing control tissue , E . histolytica wild type and the gene silenced trophozoites were performed . Tissues were fixed in 10% formaldehyde in phosphate buffered saline ( PBS ) for 48 hours and then embedded in paraffin . Three sections ( 5 µm thickness ) were cut from paraffin blocks and stained with standard haematoxylin-eosin ( H/E ) reagent . The trophozoites were immunostained with a 1∶200 diluted rabbit serum raised against two internal peptides in the heavy chain HGl2 of the Gal/GalNAc lectin ( H2N-CFNNENKDFIDQYNTN-COOH and H2N-CLIKRCNKTSKTTYWE-COOH ) . For each experiment , a representative image was shown . The specimens for scanning electron microscopy ( SEM ) were fixed in 2 . 5% glutaraldehyde and 2% paraformaldehyde and 0 , 05% calcium chloride in 0 , 08 M cacodylate buffer ( pH 7 , 2 ) overnight at 4°C . Samples were washed three times for 5 min in 0 . 1 M cacodylate buffer ( pH 7 . 2 ) , post fixed for 1 h in 1% osmium acid and 1 , 5% potassium iron cyanide in 0 . 08 M cacodylate buffer ( pH 7 . 2 ) , and then rinsed with distilled water and post fixed in 1% osmium tetraoxide for 1 h at RT . The samples were then rinsed in CaCl2 0 , 05% in 0 . 08 M cacodylate buffer ( pH 7 . 2 ) . Samples were dehydrated through a graded series of 25 , 50 , 75 and 95% acetone solution ( 10 min each step ) and then 100% acetone three times 15 min . After drying in a critical point drier , the specimens were coated with gold palladium and examined under a Hitachi HH-2R microscope . All LDH and cytokines concentrations are expressed as means and standard deviations ( SDs ) . Inter-group differences ( p value ) were evaluated in Student's unpaired t-test using GraphPad software ( available online at http://www . graphpad . com ) . The significant threshold was set to p<0 . 05 .
In order to determine the impact of pathogenic and non-pathogenic Entamoeba on cell viability during organotypic culture of human colonic tissue , we quantified the change over time in lactate dehydrogenase ( LDH ) enzyme activity in the supernatant . The human colonic segment was incubated ( in KREBS medium and at 37°C ) with pathogenic E . histolytica ( the HM1: IMSS strain ) or with non-pathogenic E . dispar trophozoites for up to seven hours . As shown in Figure 1 , the human cells incubated with E . dispar released the same levels of LDH as a control tissue in the absence of parasites . In contrast , after four hours of incubation , virulent E . histolytica trophozoites lysed human cells and prompted a significant increase in LDH concentration in the supernatant . These results indicate that ( i ) most of the cells in the human colonic tissue fragment were alive for at least the seven hours of culture and ( ii ) human cells were killed in the presence of the virulent E . histolytica but not in the presence of the non-pathogenic E . dispar . To ensure that a mucus layer protected the human colonic fragments at their luminal surface during organotypic culture , we performed histological analysis using specific staining for mucus ( Figure 2 . A ) . After seven hours of organotypic culture , the epithelium was still protected by a mucus layer ( left panel ) . In contrast , after seven hours of incubation , no mucus was found at the surface , suggesting that the trophozoites had caused the removal of the mucus ( right panel ) . In order to visualize the interaction between the pathogenic or non-pathogenic parasites with human colonic tissue , explants incubated with E . histolytica or E . dispar for two and four hours of incubation were fixed for scanning electron microscopy analysis . In Figure 2 . B , the micrographs show a luminal view of the colonic tissue . At the start of the incubation , a thick layer of mucus precludes further observation of the epithelium; nevertheless , E . histolytica trophozoites clearly adhered to the mucus , with branching filopodia in contact with the mucus and a tuft of filopodia at the rear ( Figure 2 . B . a ) . After two hours of incubation , the mucus layer was no more observable suggesting that it had been removed by E . histolytica; the epithelial surface and the crypts of Lieberkühn were visible and an accumulation of material ( composed of an agglomeration of human cells and trophozoites ) was seen in the inter-glandular region ( Figures 2 . B . b and c ) . After four hours of incubation in the presence of the virulent parasite , the epithelium was extensively damaged; the enterocytes were covered by thick microvilli that stuck together ( Figure 2 . B . d and e ) . In contrast , after four hours the mucus in the control tissue culture ( i . e . in the absence of amoeba , data not shown ) or in the presence of E . dispar , was still present on the luminal side and the epithelium was not visible ( Figure 2 . B . f ) . To find out whether the surface of the epithelium was altered in the presence of E . dispar , the mucus layer was mechanically scraped , after the SEM fixation procedure . Accumulated cells were not found and the epithelium had not visibly deteriorated ( Figure 2B . g ) Immunohistological analyses of the human colonic explants enabled us to monitor E . histolytica's penetration deeper into the tissue . After two , four and seven hours of incubation with E . histolytica and E . dispar , human colonic fragments were fixed for histology and longitudinal sections of the tissue were stained or immunostained ( using antibodies against the Gal/GalNAc lectin prepared in this work and described in materials and methods section ) and analysed . Firm adhesion by E . histolytica to the interglandular region and detachment of the enterocytes were observed after two hours of incubation ( Figure 3a ) . Next , the parasites migrated to the crypts of Lieberkühn in order to invade the mucosa ( Figure 3b ) . After four hours of incubation , the epithelial surface was completely degraded ( Figure 3c ) and the parasites had penetrated deeper into the mucosa . After seven hours , the parasites had left degraded trails behind them ( Figure 3d ) . Surprisingly , the pathogenic trophozoites did not invade the lamina propria indiscriminately but preferred to migrate along the crypts ( Figure 3b–d ) . After seven hours of culture , the mucosa architecture of the control tissue fragment had not been altered ( Figure 3e ) ; this contrasted with the architecture of the tissue in contact with E . histolytica , which was completely disorganized and , indeed , degraded ( Figure 3f ) . As expected , E . dispar was not able to degrade the mucus or penetrate the mucosa . Although these non-pathogenic trophozoites became embedded in the mucus , no alteration of the epithelium was observed ( Figure 3g ) . We next sought to establish whether or not segments of human colonic tissue which contains a variety of cell types , including enterocytes , fibroblasts and resident immune cells could develop a specific inflammatory response in the presence of pathogenic E . histolytica . To answer this question , we chose to screen and quantify ( using multiplexed cytokine bead-based assays ) cytokine levels in the supernatant of the tissue incubated with E . histolytica and E . dispar every hour for seven hours . A broad panel of cytokines has been reported as being present or absent in the various models used to study the inflammatory response during amoebiasis ( cell cultures , mice , human intestinal xenografts and patient samples ) . These include IL-1β , IL-2 , IL-4 , IL-6 , GM-CSF , IL-8 , IL-10 , IFN-γ and TNF . In our present study , IL-2 , IL-4 and IL-10 and GM-CSF were not detected in the supernatant of colonic explants cultured in the presence or absence of parasites ( data not shown ) . After four hours of incubation , the secretion of pro-inflammatory cytokines ( such as IL-1β , IL-6 , IL-8 , IFN-γ and TNF ) was significantly higher in the presence of E . histolytica but not in the presence of E . dispar , compared with the tissue control ( Figure 4 ) . Prior to four hours of incubation , no detectable differences in the supernatant concentration of each pro-inflammatory cytokine in each condition were observed . The data obtained after four and seven hours of incubation are presented in Figure 4 . The human colonic tissue segments incubated ex vivo were rapidly able to develop an inflammatory response in the presence of the pathogenic species E . histolytica but not in the presence of the non-pathogenic species E . dispar . It has been reported that Entamoeba histolytica virulence factors such as the amoebapores , the Gal/GalNAc lectin and the cysteine proteases , play an important role in ( i ) the killing of mammalian cells , ( ii ) adhesion to target cells and to mucus and ( iii ) degradation of mucus and the extracellular matrix ( For reviews , [40]–[42] . To further analyze the invasive process of E . histolytica within the human colon explants in general and the role of the above-mentioned amoebic factors in particular , we studied E . histolytica trophozoites lacking at least one of these components . We used trophozoites with defective Gal/GalNAc lectin signalling ( HGL2 ) , trophozoites silenced for the expression of amoebapore A ( G3 ) , trophozoites lacking amoebapore A and the light chain ( Ehlgl1 ) of the Gal/GalNAc lectin ( RBV ) and trophozoites lacking amoebapore A and the cysteine protease CP-A5 ( RB8 ) ( for review , [43] ) . We first examined the cytolytic activity of each the above mentioned strains of trophozoites and compared them with that displayed by trophozoites containing the empty plasmid vector pEhNeo ( in the case of HGL2 ) and with the parental WT strain ( for the gene silenced strains ) . The extent of background cell lysis ( which normally occurs during organotypic culture ) was determined by the LDH released from enteric cells incubated in the absence of parasites ( i . e . a control experiment ) . The ability of HGL2 trophozoites to lyse human cells was not affected , as shown by the similar quantities of LDH released in the presence of HGL2 and Neo ( Figure 5 , left panel ) . pEhNeo trophozoites behaved like WT E . histolytica in all our experiments ( data not shown ) . No significant release of LDH was observed after four hours of incubation with G3 , RBV and RB8 , compared with the tissue incubated in the absence of parasites ( Figure 5 , right panel ) . Interestingly , after seven hours of incubation with G3 and RBV trophozoites , the level of released LDH was higher than that of the control tissue and reached the amount obtained with the WT strain , whereas with RB8 trophozoites , the LDH concentration was similar to that of the control tissue . These results indicate that the lack of cell signalling through the Gal/GalNAc lectin and the absence of AP-A did not inhibit E . histolytica's ability to lyse cells . Moreover , in the absence of both AP-A and CP-A5 , E . histolytica RB8 was incapable of lysing human cells , at least during seven hours of interaction with the colonic tissue . The human host's inflammatory response to a pathogen is usually triggered by several factors , such as direct stimulation of the immune system by pathogen-associated molecular patterns ( PAMPs ) and/or factors released by necrotic cells or secreted ( i . e . cytokines ) by living cells . We thus decided to study the inflammatory reaction of the human colonic tissue in the presence of the strains HGl2 , G3 , RBV and RB8 by quantifying the supernatant concentrations of the representative pro-inflammatory cytokines IL1β , IL8 , IFNγ and TNF after four and seven hours of incubation . In the presence of HGL2 trophozoites , the supernatant concentrations of the four tested cytokines were significantly higher at four and seven hours than those obtained with the tissue alone but were similar to those obtained in the presence of the control ( Neo ) trophozoites ( Figure 6 , upper panel ) . It is important to note that the HGL2 parasites still display the heavy chain of the Gal/GalNAc lectin at their cell surface . Nevertheless , our data indicated that inhibition of signalling through the Gal/GalNAc lectin to the actin cytoskeleton did not affect the tissue inflammatory process . The concentrations of IL1β , IL8 , IFNγ and TNF in the supernatant of the human colonic fragment incubated with either the silenced strain G3 or RBV did not differ significantly from those obtained in the presence of the WT strain at each time point ( i . e . four and seven hours of incubation ) ( Figure 6 , middle panel ) . In contrast , there was a significant difference between the supernatant cytokine levels of colonic fragments incubated with the WT and those from the silenced parasite RB8 . The concentrations of IL1β , IL8 , IFNγ and TNF after incubation with RB8 did not differ from those obtained in organotypic culture , as shown in Figure 6 ( lower panel ) . In order to establish whether the trophozoites HGL2 , G3 , RBV and RB8 were capable of invading the human colonic barrier , we examined the tissue ultrastructure and ascertained the presence of the trophozoites . After two , four and seven hours of incubation with either HGL2 , pEhNeo , G3 , RBV , RB8 or WT trophozoites , the tissues were fixed and processed as described for histological analysis and immunostaining ( with anti-Gal/GalNAc lectin antibodies ) . After two and four hours of incubation , there were no major differences between the strains regarding the degradation of the epithelial surface ( data not shown ) . After seven hours of incubation , HGL2 , G3 and RBV trophozoites had penetrated into the lamina propria , as had the pEhNeo and WT trophozoites ( Figure 7 ) . Strikingly , the RB8 trophozoites were unable to penetrate the mucosa , although they were capable of reaching its surface and disorganizing its architecture ( Figure 7 ) .
Like other enteric infections , one of the main drawbacks of experimental amoebiasis is the lack of an adequate animal model that is capable of reproducing the invasive lesions which occur in the large intestine . As humans are the only hosts known to develop amoebiasis , we decided to use human colon explants as an ex vivo model for experimental analysis of the major pathogenic factors leading to this infectious disease . The ex vivo human colonic model enables us to study the early stages of infection , the integrity of the colonic tissue during the invasive process and the host immune response . Advantageously , using human colon explants means that there is no need to extrapolate from rodent models to humans and that all experiments are performed with an integrated model combining human cells and molecules present in the colonic mucus and the enteric mucosa and submucosa . In all , we used 32 human colon samples in the present experiments originating from patients of different gender or age and isolated from different parts of the colon , but we did not find any major change in the tissue responses during our experiments . By using a cytotoxicity assay , we established that no significant lysis of human cells occurred during at least seven hours of organotypic culture or in the presence of the non-pathogenic E . dispar . In contrast , a significant level of cell lysis was observed after four hours of interaction with a pathogenic strain of E . histolytica , indicating that the human cytolysis was specifically due to the presence of virulent parasites . The time course of E . histolytica's invasion of human colon explants was established using SEM and histological analysis . We observed that E . histolytica trophozoites caused the removal of the mucus and reached the epithelial surface within two hours , after which time the parasites invaded the mucosa , detached the enterocytes and migrated along the crypts of Lieberkühn . The specific attachment of trophozoites to the interglandular region of the human colonic epithelium ( and not the luminal surface ) has also been described in a rodent closed caecal loop model [44] . Shedding of apoptotic cells in the interglandular region suggests that this site is preferentially targeted by E . histolytica and is a fragile region that could facilitate penetration of the virulent parasite or the phagocytosis of apoptotic cells . Furthermore , the fact that the trophozoites did not cross and migrate everywhere in the lamina propria but followed the trail formed by the epithelium suggests that in order to migrate , the parasites should require a signal present in the epithelial basement membrane's dense extracellular matrix . The inflammatory response and cytokine release following host cell-parasite interaction have been previously demonstrated in vitro with cultured cell monolayers [45] , [46] , in vivo in several animal models [17] , [26] , [29] and most recently in patients infected with E . histolytica and E . dispar [47] . There is increasing evidence that the parasite's presence evokes an immune response ( characterized by the secretion of pro-inflammatory mediators ) by the intestinal epithelial cells and in which the latter act as antigen-presenting cells [15] , [45] . Histological analysis of human colonic biopsies has revealed slight infiltration of neutrophils , macrophages and dendritic cells into the submucosa at the start of the ulceration process . An increase in the neutrophil , plasma cell , eosinophil , macrophage and T cell counts is observed as the infection progresses [48] . In the present study , we used a histological technique to observe the presence of resident immune cells ( such as monocytes and T-lymphocytes which are capable of contributing to the tissue inflammatory response during amoebiasis ) within the lamina propria of the colonic explants . In order to analyze the relevance of the explant's inflammatory response , we measured the supernatant concentration of a panel of cytokines . We found that pro-inflammatory cytokines ( IL-1β , IL-6 , IL-8 , IFN-γ and TNF ) were significantly and specifically secreted in the presence of E . histolytica but not in the presence of E . dispar . These results are in agreement with previous findings in the SCID-HU-INT model , in which IL-1β and IL8 are produced in response to E . histolytica trophozoites introduced into the engrafted human intestinal segments [17] . The release of pro-inflammatory cytokines by the epithelium appears to be an effective means of initiating a mucosal inflammatory response . Other cytokines ( such as IL-2 , IL-4 and IL-10 ) were not detected in the supernatant of the colonic explants cultured in the presence or absence of parasites . These findings are also in agreement with the earlier reports showing low IL-10 production during E . histolytica infection in both susceptible mice and a human epithelial cell line [2] , [49] . Moreover , Hamano et al ( 2006 ) [14] successfully produced E . histolytica infections in C57BL/6 IL-10 deficient mice . The high levels of pro-inflammatory cytokines induced here by E . histolytica trophozoites demonstrated that the ex vivo human model can be exploited to study the initiation of inflammatory responses at early stages in amoebiasis and that the explants' responses were specifically induced by amoebic virulence factors . In the present study , we investigated the role of key virulence factors such as the Gal/GalNAc lectin , amoebapore A and CP-A5 , in the invasive process and inflammatory responses in the ex vivo human model . We analysed the interaction between the human colon explants and four E . histolytica sub-strains: a strain affected in the Gal/GalNAc lectin signalling HGL2 , [20] , strains silenced for the expression of amoebapore A ( G3 ) [35] , the amoebapore A and the Gal/Gal/NAc lectin light subunit 1 ( RBV ) [34] and the amoebapore A and CP-A5 ( RB-8 ) [34] . The strains with impaired Gal/GalNAc lectin ( whether through the heavy chain ( HGL2 ) or the light chain 1 ( RBV ) ) were not inhibited with respect to the invasive process , suggesting that other molecules are required to adhere to the epithelium and then to transduce signals to the cytoskeleton for migration during intestinal amoebiasis . Although the Gal/GalNAc lectin has been described as a key component in binding mucus and target cells , other molecules like EhADH 112 ( which binds red blood cells [50] , [51] and KERP1 ( which adheres to enterocytes ) [52] could have a role in trophozoite adhesion to and then migration through the colonic barrier . These results emphasize the point that the generation of intestinal amoebiasis or hepatic amoebiasis are not induced by the same virulence factors , as neither HGL2 nor RBV trophozoites were able to develop large liver abscesses in the hamster model [20] , [34] , [53] but were still capable of intestinal invasion . This is also true of amoebapores , since an amoebapore A-deficient strain was incapable of inducing liver abscess formation in a severe combined immunodeficiency ( SCID ) mouse model but was still able to cause inflammation and tissue damage in human colonic xenografts patched in the same model [26] . The results obtained in the present ex vivo model also demonstrate that the amoebapore-deficient G3 strain was not inhibited in terms of the invasive process or induction of an inflammatory response . For in vitro growing conditions , the amoebapore silenced strain , G3 , has been found to have numerous off-target silenced genes in comparison to the parent strain HM-1 ( I . Bruchhaus and D . Mirelman , unpublished data ) . Nevertheless , our results show that the G3 strain as well as the RBV strain had a similar behaviour as the parent HM-1 strain in their ability to invade the colonic mucosal surface and to trigger an inflammatory response . Cysteine proteases have been shown to have an essential role in both hepatic and intestinal amoebiasis . Trophozoites in which only 10% of cysteine protease activity was retained fail to induce liver abscess and intestinal epithelial cell inflammation [29] , [54] . These trophozoites were also ineffective at crossing the protective mucus layer produced by cell lines in culture [55] . Recently , Bracha et al . [34] , have demonstrated the role of CP-A5 in the development of liver abscess using a strain silenced for expression of this protease . Interestingly , RB8 strain was able to cross the mucus barrier in our ex vivo colonic model but was unable to migrate within the mucosa or evoke an inflammatory response during the seven hours of incubation . The behaviour of the trophozoite lacking CP-A5 suggested that ( i ) other proteases than CP-A5 , which are produced by E . histolytica RB8 appear to be involved in the removal of the colon mucin gel; ( ii ) CP-A5 may have an essential role in degradation of the extracellular matrix . In agreement with this hypothesis , it has been shown that CP-A5 possesses collagenase activity [56] . Furthermore , it has been shown in vitro that E . histolytica is attracted by TNF and IL8 [10] , [57]; one can hypothesise that in the absence of inflammation , trophozoite migration was not directed towards the human tissue . RB8 trophozoites were not able to induce human cells lysis , as demonstrated by the absence of LDH release . However , when examining the histological sections , we observed detached human cells - suggesting that cells were nevertheless dying and that the lack of an inflammatory response could be linked to the lack of cell lysis by the RB8 strain lacking CP-A5 . In a transcriptomic comparison for proteinases gene expression between G3 and RB8 strains growing in vitro , the only gene that was silenced in the strain RB8 , was the CP-A5 gene ( I . Bruchhaus and D . Mirelman , unpublished data ) . This has been also shown at the protein level , using a radiolabelled inhibitor of cysteine proteases , that covalently bound to all the cysteine proteases of E . histolytica and it revealed that only CP-A5 was not present in RB8 [34] . However , at this point we do not have information on gene expression of G3 or RB8 strains in an in vivo invasive situation in which a more complex regulation of gene expression can occurs suggesting that the absence of CP-A5 and/or the expression of non yet identified genes could account for the phenotype obtained for the CP-A5 silenced trophozoites in contact with human colon fragments . Further studies are in progress , to analyze additional roles of CP-A5 as well as the possible impact of other amoebic molecules and the effects of colonic components ( such as the bacterial flora ) on tissue invasion parameters . In conclusion , we have shown that the human colonic explant is an integrated , improved model that enables assessment of the interplay between E . histolytica and colonic tissue at the early step of infection . This model will help us better understand intestinal amoebiasis in general and the molecular mechanism of invasion by E . histolytica and the inflammatory responses evoked by the human tissue in particular . By using this model , we expect to identify key molecules in the host-pathogen interaction and which may have a role in the switch between commensal and virulent forms of E . histolytica .
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Entamoeba histolytica is the causative agent of amoebiasis , a human disease . Like other enteric infections , the lack of animal models enhances the difficulty of studying the development of amoebiasis . To date , no experimental model has been developed that reproduces the invasive intestinal amoebic lesions seen in human colon . We present the first study that examines , using human colon explants , the early steps of the human colonic barrier invasion by E . histolytica . With this ex vivo integrative model we have investigated both parasite behaviour and the human tissue response . Remarkably , in this model E . histolytica was able to cross and destroy the intestinal barrier evoking a tissue inflammatory response , while E . dispar , a non-pathogenic species , was unable to penetrate nor induce tissue responses . Furthermore , we have explored the role of three virulence factors during the invasive process , using gene-silenced E . histolytica trophozoites , particularly the kinetics of invasion , tissue destruction and induction of an early inflammatory responses . This is , to our knowledge , the first time that their role is highlighted in a complex human system . Our study provides new insights in the molecular mechanisms involved in the early steps of human colon invasion by E . histolytica .
|
[
"Abstract",
"Introduction",
"Materials",
"And",
"Methods",
"Results",
"Discussion"
] |
[
"infectious",
"diseases",
"microbiology/immunity",
"to",
"infections",
"infectious",
"diseases/neglected",
"tropical",
"diseases",
"cell",
"biology",
"molecular",
"biology",
"microbiology/parasitology",
"infectious",
"diseases/protozoal",
"infections",
"infectious",
"diseases/tropical",
"and",
"travel-associated",
"diseases",
"infectious",
"diseases/gastrointestinal",
"infections",
"cell",
"biology/cytoskeleton"
] |
2009
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An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis
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The trimeric SARS coronavirus ( SARS-CoV ) surface spike ( S ) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 ( ACE2 ) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition . Here , we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy ( cryo-EM ) . The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding “up” conformation . In addition , we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions , which may mimic different conformational states of the S glycoprotein during virus entry . Disassociation of the S1-ACE2 complex from some of the prefusion spikes was observed and characterized . We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state observed on electron micrographs . Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment . However , binding to the receptor opens up the receptor-binding domain of S1 , which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition .
Coronaviruses are a family of large , enveloped , positive-stranded RNA viruses that cause upper respiratory , gastrointestinal and central nervous system diseases in humans and other animals [1] . Human coronaviruses HCoV-OC43 , HCoV-229E , HCoV-NL63 and HCoV-HKU1 circulate in humans and cause mild respiratory diseases [2] . However , the outbreak of SARS-CoV in 2002 and MERS-CoV in 2012 showed that coronaviruses can cross the species barrier and emerge as highly pathogenic viruses [3] . The high fatality rate and wide spread of these new emerging coronaviruses indicate that they are a severe threat to global health . The spike ( S ) glycoprotein of the coronavirus is a class I viral fusion protein located on the outer envelope of the virion that plays a critical role in viral infection by recognizing host cell receptors and mediating fusion of the viral and cellular membranes [4] . The coronavirus S glycoprotein is synthesized as a precursor protein consisting of ~1 , 300 amino acids that is then cleaved into an amino ( N ) -terminal S1 subunit ( ~700 amino acids ) and a carboxyl ( C ) -terminal S2 subunit ( ~600 amino acids ) . Three S1/S2 heterodimers assemble to form a trimer spike protruding from the viral envelope . The S1 subunit contains a receptor-binding domain ( RBD ) , while the S2 subunit contains a hydrophobic fusion peptide and two heptad repeat regions . Triggered by receptor binding , proteolytic processing and/or acidic pH in the cellular compartments , the class I viral fusion protein undergoes a transition from a metastable prefusion state to a stable postfusion state during infection , in which the receptor-binding subunit is cleaved , and the fusion subunit undergoes large-scale conformational rearrangements to expose the hydrophobic fusion peptide , induce the formation of a six-helix bundle , and bring the viral and cellular membranes close for fusion [5] . Coronavirus S glycoprotein features two distinct protease cleavage sites . The S1/S2 cleavage site of the SARS-CoV S glycoprotein is located after residue 667 of the precursor protein , whereas the S2’ cleavage site of the SARS-CoV S glycoprotein is on the S2 subunit and is 130 amino acids from the N terminus of the S2 subunit [6–8] . The S1/S2 cleavage site is located in a flexible loop of residues 660–675 that is completely exposed in the prefusion S1-S2 trimer spike . The S2’ cleavage site of the SARS-CoV S glycoprotein is highly conserved among coronaviruses and is completely buried in the prefusion SARS-CoV S glycoprotein [6–8] . Cleavage of the S2’ site by host cell proteases is required for successful infection by SARS-CoV [8 , 9] . However , the mechanisms involved in exposure and cleavage of the S2’ cleavage site are not well understood . Structural biology studies , especially recent cryo-electron microscopy ( cryo-EM ) studies , have advanced our understanding of the role of the coronavirus S glycoprotein in virus entry . S glycoprotein structures in the prefusion state have been reported for members from the Alphacoronavirus genus ( HCoV-NL63 ) , Betacoronavirus genus ( mouse hepatitis virus ( MHV ) , HKU1 , SARS-CoV and MERS-CoV ) , Gammacoronavirus genus ( IBV ) , and Deltacoronavirus genus ( PdCoV ) [7 , 10–17] . Prefusion S glycoproteins adopt a similar mushroom-like homo-trimer architecture , of which the stem is mainly composed of three S2 subunits , and the top cap consists of three interwoven S1 subunits . A recently reported cryo-EM structure of the MHV S glycoprotein in its postfusion state shows an elongated cone-shaped structure that is significantly different from the prefusion structure and suggests that dramatic conformational changes occur during the prefusion to postfusion transition [18] . The prefusion SARS-CoV S1 subunit is structurally organized into four distinct domains: NTD , CTD1 , CTD2 and CTD3 [13] . Among these , CTD1 is the receptor-binding domain , and one CTD1 in the trimer adopts an “up” conformation as a prerequisite for the binding of SARS-CoV to the cellular receptor angiotensin-converting enzyme 2 ( ACE2 ) [13] . Similar observations of a protruding “up” CTD1 have also been reported for MERS-CoV S glycoproteins [7 , 16] . Although crystal structures of CTD1 in complex with ACE2 have been reported [19 , 20] , the structure of the trimeric coronavirus S glycoprotein in complex with the cellular receptor has not been reported . The mechanisms involved in the conformational changes of the S glycoprotein during coronavirus infection , especially for the highly pathogenic SARS-CoV and MERS-CoV , are not completely understood . Here , we report the SARS-CoV S glycoprotein structures observed by cryo-EM in different stages , including the SARS-CoV S glycoprotein structures in ACE2-free and ACE2-bound states after trypsin cleavage of the S1/S2 site and acidic pH treatment . We also observed and characterized the disassociated S1-ACE2 complex and the postfusion S2 trimeric core . These results collectively enrich our understanding of the SARS-CoV S glycoprotein and its conformational rearrangements during virus entry .
By following a similar procedure to those described in previous studies [13] , we prepared a SARS-CoV S glycoprotein mutant , of which the S1/S2 cleavage site was impaired by mutating Arg667 to Ala ( S1A Fig ) . The mutated S glycoprotein was mixed with ACE2 at a molar ratio of approximately 1:4 , and the mixture was further purified by gel-filtration chromatography to isolate the complex . Fractions of the two elution peaks were collected by gel-filtration chromatography , and the fractions containing the complex were subjected to EM analysis . Cryo-EM analysis of the complex sample showed two major types of particles: the S glycoprotein alone and S glycoprotein bound to ACE2 ( S1B Fig ) . However , the percentage of the S-ACE2 complex particle was less than 7% . We then prepared wild-type S glycoprotein in insect cells . SDS-page gel analysis of the purified sample showed that only a trace of the S glycoprotein had been proteolytically processed by host proteases . By following a published protocol [21] , the sample was further treated with trypsin that completely cleaved the intact S glycoprotein into the S1 and S2 subunits ( S2A Fig ) . Native page gel analysis of the cleaved S glycoprotein sample showed that the S1 and S2 subunits remained associated after cleavage ( S2B Fig ) . Cryo-EM analysis of the cleaved S glycoprotein showed an intact trimer in both the neutral-pH ( pH 7 . 2 ) and the low-pH ( pH 5 . 6 ) buffer . The maps are consistent with the prefusion S trimer structure previously determined using the Arg667Ala mutant ( S2C–S2F Fig ) [13] . These results indicate that cleavage of the S1/S2 site does not significantly change the structure , which is consistent with the conclusions of previous biochemical and structural studies [21] . The S-ACE2 complex was then prepared using trypsin-cleaved and low-pH-treated S glycoprotein and ACE2 . Cryo-EM analysis of the complex showed a significant increase in complex particles ( 49% of the total particles ) ( S3 Fig , S1 Table ) . After 3D classification and refinement , three conformational states of the S-ACE2 complex were captured and determined at resolutions of 5 . 4 Å , 4 . 2 Å and 4 . 5 Å . Two conformational states of the ACE2-free S glycoprotein were also determined at resolutions of 3 . 6 Å and 3 . 9 Å ( S3 and S4 Figs , S2 Table ) . Our cryo-EM analysis of the SARS-CoV S glycoprotein and ACE2 complex sample captured three ACE2-bound and two ACE2-free conformational states of the trypsin-cleaved and low-pH-treated SARS-CoV S glycoprotein . The ACE2-bound states showed that the SARS-CoV S glycoprotein binds one ACE2 receptor utilizing only the “up” CTD1 ( Fig 1 , S5 and S6 Figs ) . The “up” CTD1 and the bound ACE2 are flexible , as shown by a 3D classification analysis that yielded three major ACE2-bound conformational states , in which the “up” CTD1s had different “up” angles ( the angle between the long axes of the “up” CTD1 and the horizontal plane ) of 51 . 2° , 73 . 3° and 111 . 6° ( Fig 1A–1C ) . The structure of one of the two ACE2-free states had one CTD1 in the “up” position and was determined at a resolution of 3 . 9 Å ( Fig 1D ) . This conformation was designated as the unbound-up conformation , which is ready for receptor binding and represents a receptor-binding active state . The structure of the other ACE2-free state was determined at a resolution of 3 . 6 Å with C3 symmetry imposed ( Fig 1E ) . This ACE2-free state had all three CTD1s in the “down” position and was designated as the unbound-down conformation that is not accessible for receptor binding . We also prepared a complex consisting of ACE2 and the trypsin-cleaved SARS-CoV S glycoprotein without low-pH treatment . Cryo-EM analysis showed similar ACE2-bound , unbound-up and unbound-down conformations ( S7 Fig ) . Collectively , these results showed different conformational states of the SARS-CoV S glycoprotein and confirmed that the “up” conformation of CTD1 is required for ACE2 binding . Decreasing the pH does not induce significant conformational changes of the S glycoprotein , even after complete cleavage of the precursor S glycoprotein . Interestingly , only one of the three CTD1s in the S glycoprotein was observed in the “up” position in all analyzed structures . Simultaneous observation of the ACE2-bound , unbound-up and unbound-down conformations of the S glycoprotein in the trypsin- and low-pH-treated sample provided an opportunity to investigate possible conformational changes induced by receptor binding . Structural comparisons of the CTD1s from different ACE2-free conformational states showed that the “up” angles of the CTD1s were between 50° and 70° , while the angle for the CTD1s in the “down” position was approximately 22 . 7° ( Fig 1D and 1E ) [13] . However , the “up” angles of the CTD1s from the ACE2-bound conformations were in the range of 50° to 111 . 6° . Approximately 19% of the ACE2-bound particles had their CTD1s open to 111 . 6° ( Fig 1C ) , which was not observed in any of the ACE2-free conformational states . This result indicates that receptor binding can open up CTD1 . Further comparisons were performed for CTD2 , which is located underneath CTD1 . CTD2 has close contact with the S2 stem region and is connected to CTD1 through two anti-parallel short hinge linkers ( residues 315–322 and residues 512–523 ) ( Fig 2A ) . Cross-correlation coefficients ( CCs ) between the CTD2s were calculated with the EM maps aligned using the S2 region or the CTD2 region ( Fig 2B ) . The CCs ( average CC between the CTD2s: 0 . 96 ) calculated with the maps aligned using CTD2 were much higher than those ( average CC between the CTD2s:0 . 92 ) calculated with the maps aligned using S2 , indicating rigid body movement of CTD2 ( Fig 2A and 2B ) . Model-based structural comparisons showed similar results . The CTD2s of the unbound-up and ACE2-bound conformations exhibited a hinge motion away from the spike axis compared to CTD2 in the unbound-down conformation ( Fig 2C ) . These results indicate that the hinge linker between CTD1 and CTD2 underlies the flexibility of CTD1 . Upon binding to the receptor ACE2 , CTD2 tended to exhibit a hinge motion away from the S2 stem . The “down” to “up” conformational switch of one CTD1 and its binding to one ACE2 did not induce significant conformational changes in the stem region of the prefusion S glycoprotein . Exposure of the S2’ cleavage site is assumed to occur after receptor binding [8] . We examined the S2’ cleavage site in different conformational states of the S glycoprotein . The S2’ cleavage site located in a surface pocket of the stem around Arg797 and covered by a “C”-shaped loop ( residues 787–796 ) is inaccessible in both the unbound-down and the ACE2-bound SARS-CoV S glycoprotein structures ( Fig 2D ) . These results indicated that neither the “down” to “up” conformational change of one CTD1 and its binding to one ACE receptor nor a decrease in the pH drive exposure of the S2’ cleavage site . Rosette-shaped particles that did not belong to any of the prefusion S glycoprotein states were observed in the EM micrographs of a size-exclusion chromatography elution peak before the S-ACE2 complex peak ( Fig 3A and 3B ) , and 2D and 3D cryo-EM image analysis of the selected particles generated a dumbbell-shaped density map for the petals of the rosette ( Fig 3C and 3D ) . The shape and size of the density map were consistent with the recently determined postfusion MHV S2 trimer structure [18] . Based on the postfusion MHV S2 trimer model , a homologous model of the SARS-CoV S2 trimer in the postfusion state was produced using SWISS-MODEL ( S9A and S9B Fig ) . Fitting of the SARS-CoV postfusion S2 trimer model onto the EM map showed good agreement ( Fig 3C ) . The uninterpreted density at one distal end of the dumbbell-shaped density map should be the exposed fusion peptide that is disordered and mediates the aggregation of the postfusion S2 trimers into the rosette-shaped particles . The S2’ cleavage site is not resolved and should be completely exposed based on the postfusion S2 structure model ( S9C Fig ) . When preparing the trypsin-cleaved and low-pH-treated S and ACE2 complex , an additional small peak in the middle of the elution profile was observed ( Fig 4A ) . The elution position of the peak indicated a protein or protein complex with a molecular weight of approximately 150 kDa . Subsequent biochemical and cryo-EM analyses of the peak showed a complex of one S1 subunit monomer and one ACE2 molecule ( Fig 4B–4D , S10 Fig ) , suggesting that binding of ACE2 to the S glycoprotein could trigger the dissociation of one S1 from the trimer together with ACE2 . Analysis of the S trimer structure in the prefusion state showed that the “C”-shaped loop ( residues 787–796 ) covering the S2’ cleavage site was clipped by the linker downstream of the S1/S2 cleavage site of an adjacent protomer ( Fig 4E ) . Disassociation of one S1-ACE2 complex from the spike would release the clipping linker and could induce conformational changes of the “C”-shaped loop , exposing the S2’ site for protease cleavage and promoting the formation of a postfusion S2 trimer ( Fig 4E ) . Of note , we observed a similar size-exclusion chromatography profile and rosette-shaped particles in the EM image of the complex consisting of ACE2 and the trypsin-cleaved S glycoprotein without low-pH treatment ( S11 Fig ) . This finding indicates that low-pH treatment of the cleaved S glycoprotein is not required for the disassociation of S1-ACE2 or for the formation of rosette-shaped particles of the postfusion S2 trimer . We also incubated the S glycoprotein with ACE2 first and then used low-pH buffer and trypsin to treat the sample . Size-exclusion chromatography analysis of the sample showed a similar elution profile with four peaks consisting of S2 , S-ACE2 , S1-ACE2 and excess ACE2 , respectively ( S12 Fig ) .
Proteolysis is key to coronavirus entry . Unlike the S glycoproteins of MERS-CoV and MHV , the SARS-CoV S glycoprotein is not cleaved at the S1/S2 site during virus packaging in cells and , hence , remains uncleaved on mature virions [22 , 23] . However , cleavage of the precursor S protein at the S1/S2 cleavage site by extracellular or endosome proteases is required for a functional SARS-CoV S glycoprotein [24] . Previous studies showed that blockage of the pH decrease in endosomes slightly reduced SARS-CoV infection , which indicated that low pH is not an essential factor for virus entry [25 , 26] . Here , our results showed that the prefusion architecture , either in the unbound or ACE2-bound state , was retained in the cleaved S glycoprotein , and the conformational heterogeneity of CTD1s still existed . Similar results were also obtained for the cleaved with/without low-pH treatment S glycoproteins . Therefore , although cleavage at the S1/S2 site is required for a functional S glycoprotein , it does not significantly affect the overall architecture and conformation heterogeneity . However , we did observe significant differences between the mutant and wild-type cleaved S glycoproteins in binding ACE2 , indicating that the S1/S2 cleavage site might affect CTD1 receptor binding , although the mechanism is still under investigation . Receptor binding plays critical roles in facilitating virus-cell attachment and in determining tissue and host tropism [4] . Our complex structures confirmed that the conformational switch of CTD1 from the “down” to “up” position is a prerequisite for receptor binding . Structural comparisons showed that binding of the receptor further opens CTD1 . In addition , observations of the S1-ACE2 complex and the postfusion S2 assembly in the receptor-present sample suggested that receptor binding can open one CTD1 and trigger the release of all the S1 subunits from the spike . We did not observe S glycoprotein particles with two remaining S1 subunits in any sample , which suggests that disassociation of one S1-ACE2 from the S trimer could cause sequential disassociation of the S1 subunits from the spike trimer . A similar disassociation of the S1 subunits from the spike was also observed for MERS-CoV , even without binding the receptor [7 , 16] . Although the virus uses a similar CTD1 “up” mechanism for receptor binding , the MERS-CoV S glycoprotein , which can have all three CTD1s of a trimer spike in the “up” conformation , is significantly different from the SARS-CoV S glycoprotein , in which only one “up” CTD1 was observed . In addition , disassociated trimeric S1 particles were observed for MERS-CoV [16] , suggesting simultaneous disassociation of all three S1 subunits from the MERS-CoV S glycoprotein . Structural modeling showed that sufficient space exists for the other two CTD1s in the “down” position to point “up” and to bind with ACE2 for most of the S-ACE2 complexes ( S13 Fig ) . However , we did not observe more than one ACE2 or more than one “up” CTD1 in one spike . We speculate that a conformational switch producing more than one “up” CTD1 may destabilize the cleaved S glycoprotein and trigger immediate disassociation of the S1 subunits and the S1-ACE2 complex . Additionally , disassociation of the SARS-CoV S1 subunit should be receptor binding-dependent , since no free S1 was observed when ACE2 was missing . The prefusion S2 subunits assemble to form a nine-helix bundle in the central core region . The three helix fragments ( H1 , H2 and H3 ) of each S2 subunit are connected through two short linkers ( helix linker 1: residues 921 to 927; helix linker 2: residues 949 to 969 ) ( Fig 5 ) . The postfusion S2 subunits are a six-helix bundle with a 160 Å-long central helix core . The pre- to postfusion transition of the S2 subunits requires a 180° flip of prefusion helix fragments H1 ( residues 902–920 ) and H2 ( residues 928–948 ) that fuse with helix fragment H3 ( residues 970–1015 ) to form the postfusion central helix core . Helix linker 2 is critical for the formation of the postfusion helix core , as has been shown in a previous study , which indicated that substitution of the residues in this region with prolines retains the S glycoprotein in the prefusion conformation and prevents conformational rearrangement [7] . The “down” CTD1s are located immediately above the S2 subunits and have direct contact with helix linker 2 ( Fig 5 , left ) . The direct contact should stabilize the prefusion S2 subunit and prevent it from transitioning into the postfusion state . Opening of CTD1 , especially by binding the receptor , would remove the steric restraints on helix linker 2 , triggering the release of the S1 subunits and probably simultaneously allowing the extension of prefusion S2 helixes to form the postfusion S2 long helix bundle ( Fig 5 ) . These new data allow us to reorganize and optimize the current model for SARS-CoV entry ( Fig 6 ) . However , some details are still missing , including the exact function of the S2’ cleavage site and the time point when this site is cleaved , which may be illustrated by further investigation .
A human codon-optimized gene coding the SARS-CoV S glycoprotein ectodomain ( NCBI Accession NP_828851 . 1 ) residues 1–1195 fused with a C-terminal strep tag for purification was cloned and inserted into a pFastBac-Dual vector ( Invitrogen ) . The recombinant SARS-CoV S glycoprotein ectodomain was generated in Hi5 insect cells , purified by Strep-Tactin Sepharose ( IBA GmbH ) and concentrated to less than 100 μl for gel-filtration chromatography using an increase Superdex 200 column ( GE Healthcare ) pre-equilibrated with HBS buffer ( 10 mM HEPES at pH 7 . 2 , 150 mM NaCl ) . Human ACE2 extracellular domain ( residues 19–615 ) with an N-terminal gp67 signal peptide for secretion and a C-terminal 6 × his tag for purification was inserted into a pFastBac-Duel vector ( Invitrogen ) . The construct was transformed into bacterial DH10Bac component cells using Cellfectin II Reagent ( Invitrogen ) . After 5 days of incubation at 27°C , the low-titer viruses were harvested and amplified to P2 and then used to infect Sf9 cells . The cell culture was collected by centrifugation at 48 hours post infection . The supernatant was concentrated , loaded into nickel ( Ni ) -charged resin ( GE Healthcare ) , eluted with 0 . 5 M imidazole in Buffer A ( 50 mM Tris , pH 8 . 8 , 40 mM NaCl ) and further purified using an increase Superdex 200 high-performance column ( GE Healthcare ) pre-equilibrated with Buffer A . Fractions containing ACE2 were collected , applied directly to a pre-equilibrated Resource Q column ( GE Healthcare ) , and then eluted with a 0 . 05–1 M NaCl gradient in 40 mM Tris buffer ( pH 8 . 8 ) . Fractions containing ACE2 were then purified using an increase Superdex 200 column pre-equilibrated with HBS buffer . L- ( tosylamido-2-phenyl ) ethyl chloromethyl ketone ( TPCK ) -treated trypsin was added to the purified SARS-CoV S glycoprotein at a mass ratio of 1:100 in 50 mM Tris-HCl at pH 8 . 0 with 20 mM CaCl2 . The mixture was incubated at 18°C for more than 16 hours and then purified by gel-filtration chromatography ( Superose 6 5/150 ) running in HBS to remove trypsin . Low-pH treatment of the sample was performed by adding 1/10 volume of 1 M sodium citrate stock at pH 5 . 6 , followed by incubation at room temperature for 2 hours . To prepare the complex of the cleaved SARS-CoV S glycoprotein with its receptor , ACE2 , the cleaved S protein was mixed with ACE2 at a molar ratio of 1:3 , and then the mixture was incubated at 4°C overnight . The mixture was purified and verified by gel-filtration chromatography using an increase Superose 6 5/150 column ( GE Healthcare ) pre-equilibrated with HBS buffer . Purified S glycoprotein and ACE2 were mixed at a molar ratio of more than 1:3 and were incubated at 4°C overnight . Then , 1/10 volume of 1 M sodium citrate stock at pH 5 . 6 was added to the mixture , followed by incubation at room temperature for 2 hours . Next , the mixture was treated with trypsin at a mass ratio of 1:100 in 50 mM Tris-HCl at pH 8 . 0 with 20 mM CaCl2 at 18°C , for more than 16 hours . The mixture was purified by filtration chromatography using an increase Superose 6 5/150 column ( GE Healthcare ) pre-equilibrated with HBS buffer . For native page gel analysis , purified S glycoprotein and trypsin-cleaved S glycoprotein were with mixed with 5x native loading buffer ( 30% glycerol , 100 mM Tris-HCl , pH 8 . 0 , 1% bromophenol blue ) . The native page gel consisted of an acrylamide gradient ranging from 4% to 15% . Uncleaved S glycoprotein , cleaved S glycoprotein , S1-ACE2 and ACE2 were prepared as previously described in this paper . The samples were mixed with loading buffer and boiled at 100°C for 10 minutes . The samples were separated by electrophoresis on a 12% ( w/v ) sodium dodecyl sulfate-polyacrylamide ( SDS-page ) gel and were then transferred to a nitrocellulose membrane . The membrane was blocked for 1 hour at room temperature with a non-fat milk buffer and then incubated with mouse monoclonal anti-strep tag antibodies ( HX1816 , Huaxingbio Biotechnology , Beijing , China ) , mouse monoclonal anti-6x his tag antibodies ( HX1804 , Huaxingbio Biotechnology , Beijing , China ) , or mouse anti-CTD1 serum for 1 hour at room temperature . Then , the membrane was incubated with goat-anti-mouse IgG ( HX2032 , Huaxingbio Biotechnology , Beijing , China ) for 45 minutes at room temperature . The blots were visualized by using ECL chemiluminescence reagents ( HX1868 , Huaxingbio Biotechnology , Beijing , China ) . An Amersham Image 600 device ( GE Healthcare ) was used to analyze the results . Mouse monoclonal anti-strep tag antibodies and mouse monoclonal anti-6x His tag antibodies were diluted at 1:5000 , and mouse anti-CTD1 serum ( provided by Panpan Zhou from Linqi Zhang’s lab at Tsinghua University ) was diluted at 1:1000 . For negative-staining sample preparation , 3 μl aliquots of samples at a concentration of ~0 . 02 mg/ml were applied onto a glow-discharged grid with a continuous carbon layer ( Beijing Xinxing Braim Technology Co . , Ltd . ) . Excess sample was removed using filter paper after 1 minute of incubation on the carbon grid . The grid was immediately washed twice and incubated with 3 μl of 1% uranyl acetate ( UA ) solution for an additional minute . The grid was then blotted with filter paper to remove UA , air-dried at room temperature , and examined under an FEI Tecnai Spirit electron microscope operating at an acceleration voltage of 120 keV . Images were collected using a Gatan 895 4 k × 4 k CCD camera at a nominal magnification of 49 , 000× with a pixel size of 0 . 227 nm . For cryo-EM sample preparation , 3 μl aliquots of samples at a concentration of ~0 . 2 mg/ml were applied to glow-discharged holey carbon grids ( Quantifoil , Cu 200 mesh , R1 . 2/1 . 3 ) or grids with a layer of continuous ultrathin carbon film ( Ted Pella , Inc . ) . The grids were blotted and then were plunged into liquid ethane using an FEI Vitrobot Mark IV . The concentration of the S2 rosette sample was low . Aliquots ( 3 μl ) of the S2 rosette sample were applied to the grids , and excess sample was removed using filter paper after 30 s . This procedure was repeated two times . When the sample was applied to the grid for the third time , the grids were blotted and then submerged in liquid ethane as described above . Images of the S2 rosette , trypsin-cleaved S glycoprotein , trypsin-cleaved and low-pH-treated S glycoprotein , and the complex of ACE2 and trypsin-cleaved S glycoprotein ( S11A Fig , S-ACE2 peak ) were collected in frame stacks using an FEI Tecnai Arctica electron microscope operating at an acceleration voltage of 200 keV and equipped with a Falcon II direct electron detector . Images were recorded at a defocus range of -1 . 5 μm to -4 μm with a pixel size of 1 . 27 Å . The exposure time was 1 . 2 s , with a total exposure dose of ~60 electrons per Å2 over 19 frames . Images of the ACE2 and cleaved and low-pH-treated S glycoprotein complex ( Fig 4A , S-ACE2 peak ) and the disassociated ACE2 and S1 complex ( Fig 4A , S1-ACE2 peak ) were collected using an FEI Titan Krios electron microscope operating at an acceleration voltage of 300 keV and equipped with a Gatan K2 Summit direct electron detector . Images were recorded at a defocus range of -1 . 5 μm to -4 μm with a pixel size of 0 . 66 Å . Each image was dose-fractionated into 32 movie frames at a dose rate of 8 . 2 counts per physical pixel per second , with a total exposure time of 8 s and a frame exposure time of 0 . 25 s , resulting in a total dose of ~50 electrons per Å2 . Data collection and image processing statistics are listed in S1 and S2 Tables . Movie frames of the K2 image stacks collected for the ACE2 and trypsin-cleaved and low-pH-treated S complexes ( Fig 4A , S-ACE2 peak ) were 2 × 2 down sampled , resulting in a pixel size of 1 . 32 Å , and then were aligned using the MOTIONCORR2 program [27] before further processing . The CTF parameters were determined with the Gctf program [28] . Particles were automatically selected using the Gautomatch program [29] . Particles were visually inspected to remove false positives . 2D and 3D classifications and refinements were performed using RELION 1 . 4 [30] . The density map of SARS-CoV S glycoprotein in the prefusion state ( EMDB ID: EMD-6732 ) was low-pass filtered to 40 Å and was used as an initial model for 3D analysis . In total , 1 , 033 , 788 particles selected from 2 , 813 micrographs were subjected to several rounds of 2D classification , and 688 , 289 selected particles were then subjected to 3D classification , yielding three classes of S glycoproteins with ACE2 bound and two classes of S glycoproteins free of ACE2 . One of the ACE2-free classes was three-fold symmetrical , with all three CTD1s in the “down” conformation ( unbound-down ) , and the other ACE2-free class was asymmetrical , with one CTD1 in the “up” conformation and two CTD1s in the “down” conformation ( unbound-up ) . The particles from the symmetrical unbound-down class were sorted according to their LogLikeliContribution values , and particles with higher values were selected and subjected to 3D auto-refinement with C3 symmetry imposed , yielding a density map of 3 . 6 Å . Particles from the unbound-up class were further selected through 3D classification with a mask excluding the flexible “up” CTD1 . Selected particles were used for auto-refinement and reconstruction , which resulted in a final density map of 3 . 9 Å . Similar procedures were also used for the reconstructions of the ACE2-bound particles , yielding three ACE2-bound reconstructions with resolutions of 5 . 4 Å , 4 . 2 Å and 4 . 5 Å . Movie frames of the K2 dataset of the ACE2-S1 complex ( Fig 4A , S1-ACE2 peak ) were aligned , and the CTF parameters were determined using a similar strategy to that used for the ACE2 and trypsin-cleaved or low-pH-treated S glycoprotein complexes . A total of 241 , 551 particles were automatically selected using the Gautomacth program . Then , 2D and 3D classifications and refinements were performed using RELION 1 . 4 . An initial model was generated by e2initialmodel . py using typical 2D class averages . To validate the accuracy of the reconstruction , another run of 3D autorefine was performed using the same particle dataset but with a different initial model of ACE2 . The ACE2 atomic model ( PDB ID: 2ajf ) was converted into a density map using the python script e2pdb2mrc . py [31] . Both initial density maps were low-pass filtered to 40 Å before being used for 3D analysis . The two reconstructions had a CC value of 0 . 88 ( at a contouring level of 3 σ ) , and the consistency of the two reconstructions indicated that the reconstruction was reliable . The 2D projections of ACE2 were generated with e2project . py [31] . The Falcon II datasets were processed in a similar manner to that used for the K2 dataset of the ACE2 and trypsin-cleaved or low-pH-treated S glycoprotein complexes . For the ACE2 and trypsin-cleaved S glycoprotein complex ( S11A Fig , S-ACE2 peak ) , a total of 324 , 514 particles were automatically selected using the Gautomacth program and then were 2 × 2 down sampled before further processing . The 2D classification yielded 224 , 052 selected particles , and 3D classification yielded five conformational states corresponding to ACE2-bound S glycoprotein conformation 1 , ACE2-bound S glycoprotein conformation 2 , ACE2-bound S glycoprotein conformation 3 , unbound-up S glycoprotein and unbound-down S glycoprotein . Then , 3D auto-refinements were conducted for particles of each class , yielding a density map of 19 . 7 Å for the ACE2-bound S glycoprotein conformation 1 , a density map of 9 . 0 Å for the ACE2-bound S glycoprotein conformation 2 , a density map of 18 . 5 Å for the ACE2-bound S glycoprotein conformation 3 , a density map of 9 . 0 Å for the unbound-up S glycoprotein and a density map of 8 . 3 Å for the unbound-down S glycoprotein subset . For the Falcon II dataset of the S2-rosette , a total of 34 , 149 particles were subjected to several rounds of 2D and 3D classification . An initial model was generated by e2initialmodel . py using typical 2D class averages . In total , 7 , 636 selected particles were used for auto-refinement , yielding a density map of 30 . 5 Å . For the Falcon II dataset of the trypsin-cleaved S glycoprotein and the trypsin-cleaved then low-pH-treated S glycoprotein , the density map of SARS-CoV S in the prefusion state ( EMDB ID: EMD-6732 ) was low-pass filtered to 40 Å and used as an initial model for 3D analysis . After several rounds of 2D and 3D classification , a density map of 6 . 8 Å for the trypsin-cleaved S glycoprotein and a density map of 6 . 7 Å for the trypsin-cleaved then low-pH-treated S glycoprotein were generated . Map sharpening and B-factor application were performed using the phenix . auto_sharpen [32] and relion_postprocess programs . The reported resolutions are based on the gold-standard Fourier shell correlation ( FSC ) 0 . 143 criterion . Local resolution variations were estimated using ResMap [33] . Structure modeling statistics of the S-ACE2 complex are listed in S3 Table . Atomic models were built for three density maps of the ACE2-S complex , the 3 . 9 Å density map of the unbound-up S glycoprotein and the 3 . 6 Å density map of the unbound-down S glycoprotein . For the model building of the 3 . 6 Å unbound-down S glycoprotein density map , an atomic model of the prefusion S glycoprotein ( PDB ID: 5xlr ) was used as a reference [34] . The NTDs that were missing in the initial model were built with the atomic model of the NTD crystal structure ( PDB ID: 5x4s ) . The model was refined by using ROSETTA [35] and PHENIX with secondary structure restraints and geometry restraints [32] . Manual adjustments of the model were performed in COOT [36] . For the unbound-up S model , the unbound-down S model excluding the “up” CTD1 was fitted into the 3 . 9 Å density map as a rigid body . The initial “up” CTD1 was built by fitting the crystal structure of CTD1 to the EM density . Fitting of the atomic models to the cryo-EM densities was performed by maximizing the density value around the fitted atoms [37] . The separately fitted models were combined to yield a complete model . The model was then refined using PHENIX real-space refinement with secondary structure restraints and geometry restraints and adjusted in COOT . CTD1 was defined as a rigid body during the refinement . The models of the ACE2-S complex were generated in a manner similar to that used for the unbound-up S glycoprotein model . The crystal structure of the CTD1-S complex ( PDB ID: 2ajf ) was used to build the initial model by fitting the structure as a rigid body into the EM density . Molprobity was used to evaluate the final refined models [38] . CCs between the aligned maps were calculated using the UCSF Chimera “measure correlation” command . The CC was calculated using the following formula: CC = <u , v>/|u||v| , where vector u contains the values of the first map and vector v contains the values of the second map . The calculation includes only the grid points in the first map with values above the stated contour level . To calculate the “up” angles of the CTD1s , the horizontal plane of the S glycoprotein perpendicular to the 3-fold axis and the long axis of the “up” CTD1 were generated using the UCSF Chimera “define” command , and then the angle between the axis and the plane was calculated using the UCSF Chimera “angle” command . The homologous model of the SARS-CoV S glycoprotein in postfusion state was calculated using the MHV S glycoprotein in the postfusion state ( PDB ID: 6b3o ) as reference model with SWISS-MODEL [39] . All figures were generated with UCSF Chimera [34] . The coordinates and EM maps have been deposited into the Protein Data Bank and the EM Data Bank with the accession numbers: 6ACG , 6ACJ , 6ACK , 6ACD , 6ACC , EMD-9591 , EMD-9593 , EMD-9594 , EMD-9589 , EMD-9588 , EMD-9598 , EMD-9597 , EMD-9595 , EMD-9596 , EMD-9585 , EMD-9586 , EMD-9587 , EMD-9584 and EMD-9583 .
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The global outbreak of SARS in 2002–2003 was caused by infection by a human coronavirus , SARS-CoV . Although the virus has been extensively studied with regard to epidemiology , virology , clinical features and other aspects , there are still no approved antiviral drugs and vaccines to treat and prevent infections of SARS-CoV . The spike ( S ) glycoprotein of the coronavirus , responsible for host cell attachment and mediating host cell membrane and viral membrane fusion during infection , is key to the viral life cycle and a major target for antiviral drugs and vaccines . In this study , we report the structures of different conformational states of the SARS-CoV S glycoprotein during virus entry . Specifically , we found that the S glycoprotein retains the prefusion trimer structure after trypsin cleavage and low-pH treatment . Additionally , binding with host cell receptor ACE2 promotes the release of S1 subunits from the S trimer and triggers the pre- to postfusion conformational transition . Our results provide new insights for understanding the mechanisms involved in coronavirus S glycoprotein-mediated virus entry .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
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2018
|
Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2
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The Influence Maximization Problem ( IMP ) aims to discover the set of nodes with the greatest influence on network dynamics . The problem has previously been applied in epidemiology and social network analysis . Here , we demonstrate the application to cell cycle regulatory network analysis for Saccharomyces cerevisiae . Fundamentally , gene regulation is linked to the flow of information . Therefore , our implementation of the IMP was framed as an information theoretic problem using network diffusion . Utilizing more than 26 , 000 regulatory edges from YeastMine , gene expression dynamics were encoded as edge weights using time lagged transfer entropy , a method for quantifying information transfer between variables . By picking a set of source nodes , a diffusion process covers a portion of the network . The size of the network cover relates to the influence of the source nodes . The set of nodes that maximizes influence is the solution to the IMP . By solving the IMP over different numbers of source nodes , an influence ranking on genes was produced . The influence ranking was compared to other metrics of network centrality . Although the top genes from each centrality ranking contained well-known cell cycle regulators , there was little agreement and no clear winner . However , it was found that influential genes tend to directly regulate or sit upstream of genes ranked by other centrality measures . The influential nodes act as critical sources of information flow , potentially having a large impact on the state of the network . Biological events that affect influential nodes and thereby affect information flow could have a strong effect on network dynamics , potentially leading to disease . Code and data can be found at: https://github . com/gibbsdavidl/miergolf .
In order to respond to messages and environmental changes , cells dynamically process information arriving from cell surface receptors [1 , 2] . Information is transferred , stored , and processed in the cell via molecular mechanisms , often triggering a response in the regulatory program . These types of dynamic genetic regulatory processes can be modeled and analyzed using networks . The cell cycle process in Saccharomyces cerevisiae is well studied , but not completely characterized [3] . The dynamic regulatory process is controlled by a network that processes signals . To gain further understanding of the regulatory structure , we used publicly available time series data and regulatory databases to solve the influence maximization problem ( IMP ) ( Fig 1 ) [4 , 5] . Recently , the influence maximization problem ( IMP ) has received a great deal of interest in social network analysis and epidemiology as a general method for determining the relative importance of nodes in a dynamic process [6 , 7] . Use case examples are found in modeling the spread of infectious disease in social networks and in identifying optimal targets for vaccination ( or advertisements ) [8] . The IMP is a search over sets of nodes that , when acting like sources in a diffusion process , cover as much of the network as possible [9 , 10] . Diffusion on graphs is part of a general class of problems where some quantity flows from source nodes , across the edges of a graph , draining in sink nodes . Various forms of network flow methodologies have found success in algorithms such as Hotnet , ResponseNet , resistor networks , and others [11 , 12 , 13] . Diffusion , like the propagation of infection , does not follow algorithmically defined paths on graphs , such as shortest paths , but instead flows on all possible paths . In this work , we use a diffusion algorithm that is modeled using a random walk , where transition probabilities are proportional to edge weights . The random walk produces an expected number of visits to each node . If the expected number of visits is greater than a given threshold ( here 0 . 0001 ) , the node is considered to be ‘covered’ , and the network cover is a count of ‘covered’ nodes . The goal of the IMP is to maximize this network cover with a fixed number of nodes . In our application of the IMP to genetic regulatory networks , the diffusion process represents a flow of information on the network , which opens up many applications in biology [14 , 15 , 16] . Directional information flow can be described quantitatively using the model free method , transfer entropy ( TE ) [15] . Since processes in biology are not instantaneous , time lags are introduced , representing a lag between the transmission and reception of information . As an example , the expression of transcription factors , their subsequent binding to promoter regions , and ultimately , the induction of transcription can take substantial amounts of time . In this case , we use ant optimization to search for sets of source nodes that lead to diffusion generated network covers that score highly [17] . Typically , ant optimization is used for path finding , but it can also be applied to combinatorial , subset selection problems [18 , 19] . In ant optimization , ants construct potential solutions as sets , which are scored and reinforced , encouraging good solutions in later iterations . In this work , the result of the optimization procedure is an optimal , or nearly optimal , set of nodes that maximizes network cover after applying the diffusion [15] . In application to biological networks , the IMP essentially remains an unexplored area of research [20] . Each run of the IMP returns a solution set of size K . Using both ‘fast’ and ‘slow’ parameter sets for the ant optimization , we have run the IMP for values of K from 1 to 50 , resulting in 50 solutions , one set for each value of K . Genes were ranked by counting the number of times a given gene appeared in a solution set . A highly influential gene would appear in the solution for many values of K , regardless of the solution set size , implying that topologically , the gene is in an optimal position as a source of information , enabling contact to a large portion of the network . Optimization can proceed at different rates; more restarts , more ants , a slow pheromone evaporation rate , and a high number of local optimization steps may result in more robust and repeatable results , but more iterations might be needed and the run time can be longer . On the other hand , few restarts with a small number of ants and a fast evaporation rate , plus fewer local optimization steps , leads to more stochastic results and a shorter run time . The slow-and-steady approach can consistently get stuck in non-optimal minima , whereas the highly stochastic results can sometimes 'jump' out of non-optimal minima . In order to explore results and convergence behavior , both fast and slow parameter sets were used . Our results from either parameter set were in excellent agreement regarding influence rankings , reducing concerns about the stochastic nature of ant optimization . To better understand topologically where the influential genes are situated , we compare the IMP solution sets to gene sets derived from other centrality metrics , such as degree centrality [21] , betweenness-centrality [22] , where shortest paths are considered , and PageRank [23] , the algorithm used in web search . This analysis produced a ranked list of genes that agrees with previous studies of cell cycle regulators and models , giving credence to the method as a fairly general approach to analyzing large scale biological network dynamics .
The yeast genetic regulatory network was constructed starting with 26 , 827 genetic regulatory edges from YeastMine and statistically filtering out edges [4] . Regulatory processes in biology are not instantaneous , so time lags are introduced to account for propagation time ( S1 Fig ) [24] . Further , genetic regulatory interactions are directional; transcription factors act on genes , and not the other way around . So , although correlation is easy to compute and is sometimes used to estimate the activity of regulatory edges , there are more appropriate metrics to use with time series data , such as transfer entropy . Transfer entropy ( TE ) is a model-free method that attempts to quantify information transfer between two variables in a directional manner . At present , computing transfer entropy is not trivial , and there is active research in comparing and deriving methods for approximating the value . In this work , we used a Gaussian kernel density based approach , which has been previously shown to be relatively accurate [25 , 26] . Using time series data for 5 , 080 measured genes and 26 , 827 genetic regulatory edges from YeastMine , both time lagged Spearman's correlation and transfer entropy were computed for all regulatory edges . Permutation-based statistics were applied to assess the significance of TE . Edges were accepted if empirical p-values was less than or equal to 1 ( pn+1 ) where pn is the number of permutations ( pn = 50 , 000 ) . Spearman's correlation tests were performed on each time lag ( 0–5 time steps ) . The maximum ρ was kept , and at FDR 1% , this resulted in 12 , 555 edges , containing 3 , 939 nodes . Significant edge weights had a median correlation of 0 . 58 . Most of the edges ( 52% ) showed a maximum correlation when using a time lag of zero . The metric of interest , time lagged TE , resulted in 2 , 084 significant edges containing 1 , 409 nodes with median values of 0 . 499 ( Fig 2 ) . The overlap between the correlation and TE networks is moderate; only 16% of the edges in the correlation network are shared with the TE network ( 1 , 988 of 2 , 084 edges in the TE network or 97% ) , and while most TE nodes are found in the correlation network ( 95% ) , only 35% of the correlation nodes are found in the TE network . When comparing Spearman's and TE weights on matched edges , the correlation between matched edge weights was moderately weak ( Spearman's correlation 0 . 43 ) . Additionally , the mean node degree distribution in the correlation network is much higher than that of the TE network . For example , SFP1 has degree 923 in the correlation network , compared to 76 in the TE network ( summing both in- and out-edges ) . The high node degree in the correlation network suggests that correlation testing may be overly permissive , with less informative edge weights . Clauset , Shalizi , and Newman’s method for statistically determining whether a network is ‘scale-free’ showed that the TE network is not [27] . Using the TE network , the result showed alpha = 2 . 17 , which is consistent with power law networks . However , the goodness of fit test using the Kolmogorov-Smirnov statistic produced a p-value of 0 . 011 , indicating that only a small fraction of the simulated scale-free distributions are "close" to the observed degree distribution . In the rest of the analysis , only the transfer entropy network is used , since it is clear that the correlation-based network is not a super-set of the transfer entropy network , does not agree in the weighting , and is likely overly permissive with regard to active interactions . Using transfer entropy to quantify information flow , if an upstream node transfers information to a downstream node , respecting edge directions , the downstream node is said to be 'influenced' . The area of influence can be found by application of a diffusion process , where the flow follows edges with greater information transfer ( edges with greater weights ) , ‘visiting’ nodes and resulting in a cover on the network . The maximization problem involves finding a set of nodes with size K , that when treated as sources , influences the largest proportion of the network , which is to say , that after the diffusion process is applied , no other set would lead to a greater network cover . The Influence Maximization Problem ( IMP ) was solved over a range of set sizes , K = 1 to 50 . Since ant optimization is stochastic and can result in variable solutions , two different parameter sets were used ( S1 Text ) . First a ‘slow’ parameter set was used ( best of 8 restarts , 64 ants , 32 local optimization steps , evaporation rate 0 . 2 ) . The range of K was run three times , for a total of 150 ant-optimization runs . A count was made on the number of times genes were selected across solutions . As an example , if a gene appeared in 46 solutions , on average , for K = 1 to 50 , it would be considered a high-ranking gene . The influence score , representing a network cover , increased quickly for small values of K , gradually leveling out . With K = 44 source nodes ( 3% of the network ) , a maximum network cover of 1 , 308 nodes ( 93% ) was produced . Beyond K = 44 , the score increased by single digits through the addition of single nodes ( see S2 Fig ) . Regarding the rate of change in network cover , from K = 1 to K = 2 , the total network cover increased 12% . However , after that , the rate of increase drops quickly . Between K = 14 to K = 15 , the network cover increased at a rate of less than 1% , and after K = 24 , for each additional node added to the set of sources , the increase in network cover dropped to less than 0 . 5% . The top ranked gene FKH1 , was selected on average 49 ( out of 50 possible ) times , followed by two genes , SFP1 and TFC7 , that were selected on average 47 and 46 times respectively . Overall , 52 genes were selected in at least one run . A second parameter set , the ‘fast’ set , used 4 restarts , 16 ants , 8 local optimization steps , evaporation rate 0 . 2 . For each value of K , 49 optimizations were run , for a total of 2 , 450 result sets . We found that faster optimization runs lead to more variation in the results . However , using the same ranking method , counting the number of times a gene was selected , resulted in excellent agreement with the ‘slow’ parameter set ( S1 Text , S3 Fig ) . The set of genes in the top 15 ranked influencers are identical across parameter sets . The top 15 influencers from both parameter sets are found in Table 1 . To provide a basis for comparison to the ranked influencers , 13 different centrality measures were computed on the TE network . Brief descriptions of each centrality metric can be found in supplementary text ( S1 Table ) . As stated earlier , after K = 24 , the increase on network cover had dropped below 0 . 5% , making this a reasonable stopping point in selecting the most influential genes . To compare with other metrics , the top 24 genes were selected for each centrality measure . A Jaccard index was computed for each pair of centrality measures ( Fig 3 ) , and although some clustering is observed among centrality metrics , especially among node-degree related measures , there remains substantial disagreement in top ranked genes . The top ranked influential genes are not found among highly ranked genes in eigenvector based centrality measures including authority , eigenvector centrality , and alpha centrality . However , eigenvector related measures of centrality revealed important genes that are not found in other lists . For example , the well-known cell cycle regulatory gene CLB2 was selected by alpha centrality and authority , while it was not found using influence ranking or betweenness . Overall , no ranked list contained a definitive set of cell cycle related regulators . Across measures , gene set enrichment showed a wide variety of associations with biological processes , illustrating differences in the gene rankings ( S2 Text ) . We have found that within the regulatory network structure , the influential genes tend to be situated upstream of genes selected by other centrality measures ( Fig 4 , S4 Fig ) . For example , the influencer genes act as regulators for genes selected by alpha centrality , while no genes selected by alpha centrality regulate the influencer genes . The same is found for the eigenvecteor centrality and betweenness sets . In some cases , there is a fair amount of overlap in the top-level regulators , such as among the high degree nodes and the articulation set . But , overall , we see the influencers stay as top-level regulators to genes selected by other centrality measures . This can be quantified by computing the fraction of reachable genes , starting at a given measure , and excluding overlapping genes ( Fig 5 ) . For example , starting at the set of influential genes , 79% of the betweenness selected genes can be reached , while starting at the betweenness genes , only 12% of influencers can be reached . Starting at the influencer genes , 41% of degree central nodes can be reached , while only 12% of influencers can be reached from the degree central nodes . Starting from every centrality measure , the fraction of reachable nodes is fewer , compared to starting from the influential genes . On average , 54% of “central genes” ( excluding subgraph centrality ) can be reached when starting at the influential genes , compared to 8% of reachable influential genes , after starting from “central genes” of other measures . Subgraph centrality forms a strong intersection with the influential genes , resulting in no connections between sets . These influential genes are , in a sense , topologically central and connect to important genes found by other centrality measures . Since the yeast cell cycle has been the subject of many studies , we have data and results from other projects which we can use in the evaluation of the algorithm . First , we examined the experimental outcomes for yeast genetic experiments found in the SGD [28] . In order of influence ranking , large-scale genetic survey phenotypes are listed in Table 2 , as well as PubMed Central IDs for papers showing evidence of cell cycle regulation . If a direct cell cycle related phenotype was found , it was reported in Table 2 . But given the close connection between lifespan , metabolism and the cell cycle , if no direct cell cycle phenotype was found , then a related phenotype was reported . It should be noted that even MBP1 , which is clearly involved in the G1/S transition , does not have a phenotype listed that directly mentions the cell cycle . Nearly all ranked genes have phenotypes that are in some way related to cell cycle , metabolism , or longevity . To compute gene set enrichment , over-representation testing was performed using the ConsensusPathDB service , which utilizes a hypergeometric test over a large collection of pathways and gene ontology ( GO ) terms [29] . P-value adjustment is done using FDR correction and a background of 4 , 766 genes was used relating to the array used . Gene set enrichment showed that the influence ranked genes were significantly associated with cell cycle related pathways and cell cycle related GO categories . The “regulation of transcription involved in G1/S phase of mitotic cell cycle” GO term ( GO:0000083 ) had a q-value of 1 . 1e-4 , the "regulation of transcription involved in G2/M-phase of mitotic cell cycle" GO term ( GO:0000117 ) had a q-value of 1 . 08e-3 and the cell cycle phase ( GO:0022403 ) had a q-value of 0 . 008 . The KEGG pathway “Cell cycle—yeast—Saccharomyces cerevisiae ( budding yeast ) ” had a q-value of 0 . 02 . In Eser et al . , the source of the data , 32 hypothesized cell cycle regulators were named [5] , five of which are found in the 24 top ranked influencer list . Comparing the top ranked influential genes , we see again that the influential genes are immediately upstream of the Eser TFs ( Fig 6 ) , where in total , out of 27 TFs in the network , 15 cell cycle regulators overlap with the influential list , or are regulated by influential genes . Two more , SWI6 and BAS1 were selected as low ranking influential genes ( ranks 33 & 34 ) . Therefore , the influential ranked list contained or regulated 63% of the available Eser genes . Recently a cell cycle model by Tyson et al . that successfully accounts for 257 of 263 phenotypes [30] was published . In total , 29 genes were extracted from the model where complexed genes were considered separately ( e . g . SWI6 and SWI4 were used instead of SBF ) . The full YeastMine network scaffold contained 28 of the 29 genes ( CDC55 was not present ) , and 20 genes were in the TE network . Three genes from the model were ranked as influencers ( MBP1 , SWI4 and SWI6 ) . While most of the Tyson model genes are not ranked influencers , they are immediately regulated by influential genes . SWE1 is regulated by 4 ranked genes . CDC20 is regulated by 2 ranked genes . CLB5 is regulated by 2 ranked genes . SIC1 is regulated by 1 ranked gene . So , in almost all cases , the Tyson model genes are not regulated by a single influencer , but by multiple influencers . This shows that even though the mechanistic modelers have different goals–the derivation of small models consisting of well-known elements on multiple levels ( protein level and others ) that produce a desired behavior , such as cell cycle timing , and timing changes with given mutations–there is a clear relation to the influential genes .
Transfer entropy has been shown to be useful in quantifying information transfer . Here , we showed that using time lagged transfer entropy , along with a permutation testing framework , leads to biologically salient network structures . Even though the network was constructed by considering all possible regulatory edges , it recovers much of the structure and functional enrichment that one would expect , as demonstrated by the lists of genes returned by commonly used centrality metrics , such as betweenness and degree . Edges with the highest weights , implying greatest information transfer , include ( SWI4 → SPT21 , TE = 1 . 57 ) , ( TFC7 → MSL1 , TE = 1 . 36 ) , ( FKH2 → ALK1 , TE = 1 . 34 ) , ( TFC7 → CHL1 , TE = 1 . 27 ) and ( SWI4 → RNR1 , TE = 1 . 27 ) . The source nodes are well-known , multi-functional transcription factors , while the target nodes have more focused functions . SPT21 has a role in regulating transcription through chromatin silencing . MSL1 is involved in mRNA splicing through interactions with the U2 small nuclear RNA . ALK1 is involved in proper spindle positioning and nuclear segregation following mitotic arrest . CHL1 is related to the cohesion of sister chromatids during mitosis . Finally , RNR1 plays an essential role in the cell cycle , assisting with DNA replication and repair . More well-known cell cycle interactions also have high TE edge weights . These include SWI4-SWE1 ( TE ranked 7th highest out of 2 , 084 ) , NDD1-SWI5 ( ranked 17/2084 ) , RAP1-FKH2 ( ranked 20/2084 ) , and SWI4-YHP1 ( ranked 30 / 2084 ) . Yeast is often used as a model organism in the study of aging . Interestingly , the top two most influential genes , FKH1 and SFP1 have both been related to lifespan [31–34] . The close ties of sources and edge weights to the cell cycle process show that the general dynamics of the cell cycle were captured , reinforcing the usefulness of transfer entropy in biological investigations . Some well-known cell cycle regulators , such as NDD1 , were not selected by influence maximization . In cases such as this , it can often be explained by exploring the immediate neighborhood . In the TE network , NDD1 has upstream regulators FHL1 , STB1 , SWI4 and SWI6 ( three of which are ranked influencers ) . NDD1 itself targets 18 other genes , all with no influence ranking . Among the targets , we found ALK1 , which is also a target from FKH2 as mentioned earlier , as well as CLN1 , which is also targeted by three influencers FKH2 , SWI4 , and SWI6 . So , although NDD1 is famous as a cell cycle regulator , when solving the IMP , there are more optimal sources that target the same downstream genes . When we considered the ranking of influential genes , we saw that high-ranking genes were also more likely to be ranked by other centrality metrics . But there are several notable exceptions . SWI4 and SWI6 were relatively low ranked influencers , but were highly ranked by other metrics . These examples are notable due to their established role in the cell cycle and regular inclusion in models . Proteins SWI4 and SWI6 are members of the SBF complex , interacting with the MBF complex ( SWI6-MBP1 ) to regulate late G1 events . The “low” influence ranking was due to higher ranked influencers being upstream in the regulatory network . Therefore , they were only selected as K , the set of requested influencers , grew large enough . Network control is one goal in the study of dynamic networks [35 , 36] . Given that influential nodes seem to have a topologically advantageous position , one could speculate that influential genes might be useful selections for network control . Biological events that impact the influential nodes , thereby affecting normal information flow , could have a strong effect on the network , potentially leading to disease states . Discovering the minimum sets of biological entities that hold the greatest influence in the network context could lead to further understanding of how network dynamics is associated with disease .
Eser et al . [5] generated time series expression data from two replicates of synchronized yeast producing metabolically labeled RNA levels every five minutes over 41 time points . The expression series spans three cell cycles , which progressively dampen in wave amplitude , as yeast synchrony is lost . Using a model for detecting periodicity in gene expression , 479 genes were labeled as statistically periodic . Additionally , 32 transcription factors were predicted to be cell cycle regulators . YeastMine , the database of genetic regulatory interactions in yeast ( May 2015 ) [4] provided regulatory edges . Using 6 , 417 yeast genes , 26 , 827 genetic regulatory edges were collected . Edge weights were computed using a variation of transfer entropy , as described below . The Saccharomyces Genome Database ( SGD ) was used to reference experimental phenotypes and gene annotations [28] . Given two genes connected by an edge , the edge weight was computed in two ways . First , time lagged Spearman's correlation was used with time lags of 0 to 5 steps ( 0 to 25 mins . ) , keeping the maximum . Second , time lagged transfer entropy ( TE ) was used , similar to what is described in [37 , 38] . TE is computed at each time lag along with a robust distance comparing the observed TE to TEs generated from permuted data . The TE and lag time is returned that maximizes this distance . Time lagged Spearman's correlation is computed by taking two time series , or numeric vectors x = {x1 , x2 , … , xn} and y = {y1 , y2 , … , yn} , and computing the correlation on sub- sequences {x1+k , …xn−1 , xn} and {y1 y2 , …yn−k} , where k is some integer representing the time lag between variables . Transfer entropy ( TE ) is an information theoretic quantity that uses sequence or time series data to measure the magnitude of information transfer between variables [25 , 38] . Transfer entropy is model-free , directional , and shown to be related to Granger causality [39] . In TE , given two random variables variables X and Y , where X is directionally connected to Y ( or X → Y ) , we would like to know if prior states X help in the prediction of Y , beyond knowing the prior states of Y . Given two sequences x and y , we describe transfer entropy as Tx→y ( k ) =∑yt , yt−1 , xt−kP ( yt , yt−1 , xt−k ) logP ( yt , yt−1 , xt−k ) P ( yt−1 ) P ( yt−1 , xt−k ) P ( yt , yt−1 ) , where xt−k indicates value of the sequence at time step t − k . To perform the computation , first x and y are mean-centered and scaled to be within the range [−1 , 1] . A Gaussian kernel density estimate ( KDE ) is fit with a bandwidth given by “Scott’s rule” . Then , a three-dimensional grid is generated by equally spacing some number of points between −1 and 1 in each dimension . Using the grid , points are sampled from the KDE , creating a joint probability distribution , which is normalized in order to sum to 1 . The required distributions are marginalized from the joint distribution by summing across the grid . Smaller grid sizes provide a finer grained probability distribution , but slow the computation without changing the values substantially . A three-dimensional grid of 103 points was found to be a good compromise between computation time and accuracy . A permutation test was performed to assess statistical significance of the transfer entropy , Tx→y . The sequence x was split into a list of subsequences with length 3 and permuted 50 , 000 times . A robust distance , ( Tx→y−Median ( Tx→yperm ) ) /MAD ( Tx→yperm ) , was computed where Tx→y is the observed transfer entropy and Tx→yperm is the set of TEs resulting from permuted sequences , and the MAD is the median absolute deviation . The time lag maximizing the robust distance is selected and a p-value is computed by taking a count on the number of times the permuted TE was greater than the observed TE , giving an empirical p-value . Edges were accepted if empirical p-values were less than or equal to 1/ ( pn + 1 ) , where pn is the number of permutations ( pn = 50 , 000 ) . The IMP maximizes a network cover based on diffusion . The diffusion model , and most of the nomenclature , is described in [15] . The diffusion models are Markov chains with absorbing states [40] . In the model , vertices are first partitioned into sets S ⊆ V and T ⊆ V , where V is the set of all vertices . The set S contains sources , which in the model are generating information flowing through the rest of the network ( nodes in T ) until reaching a dead end or absorbing back into S . The stochastic matrix , defining the probability of moving from one vertex to another , is defined as pij=wij∑jwij , where edge weights wij are the weights on outgoing edges . Sets S and T partition the stochastic matrix as P=[PSSPSTPTSPTT] , where PSS defines the transition probabilities from nodes in S to S , and PST defines transition probabilities from S to T , and so on . Although the matrix is square , it is not symmetric , given the directed edges . Ultimately , we wish to compute the expected number of visits from a node vi ∈ S , to a node vj ∈ T , defined as matrix H . At time step t , information can travel from vi ∈ S to vj ∈ T directly , or it would already be at adjacent node vk , and would travel from vk ∈ T to vj ∈ T in the next time step . So , at time point t , the estimated number of visits from vi ∈ S to vj ∈ T is given as hij ( t ) =pij+∑k∈Thik ( t−1 ) pkj , where pij is the transition probability of vi ∈ S to vj ∈ T , hik ( t−1 ) is the expected number of visits that have already taken place at time ( t − 1 ) , from vi ∈ S to vk ∈ T , and pkj is the probability of the transition from vk ∈ T to vj ∈ T . The matrix form of the equation is H ( t ) =PST+H ( t−1 ) PTT . In the long run , at steady state , when H ( t ) ∼ H ( t−1 ) , the equation reduces to H ( I − PTT ) = PST , where I is the identity matrix . By taking the transpose of both sides , we have ( I−PTT ) ′H′=PST′ . This form lets us avoid the matrix inverse when solving for H , which can be expensive or impossible to compute given that the directed network is represented as an asymmetric matrix . Fortunately , the appropriate iterative solvers are available in the Python SciPy sparse linear algebra library and are robust enough to handle singular matrices . To compute a measure of influence on the network , after solving for H the expected number of visits on nodes , the influence is summarized as the “influence-score” , Ωs=∑i∈S{∑j∈TΙ ( hij>θ ) } where hij is the number of visitations ( using matrix H ) from node vi ∈ S to connected nodes vj ∈ T . Indicator function I ( hij > θ ) is equal to 1 if the number visitations is greater than a threshold θ . The sum of edge weights , ∑i∈S wi , is used as a tie-breaker in the case of degenerate solutions . Degenerate solutions refer to the situation where different solution sets produce an identical cover on the network . In that case , we would like to give preference to the solution that contains nodes with higher overall edge weights , indicating greater degree of information transfer to the network , and potentially greater influence . This influence score is equivalent to computing the cover on nodes in T . In this work , θ = 0 . 0001 is used , which was selected after observing values in H . An implementation of the hypercube min-max ant optimization algorithm was used to search for solutions to the Influence Maximization Problem [41 , 42] . Ant optimization is based on the idea of probabilistically constructing potential solutions to a given problem , in this case a subset selection problem , and reinforcing good solutions with a "pheromone" weight deposited on solution components , ensuring that good solutions become increasingly likely in later iterations . Since the algorithm is stochastic , and results can vary , the optimization is repeated for a defined number of runs . The main results were produced using a ‘slow’ parameter set , using 8 restarts per value of K , 64 ants , and 16 local optimization steps ( full parameterization is given in S1 Text ) . Each convergence ( before restarting ) takes a number of iterations where ants construct solutions , perform a local search , score the solutions using the influence score , and reinforce the components in that order . As a run progresses , the pheromone values move to either one or zero , indicating whether the component was selected . The goal of the optimization is to find the subset S ⊆ V of vertices such that Sopt=argmax{S⊆V:|S|=K}Ωs . At the start of each iteration , ants construct potential solutions , a subset of vertices , by sampling from nodes using probability distribution qi=uiαriβ∑uiαriβ , where qi is the probability for sampling any node vi , with the sum of outgoing edges giving node weight ui and pheromone weight ri . The α and β parameters are used to give importance to either node weights or pheromones . Solutions are constructed by sampling one node at a time . After each sample , the probabilities are renormalized . Here , α and β are set to 1 . Local search is performed by stochastic hill climbing , where we try alternative solutions produced by random single bit flips . If a better score is found , the solution is replaced , and carried forward . Local search has a fairly strong effect on the quality of the solutions , and even a small number of hill climbing steps tends reduce the time required for convergence . Next , using the influence score function , each potential solution is scored , with the best solution kept and compared to solutions found in earlier runs . As part of the Min-Max algorithm , three solutions are kept throughout the run: the iteration-best , the restart-best and the overall-best . The pheromone updates use a weighted average over the three solutions . At the beginning of the run , the pheromone updates are entirely from the iteration-best solution , but gradually , the updates are increasingly influenced by the restart and overall-best solutions , which is done to avoid local minima . The weighted average pheromone would be ravg = f1bi + f2br + f3bb where bi is the iteration best , br is the restart best , bb is the best overall , and fractions f1 + f2 + f3 = 1 . The pheromone updates are defined as r ( t+1 ) = r ( t ) + d ( ravg − r ( t ) ) , where r ( t ) is the pheromone weights at time t , d is the learning rate , and ravg is the average over the three solutions . Eventually , the pheromone weights become sufficiently close to zero or one , and the rate of change among the weights slows . When the difference in sums over the last solution ( all r ) and the next solution is less than 0 . 0001 , the solution is returned along with the influence score . BioFabric , R and the R packages igraph , pheatmap and ggplot2 were used for visualization and analysis [43 , 44 , 45 , 46] . Cytoscape 3 . 5 . 1 was used for visualizing graphs [47 , 48] . Pathway and GO term enrichment was generated using the CPDB from The Max Planck Institute for Molecular Genetics [49] . SciPy was used in the software implementation [50] .
|
The Influence Maximization Problem ( IMP ) has been applied in fields such as epidemiology and social network analysis . Here , we apply the method to biological networks , aiming to discover the set of regulatory genes with the greatest influence on network dynamics . Fundamentally , since gene regulation is linked to the flow of information , we framed the IMP as an information theoretic problem . Dynamics were encoded as edge weights using time lagged transfer entropy , a quantity that attempts to quantify information transfer across variables . The influential nodes act as critical sources of information flow , potentially affecting the global network state . Biological events that impact the influential nodes and thereby affecting normal information flow could have a strong effect on the network , potentially leading to disease .
|
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"Abstract",
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"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
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"genetic",
"networks",
"gene",
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2017
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Solving the influence maximization problem reveals regulatory organization of the yeast cell cycle
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Plants are constantly exposed to a large and diverse array of microbes; however , most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens . The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking , endocytosis and the spatial distribution of organelles and protein complexes . For years , the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes , based largely on static images and pharmacological studies . To date , however , there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria . Here , we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv . tomato DC3000 . Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i . e . the perception of microbe-associated molecular patterns ( MAMPs ) during pattern-triggered immunity ( PTI ) and perturbations by effector proteins during effector-triggered susceptibility ( ETS ) . We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI . Notably , treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells , and this actin response required the host-cell MAMP receptor kinase complex , including FLS2 , BAK1 and BIK1 . Finally , we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria .
Actin filament arrays in plant cells undergo constant remodeling and can respond rapidly to a diverse array of extracellular stimuli . Even in unstimulated epidermal cells , architectural rearrangements occur within seconds due to myosin-dependent translocation , remarkably fast filament assembly , and the destruction of filaments by prolific severing activity [1] , [2] . This incessant remodeling of the actin cytoskeleton expends an enormous amount of energy , yet the physiological relevance of this is poorly understood . The actin cytoskeleton is a major signaling target and changes dramatically in response to numerous abiotic and biotic stimuli; the responses however are quite varied , ranging from filament bundling , to massive actin depolymerization , to assembly of new filament arrays [2]–[4] . For example , cells that are gently prodded with glass or tungsten needles generate extensively bundled filament arrays directly under the site of mechanical stimulation; yet , once the stimulus is removed the bundling is abrogated [5] . This is thought to mimic the efforts of fungi and oomycetes to gain entry into plant cells and , as such , it has been commonly assumed that attempted or actual penetration is responsible for eliciting changes in the host-cell actin cytoskeleton , rather than activation of host-cell defense signaling following the recognition of ‘non-self’ . Actin filament arrays undergo a markedly different response upon recognition of ‘self’ pollen grains by a flower's stigma . Poppy pollen , for example , initiates a self-incompatibility ( SI ) response , resulting in massive depolymerization of actin filaments within minutes of stimulus perception , effectively inhibiting pollen tube growth and blocking fertilization [6] . In contrast with this signal-mediated destruction of actin filaments , the interaction between mutualistic bacteria and plant cells generally results in the development of bright phalloidin-decorated spots in host cells [7] – suggestive of actin polymerization . Other changes to actin during mutualistic interactions have been described , including filament reorientation from longitudinal to transverse arrays and increased numbers of actin bundles at the tip of root hairs; these responses can be reproduced with the application of purified Nod-factors from mutualistic bacteria onto host plant cells [8] , [9] . Furthermore , nap1 mutant root hairs , which are incapable of proper nodule formation , lack the ability to elicit changes to the actin cytoskeleton when Nod-factors are applied because these mutants are deficient for ARP2/3-dependent actin polymerization [10] , [11] . On the other hand , certain signals from pathogenic fungi , like the Verticillium dahlia ( VD ) toxin , can stimulate dose-dependent destruction and relocation of cortical actin filaments to the perinuclear region [12] . Thus , biotic and abiotic signaling cascades produce a myriad of responses that can lead to dramatically different outcomes for actin organization and dynamics . Plants are constantly exposed to a large number of fungal and bacterial microbes , however , most plants are immune to the majority of potential invaders due to a multilayered defense system . The initiation of plant immunity relies on structural defenses ( i . e . the presence of trichomes , the closing of stomata to prevent bacterial entry , and the thickness and composition of the cell wall and cuticle ) and inducible measures to guard the plant from various microbes [4] . These inducible processes can be classified in two nodes of defense signaling: pattern-triggered immunity ( PTI ) and effector-triggered immunity ( ETI ) . PTI is a broad based immunity initiated through the host-cell recognition of conserved structural components , known collectively as microbe-associated molecular patterns or MAMPs , by cell-surface receptors [4] , [13] , [14] , [15] . The recognition of microbes by the receptor kinase , FLAGELLIN-SENSING 2 ( FLS2 ) , is one of the best-studied PTI pathways in Arabidopsis . Upon perception of bacterial flagellin or the synthetic 22-amino acid peptide flg22 , FLS2 associates with another receptor-like kinase , BAK1 ( BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE 1 ) ; this association releases the cytoplasmic kinase BIK1 ( BOTRYTIS-INDUCED KINASE 1 ) to induce down-stream defense signaling [4] , [16]–[19] , and ultimately prevents infection by non-adapted pathogens [4] , [13] , [14] . Pathogens elicit PTI in their respective host plants , and thus necessarily secrete or translocate various toxins and inject effector proteins into host cells to subvert PTI [14] , [15] . For example , Gram-negative bacteria use a specialized type III secretion system ( T3SS ) to translocate collections of type III effector ( T3E ) proteins directly into host cells resulting in effector-triggered susceptibility ( ETS ) of the host plant [4] , [13] , [14] . The second node of immunity , ETI , relies on host-cell recognition and response to pathogen-specific effector proteins or their activities , to mount a defense response that is more rapid and more pronounced than PTI [4] , [13] , [14] . The plant actin cytoskeleton has been implicated in the generation and maintenance of many aspects of PTI . Major hallmarks of PTI in plant cells include endocytic uptake of receptors , changes to cytoplasmic streaming , activation of defense genes via mitogen-activated protein kinase ( MAPK ) signaling , recruitment of NADPH oxidase to the plasma membrane , an oxidative burst , directed trafficking of Golgi and endoplasmic reticulum to the site of attack , and callose deposition [3] , [20]–[22] . The importance of an intact actin cytoskeleton for each of these responses has been demonstrated with pharmacological studies . The actin cytoskeleton is further assumed to play a central role in plant defense against microbes based on static images that show actin filament bundles impinging upon sites of both compatible and incompatible fungal or oomycete attack [3] , [20] , [21] , [23] . Finally , disrupting the plant actin cytoskeleton with cytochalasin E treatment or by overexpressing actin-binding proteins allows penetration into plant cells and tissues by incompatible fungi and oomycetes [24]–[26] . Despite the growing body of evidence that suggests the involvement of the actin cytoskeleton in either PTI or ETI , no direct evidence linking specific aspects of either layer of immune signaling have been reported . However , a recent report shows that a T3E protein from Pseudomonas syringae , HopZ1a , targets the microtubule cytoskeleton to circumvent PTI [27] . Similarly , one report describes actin filament stabilization through monoubiquitination of actin during infection by either pathogenic or mutualistic bacteria , but not in response to stress or viral infection [28] . We hypothesize that the continuous rearrangements of the actin cytoskeleton in Arabidopsis epidermal cells represent a surveillance mechanism to external threats [1]; however , it is still unclear whether there are direct links between actin and PTI- or effector-mediated signal transduction cascades or which actin-binding proteins are involved . Significantly , changes in the expression of Actin Depolymerizing Factor ( ADF ) enhance non-host susceptibility in both fungal and bacterial pathosystems , but these changes do not alter the focal accumulation of profilin at pathogen-invasion sites or expression of defense genes [26] . Notably , the localization of Arabidopsis ADF4 to the nucleus correlates with the reduced expression of the hallmark PTI-defense gene , FRK1 , which implicates the actin cytoskeleton in the early onset of PTI [29] . Additionally , this adf4 knockout mutant fails to activate ETI in response to P . syringae expressing the cognate bacterial effector gene AvrPphB [30] . Collectively , these results suggest that actin organization and dynamics are strictly regulated in both PTI and ETI . To date , the timing and nature of actin-based responses in host cells during bacterial pathogen attack have not been described . Using a combination of bacterial mutants and advanced imaging of actin cytoskeleton organization in epidermal cells from Arabidopsis , we analyzed the host-cell response to the bacterial phytopathogen P . syringae pv . tomato DC3000 . We quantified the nature of specific changes in actin array architecture over a time-course of infection with both pathogenic and non-pathogenic strains of bacteria . A transient increase in the density of actin filament arrays in the cortex of epidermal cells was identified , and we demonstrate that this change did not require either the T3SS or effector proteins . Moreover , we found that infiltration of leaves with MAMPs was sufficient to elicit an increase in actin filament density . Using reverse genetics , we have also begun to dissect the plant signaling pathways required to elicit actin rearrangement during PTI . Notably , we found that FLS2 , BAK1 and BIK1 were required for the increase in actin filament density . When actin polymerization was blocked by treatment with latrunculin B , the increase in actin filament density did not occur and plants were more susceptible to infection with pathogenic and non-pathogenic bacteria . These data implicate the transient increase in cytoskeletal array density as a contributing factor during PTI and identify parts of the signal transduction machinery necessary for this response .
In this study , we focused on cytoskeletal responses in the Arabidopsis–Pseudomonas pathosystem and used seedlings expressing a well-characterized actin reporter , GFP-fABD2 . Dip-inoculated cotyledons from wild-type Col-0 and transgenic plants expressing GFP-fABD2 , a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis FIMBRIN1 [1] , exhibited necrotic lesions when inoculated with pathogenic P . syringae pv . tomato DC3000 ( hereafter referred to as DC3000; Figure S1A & C ) , whereas cotyledons inoculated with the non-pathogenic T3SS-deficient mutant hrpH did not ( Figure S1B & D ) . Furthermore , cotyledons ( Figure S1E ) and rosette leaves ( Figure S1F ) inoculated with DC3000 had a higher bacterial load than those inoculated with hrpH at 4 days after inoculation . These results confirm that DC3000 can proliferate and cause disease symptoms on seedling cotyledons expressing GFP-fABD2 , and that bacterial growth is not significantly different when bacteria are infected in cotyledons or rosette leaves . To study the response of host-cell cytoskeleton during bacterial infection , we imaged actin filament arrays in cotyledons with spinning disk confocal microscopy ( SDCM ) at various time-points after dip-inoculation with DC3000 and hrpH ( Figure 1 ) . Epidermal pavement cells from Arabidopsis cotyledons , display two populations of actin filaments in the cortical cytoplasm—dynamic , faint structures that resemble single actin filaments; and , thick , bright actin filament bundles ( Figure 1A & D ) . At 6 hours post inoculation ( hpi ) , we observed an increase in the abundance of actin filaments in the cortical array of epidermal cells inoculated with DC3000 ( Figure 1B ) or hrpH ( Figure 1C ) , compared to mock-treated material ( Figure 1A ) . At 24 hpi , we noticed little difference between the mock control ( Figure 1D ) and hrpH inoculation ( Figure 1F ) ; however , obvious actin filament bundling occurred following DC3000 treatment ( Figure 1E ) . To further investigate the timing and nature of actin responses following DC3000 inoculation , we used a set of previously validated tools for measuring actin array organization [31]–[33] . We quantified and statistically compared maximum intensity projections generated from fields of Arabidopsis epidermal cells for changes in the extent of actin filament bundling ( skewness ) and percent occupancy ( density ) following microbial infection . The bundling parameter is based on the assumption that a population of individual actin filaments will have a Gaussian distribution of pixel intensities , which becomes skewed in favor of brighter pixels , when the array of actin filaments becomes more bundled [31] . The density metric is calculated as the percent occupancy of GFP-fABD2-containing pixels in each micrograph [31] . For these analyses , we performed a time-course from 0 to 36 h after DC3000 infection by sampling at 3-h intervals ( Figure 2 ) . We observed a transient increase in the abundance of actin filaments in host cells at 0–15 hpi following DC3000 treatment ( Figure 2A ) and this occurred as early as 15–30 min after inoculation ( Figure S2 ) . Actin filament abundance was elevated by as much as 16% , with a peak at 6–9 hpi , and then significantly decreased from 24–36 hpi onward ( Figure 2A ) . We also detected significantly enhanced filament bundling at 18–36 hpi , with the most prominent bundling at 24–27 hpi ( Figure 2B ) . Mock-treated seedlings had no significant changes in actin architecture compared to untreated seedlings ( Figure S3 ) . These results revealed two distinct and statistically significant changes in actin filament organization following infection with virulent pathogen , i . e . an early and transient increase in actin filament density as well as a late increase in the extent of actin filament bundling . These observations are consistent with the immediate perception of DC3000 and response of the plant immune system followed by a subsequent suppression of PTI by the pathogen . If the rapid and transient increase in actin filament density in epidermal cells exposed to DC3000 is part of the PTI response , then we predict that the same cytoskeletal change will occur with various phytopathogens that are not adapted to Arabidopsis . To test this , we quantified actin array architecture in cotyledons at 6–9 h following infection with several non-adapted pathogens that trigger PTI in Arabidopsis , including the bean pathovar P . syringae pv . phaseolicola ( Pph ) ; Agrobacterium tumefaciens; and the rice-blast fungus Magnaporthe grisea ( Figure 3 ) . Following Pph inoculation , we observed a significant increase in actin filament density ( Figure 3A ) , but no change in the extent of filament bundling ( Figure 3B ) . Additionally , we observed increased actin filament density with A . tumefaciens ( Figure 3C ) and M . grisea ( Figure 3E ) treatments , but no change in bundling ( Figure 3D & F ) . In summary , the density or abundance of actin filaments is elevated in cotyledons following treatment with various bacterial and fungal microbes and likely represents a broad-based PTI response . Since the increase in actin filament abundance was observed in host cells in response to both adapted and non-adapted microbes ( Figure 3 ) , we predict that the increased actin filament density occurs independent of the T3SS or translocated effector proteins . Therefore , we used genetic mutants to dissect the molecular nature of P . syringae's ability to elicit the host-actin response . First , we quantified actin filament architecture in cotyledons treated with the T3SS-deficient mutant hrpH over a full time-course and observed a significant increase in percent occupancy from 0–15 hpi following inoculation ( Figure 2A ) . Actin filament density peaked at 6–9 hpi , similar to DC3000; however , no decrease in density at 24–36 hpi was observed ( Figure 2A ) . In contrast to DC3000 , no increase in filament bundling was observed at any time-point following hrpH inoculation ( Figure 2B ) . The similar responses to DC3000 and hrpH inoculations at 0–15 hpi further support the argument that the transient increase in actin filament density is PTI-based . Moreover , because hrpH does not induce bundling in epidermal cells whereas DC3000 does , it is likely that bundling is associated with effector-triggered susceptibility ( ETS ) . To further dissect whether the actin organization changes were part of a general response to bacteria or can also be influenced during ETI , we quantified the actin filament architecture over a full time-course in Arabidopsis cotyledons following inoculation with P . syringae DC3000 expressing the YopT homolog , AvrPphB ( [34]–[36]; Figure S4 ) . Similar increases in actin filament density were observed with DC3000 and DC3000 expressing AvrPphB inoculations at 0–15 hpi ( Figure S4A ) . In contrast , filament bundling was not as pronounced with DC3000 expressing AvrPphB inoculation compared to DC3000 ( Figure S4B ) and the filament density did not decline at 24–36 hpi , suggesting that these later changes in filament array architecture are part of a gene-for-gene response or ETI . Finally , we quantified cortical actin architecture in cotyledons treated with another T3SS-deficient mutant , hrcC [37] , and a D28E mutant that expresses the T3SS but lacks most T3E genes ( [38]; Figure 4 ) . Following hrcC or D28E inoculation , we observed a significant increase in percent occupancy similar to treatments with hrpH and DC3000 ( Figure 4A & C ) , but no change in filament bundling at 6–9 hpi ( Figure 4B & D ) . Sensu stricto , these results demonstrate that the early increase in actin filament density is associated with PTI . As an alternative or in addition to T3SS activity , P . syringae might alter the host-cell actin cytoskeleton through the secretion of pathotype-specific toxins , such as coronatine , a jasmonate mimic synthesized by DC3000 [39] . Following inoculation of cotyledons with the coronatine-deficient mutant , COR- [37] , we observed a significant increase in filament density in host cells ( Figure 4E ) but no change in filament bundling ( Figure 4F ) . This confirms that less virulent bacteria still elicit an increase in actin filament density and that coronatine is not necessary for DC3000 to elicit changes in the cytoskeleton of epidermal cells . To further examine the mechanism of increased actin filament density during PTI , we inoculated cotyledons with a mutant of P . syringae that has the flagellin gene deleted ( ΔfliC; [40] ) and quantified changes in actin organization . We observed a significant increase in filament density ( Figure 4G ) and no change in bundling ( Figure 4H ) . Although this increase in filament abundance was significantly less than the increase associated with DC3000 treatment , flagellin is not strictly necessary to elicit the increase in actin filament density . These results imply that the perception of other MAMPs can also lead to changes in actin organization . To investigate whether MAMPs are sufficient to elicit a change in actin organization , we challenged plants with synthetic MAMP peptides , as well as with the fungal glucosamine polymer , chitin , and monitored changes in actin architecture . We used flg22 , the N-terminal twenty-two amino acids from Pseudomonas flagellin [41] , [42]; elf26 , a twenty-six amino acid peptide from bacterial EF-Tu [43]; and flgAt , the amino terminal sequence from Agrobacterium flagellin which does not elicit a response in Arabidopsis [42] . These peptides were introduced at various concentrations into mature leaves and the actin responses at 0–3 h after infiltration were quantified ( Figure 5 & S5 ) . With flg22 , we detected a rapid and dose-dependent increase in actin filament density ( Figure 5A & S5A ) ; however , no change in the extent of filament bundling was detected at any concentration tested ( Figure 5B & S5B ) . We detected similar changes to actin filament arrays in plants infiltrated with chitin ( Figure S5G & H ) . In contrast , infiltration with elf26 or flgAt was indistinguishable from mock at all concentrations tested ( Figure 5 & S5C–F ) even though , all three MAMP peptides were able to stimulate a PTI-based defense response as demonstrated by activation of FRK1 transcripts ( Figure S6 ) . In conclusion , treatments with flg22 or chitin are sufficient to induce rapid changes in actin filament organization . To examine the role of early cellular signaling pathways during PTI , we performed actin architecture analysis on several Arabidopsis knockout mutants , including a susceptible Arabidopsis ecotype , infiltrated with either 1 µM flg22 or 1 µM chitin ( Figure 6 ) . Wild-type Col-0 plants showed significantly enhanced filament abundance following treatment with flg22 ( Figure 6B & D ) or chitin ( Figure 6C & D ) , compared with mock treatment ( Figure 6A & D ) . The homozygous flagellin receptor mutant , flagellin sensing-2 ( fls2 ) , in the Col-0 background , as well as the Fls2-deficient ecotype Wassilewskija-0 ( Ws-0 ) , both lacked a significant increase in filament abundance following treatment with flg22 ( Figure 6G & I and Figure 6L & N , respectively ) . Whereas with chitin treatment , actin filament abundance was significantly increased following treatment of epidermal pavement cells in either fls2 ( Figure 6H & I ) or Ws-0 ( Figure 6M & N ) plants . To further test which signaling pathways may involve actin , we performed actin filament density analysis on two additional DC3000-susceptible Arabidopsis knockout mutants , brassinosteroid insensitive1-associated kinase1 ( bak1-4 ) and botrytis-induced kinase 1 ( bik1 ) , which are both known to associate with the FLS2 receptor [17] , [18] . Neither bak1-4 ( Figure 6P–T ) nor bik1 ( Figure 6U–Y ) homozygous mutant plants showed significant changes from mock following treatment with flg22 or chitin . Additionally , no significant changes to filament bundling were observed following treatment with either flg22 or chitin for any of the plant lines tested . Since flg22 peptide and chitin are sufficient to elicit an increase in actin filament abundance , we tested whether Pseudomonas bacteria could elicit a similar response in Arabidopsis lines with altered defense signaling . Wild-type Col-0 , the Ws-0 ecotype , and the fls2 knockout mutant were dip-inoculated , individually , with multiple Pseudomonas mutants and changes to the host cytoskeleton at 6–9 hpi were investigated ( Figure S7 ) . Notably , the actin response was reduced in both the fls2 mutant ( Figure S7C ) and the Ws-0 ecotype ( Figure S7E ) , but not completely ameliorated , indicating that some changes to the actin cytoskeleton are independent of FLS2 . However no significant changes to the extent of actin filament bundling were observed with any Pseudomonas strain or host-plant at this time-point ( Figure S7B , D & F ) . Latrunculin B ( LatB ) is a macrolide compound from marine sponges that inhibits actin polymerization by binding to monomeric actin and preventing its assembly onto filament ends [44] . To test whether actin polymerization is necessary for the increase in filament density during the initial response to phytopathogens , we co-infiltrated Arabidopsis leaves with various concentrations of LatB and DC3000 or hrpH Pseudomonas strains . At 6–9 hpi , actin filaments in epidermal pavement cells appeared to be markedly reduced following infiltration with LatB alone ( Figure 7A , D & G ) or LatB co-infiltrated with either DC3000 ( Figure 7B , E & H ) or hrpH ( Figure 7C , F & I ) . As shown previously [1] , short-term treatments with low doses of LatB primarily affected the dynamic actin filament arrays and individual filaments , whereas only modest effects on filament bundles were observed ( Figure 7D–I ) . Using the metrics described earlier , we measured a significant decrease in actin filament density following LatB infiltration in mock-treated plants compared to the 0 µM control ( Figure 7J ) . Further , we measured a significant and dose-dependent reduction in actin filament abundance in plants that were co-infiltrated with LatB and DC3000 or hrpH ( Figure 7J ) . However , no significant changes to the extent of filament bundling were observed at this early stage of infection ( P-value = 0 . 49 , ANOVA; data not shown ) . This demonstrates that actin polymerization is necessary for the increase in filament density following infection with pathogenic and non-pathogenic bacteria during the PTI response . If actin polymerization is an important component of PTI , then blocking this aspect of the host-cell response should enhance susceptibility to pathogens . To test this , we again co-infiltrated Arabidopsis plants with various concentrations of LatB and either DC3000 or hrpH and measured bacterial growth at 0 , 6 , 12 , 24 , and 48 h after treatment . As predicted , bacterial growth was significantly increased in plants co-infiltrated with DC3000 and 1 or 10 µM LatB compared to 0 µM LatB treatments ( Figure 7K ) . We also measured a significant increase in bacterial growth following co-infiltration with the non-pathogenic strain , hrpH , and LatB ( Figure 7K ) indicating that the host-actin cytoskeleton and actin polymerization are necessary for innate immunity .
The increase in actin filament density observed in Arabidopsis cotyledons is associated with a rapid PTI response; it could be detected as early as 15–30 min after inoculation with DC3000 . This response peaked at 6–9 hpi and was abrogated by 15 hpi . Further , this increase occurred in cotyledons inoculated with pathogenic and non-pathogenic Pseudomonas strains and mutants . It also occurred when seedlings were inoculated with Agrobacterium tumefaciens , and the non-host fungus Magnaporthe grisea , which indicates that the increase in actin filament density is a broad-based response to microbial perception . The increase in actin filament density is not the result of mechanical stimulation by the T3SS , as two T3SS-deficient mutants ( hrpH and hrcC ) still elicited the early cytoskeletal response . Although we observed significantly less actin filament percent occupancy following D28E inoculation compared to DC3000 , the density of filaments was still significantly higher than the mock-treated control . Since each of the P . syringae mutants utilized herein is thought to contain the same suite of MAMPs ( with the notable exception of ΔfliC ) , this significant reduction in filament abundance may imply a reduction in bacterial growth or indicate the action of T3E proteins in this response . The latter possibility seems unlikely as plants treated with P . syringae DC3000 expressing AvrPphB led to an similar actin response at early time-points and a reduced filament and bundling response at late time-points . Changes to actin filament density , usually described as increases in polymerization [9] or an increase in phalloidin-stained spots [7] , are also observed with mutualistic bacteria as well as with compatible and incompatible fungal and oomycete interactions [3] , [20]–[23] . Despite the commonly accepted dogma that actin responses are caused by the mechanical stress of invading pathogens [5] , [21]–[23] , our data indicate that host-cell penetration by an invading pathogen is not necessary to elicit changes to the actin cytoskeleton . Here , we show that Magnaporthe spores , which do not attempt to penetrate Arabidopsis epidermal cells , still elicit an increase in actin filament abundance . Previously , a Magnaporthe mutant that is deficient for the ability to penetrate cells could also stimulate rearrangement of the host cytoskeleton in onion epidermal cells , leading the authors to speculate that this was due to sensing fungal cues other than the penetration peg [45] . Unlike fungi and oomycetes which use specialized structures to penetrate host cells , phytopathogenic bacteria reside in intercellular spaces; as a result , signals indicating the presence of these bacteria may affect multiple cells , rather than a single point of invasion . Therefore , mounting a host-defense response likely requires a broad and non-localized defense mechanism using cell surface receptors . Taken together with our quantitative results , it is likely that the increase in actin filament density is a conserved , receptor-mediated response to the perception of microbes by host plant cells . Significantly , both bacterial and fungal MAMPs were capable of eliciting the increases in actin filament density in host plants . In this study , the flg22 peptide mimic of bacterial flagellin was sufficient to elicit a dose-dependent increase in actin filament density as early as 0–3 hpi . This fast response was not observed with a peptide mimic of the bacterial elongation factor EF-Tu ( elf26 ) , which indicates that the actin response may be specific to particular MAMPs , or could represent differential expression of MAMP receptors in organs , tissues and specific cell types [46] . For example , the EF-Tu receptor , EFR , may not be expressed in epidermal cells of cotyledons or rosette leaves but is still expressed in the whole organ . This could lead to lack of a detectable actin-based response in epidermal cells , whereas transcriptional hallmarks of PTI are still present in the whole organ . Another alternative is that the actin-based response in epidermal cells occurs rapidly and goes undetected over the timescales we are able to measure by SDC microscopy . In this way , a fast response would become “averaged-out” at the earliest timepoints measured . The increase in actin filament density still occurred in response to the ΔfliC mutant , indicating that additional MAMPs also trigger this response or that the presence of flagella is not completely abrogated in the ΔfliC mutant , despite lack of mobility . This increase in actin abundance occurs independently of the FLS2 receptor following DC3000 or ΔfliC treatment . Since , multiple Pseudomonas mutants are still capable of eliciting the increase in actin filament abundance 6–9 hpi in the absence of components of the FLS2 receptor complex , this further indicates that multiple or additional MAMPs are capable of altering host-actin architecture . Additionally , the application of fungal chitin on leaves ( this study ) or bacterial Nod factors on root hairs [9] also stimulated an increase in actin filament density , which further indicates that the host-actin cytoskeleton plays a general role in the perception of beneficial and pathogenic microbes . A main hallmark of PTI is signaling through MAPK and CDPK phosphorylation cascades after host-perception of various MAMPs [4] , [16]–[19] . For example , once flagellin or flg22 has bound the FLS2 receptor , BAK1 associates with FLS2 , and following this association , the cytoplasmic kinase BIK1 dissociates from the receptor complex initiating host-defense signaling [16]–[19] . As expected , Arabidopsis mutants or ecotypes with deficiencies in the flagellin-sensing pathway ( i . e . fls2 , Ws-0 ) did not display changes actin filament architecture following treatment with the flg22 MAMP peptide . However , actin architecture changes still occurred following chitin treatment , indicating that chitin-induced signaling is still intact . Further , mutants in the shared signaling nodes between the flagellin- and chitin-signaling pathways ( i . e . bak1-4 and bik1 ) did not display any significant changes to actin architecture following either bacterial or fungal MAMP treatment , indicating that actin rearrangements are conserved in several common immune pathways . Our data provide evidence that the host-actin cytoskeleton plays an important role in innate immunity because the actin polymerization inhibitor LatB promotes the growth of P . syringae DC3000 on Arabidopsis leaves . Further this growth advantage is conferred specifically during PTI as the T3SS-deficient mutant hrpH also exhibited significantly enhanced growth . It is well known that an intact actin cytoskeleton is required for receptor-mediated endocytosis of ligands including the flagellin receptor FLS2 [47] , as vesicle dynamics are reduced following treatment with either LatB or the actin stabilizer endosidin1 [48]; however , the actin cytoskeleton almost certainly plays additional roles during response to microbes . The requirement of the actin cytoskeleton for activation of NADPH oxidase at the plasma membrane , as well as Golgi , peroxisomes and endoplasmic reticulum trafficking toward sites of fungal and oomycete penetration , has been demonstrated through pharmacological studies [20]–[23] . Presumably , the trafficking of Golgi and ER is important for the production and deposition of antimicrobial compounds and fortification of the cell wall [20]–[23] . The specific targeting of defense proteins to the cell membrane is also an actin cytoskeleton-dependent process . The fungal resistance protein RPW8 . 2 prevents haustorium development and reduces oxidative damage to host cells by generating a unique membrane that fuses to the extrahaustorial matrix ( EHM; [49] ) . Targeting of RPW8 . 2 to the membrane is disrupted with cytochalasin E treatment; plants susceptible to the powdery mildew fungus had less EHM localization and are unable to activate the same proteins as resistant plants [49] . In contrast , one actin-independent mechanism in plant defense is the accumulation of a SNARE involved in membrane fusion events at the plasma membrane , PEN1 , at the fungal penetration site [20] , [23] , [50] . This differs from PEN2 and PEN3 , which are implicated in callose deposition and require the actin cytoskeleton for proper localization during fungal infection [22] , [50] , [51] . Finally , perturbations to the actin cytoskeleton using drugs and toxins have been shown to trigger or alter programmed cell death in plant cells [2] . Taken together , these observations speak to the involvement of the actin cytoskeleton at various time-points , from minutes to hours , during common biotic stress events . The regulation and turnover of the actin cytoskeleton requires the concerted activities of hundreds of actin-binding proteins that can respond to signals to polymerize or destroy actin filament networks . The growth of individual actin filaments in the cortical array of Arabidopsis epidermal cells is extremely fast , ∼2 µm/s , and most filaments exist for less than 30 s before being destroyed by prolific severing activity [1] , [33] , [52] . This constant formation and destruction of actin networks requires a huge expense of energy—on the order of millions of ATPs per second—and is thought to represent a surveillance mechanism to various biotic and abiotic stresses [1] , [12] . It is easy to imagine that changes to any number of actin-binding proteins involved in actin dynamics could result in altered filament arrays; and that targeting specific aspects of the cytoskeleton would be an excellent opportunity for successful pathogen attack . Plant actin-binding proteins respond to a plethora of second messengers in signaling cascades , including Ca2+ , phospholipids and pH [2] , [53] , [54] . A potential link between the actin cytoskeleton and specific cytosolic Ca2+ signatures following microbial infection requires additional study [2] , [55] . Further , calcium and pH fluctuations are known to occur in Arabidopsis during defense responses [56] , [57] . Several plant actin-binding proteins have different activities as pH fluctuates from alkaline to acidic [58] , [59] . Finally , there is a long history of alterations to actin filament arrays through actin-binding proteins sensing changes to concentrations and types of phospholipids like phosphatidylinositol ( 4 , 5 ) -bisphosphate ( PIP2 ) and phosphatidic acid [60] . Phosphatidic acid is also a second messenger for plant defense responses that can activate MAPK signaling and defense genes [61]–[63] and accumulates upon treatment with various MAMPs [63]–[66] . The initiation of immunity in plants requires the concerted effort of both PTI signaling and the recognition of microbe-derived proteins evolutionarily adapted to circumvent innate immunity . A hint that the host cytoskeleton is a target for effector proteins , comes from the use of the Harpin elicitor , which triggers defense responses in host and non-host plants [67] . Specifically , Harpin elicitor treatment of grapevine cells triggers host-microtubule depolymerization within 3 hours , but has variable effects on the actin cytoskeleton [67] . The first example of a bona fide phytopathogenic effector protein specifically targeting the plant cytoskeleton is HopZ1a , which depolymerizes microtubules thereby disrupting the plant secretory pathway and suppressing cell wall-mediated defenses [27] . The involvement of microtubule rearrangements during PTI that results from recognition of DC3000 is unclear since this particular pathovar of P . syringae does not elicit changes in microtubule organization [27] and lacks HopZ1 . Additionally , which cytoskeleton is targeted first remains an unanswered question , as DC3000 expressing HopZ1a did not disrupt the actin cytoskeleton at 16 hpi [27] and our data show changes to the actin cytoskeleton as early as 15 min after inoculation . Effector proteins likely target the actin cytoskeleton , as inoculations with the T3SS-deficient hrpH did not elicit the increased bundling that was obvious with DC3000 treatment . A role for actin in ETI is indicated by data from adf4 knock-out Arabidopsis plants , which are unable to elicit a hypersensitive response and are susceptible to P . syringae expressing AvrPphB [30] . Although the exact mechanism by which ADF4 mediates resistance to bacteria carrying AvrPphB is still unknown , it has been demonstrated recently that ADF4 is required for activation of resistance to DC3000 expressing AvrPphB through control of expression of the R-gene RPS5 [29] . Furthermore , this work correlated changes in the localization of ADF4 with the reduced expression of FRK1 and MAPK signaling , further implying a dual role for the actin cytoskeleton in the host response to phytopathogens . In summary , we monitored the nature and timing of changes to the actin cytoskeleton in Arabidopsis during microbial infection . We quantified two distinct actin responses—a rapid transient increase in actin filament density and a late increase in filament bundling . We demonstrate that the early transient increase in actin filament density is associated with PTI by using adapted and non-adapted microbes and treatments with MAMPs . We also established the requirement of host-cell signaling machinery including the flagellin receptor complex , FLS2 , BAK1 and BIK1 , for the increase in actin filament abundance . This is the first evidence for temporal changes in actin cytoskeleton organization during PTI elicited by a phytopathogenic bacterium , and uncovers the initial MAMP signaling cascade responsible for altering the cytoskeleton .
Arabidopsis thaliana Ws-0 , fls2 ( SALK_062054 ) , bak1-4 ( SALK_116202 ) , bik1 ( SALK_005291 ) were transformed with GFP-fABD2 [68] using the floral dip method described previously [69] . T1 plants were screened on appropriate antibiotics and by fluorescence microscopy . Multiple T2 plants ( n≥9 ) for each genotype were used for actin architecture analysis . Wild-type A . thaliana Col-0 , Col-0 expressing GFP-fABD2 [1] , and T2 plants expressing GFP-fABD2 were sown onto soil and stratified for 3 d at 4°C . Flats were transferred to a growth chamber and plants grown under long-day conditions ( 16 h light , 8 h dark ) at 21°C for 10 or 24 d . Information about the Pseudomonas mutants and strains , as well as Agrobacterium and Magnaporthe strains , used in this study are found in Table S1 in Text S1 . Various bacterial strains were grown on NYGA media ( 0 . 5% [w/v] Bacto Peptone , 0 . 3% [w/v] yeast extract , 2% [v/v] glycerol , 15% [w/v] Bacto agar ) and diluted with 10 mM MgCl2 to a concentration of 3×107 colony-forming units ( CFU ) mL−1 [70] . Ten day-old Arabidopsis seedlings were infected by gently agitating inverted seedlings in bacterial suspensions supplemented with of 0 . 02% [v/v] Silwet . MAMP peptides , flg22 [41] , elf26 [43] , and flgAt [41] all from NeoBioSci ( Cambridge , MA ) , and chitin ( Sigma-Aldrich , St . Louis , MO ) were diluted in 10 mM MgCl2 at various concentrations . For hand infiltration of peptides , and latrunculin B co-infiltration with Pseudomonas strains , leaves of similar size from 24 d-old plants were designated for analysis with a marker . Leaves were gently infiltrated with an inoculum of 1×105 CFU/mL using a 3 mL needle-less syringe until intercellular spaces were filled with solution ( ∼300–400 µL per leaf ) . After infiltration , inoculated plants were covered for 30 min and returned to the growth chamber prior to imaging . All image collection and data analyses were performed as single-blind experiments . Actin filament bundling and percent occupancy were measured using two metrics: skewness , based on the assumption that a population of actin filaments exhibits enhanced pixel intensities when bundled; and , density , an estimation calculated as the percent occupancy of signal ( actin filaments ) separated from background by setting a minimal threshold to include all actin filaments [31] . We imaged fields of epidermal pavement cells with spinning disk confocal microscopy ( SDCM ) by collecting 24 steps of 0 . 5 µm each starting at the plasma membrane . Spinning disk confocal microscopy was performed using a Yokogawa CSU-10 mounted on a Zeiss Observer Z . 1 equipped with a 100X/1 . 46 NA PlanApo objective . Illumination was from a solid-state 50-mW laser with AOTF control over excitation wavelength ( Intelligent Imaging Innovations , Denver , CO ) . The 488-nm laser emission was captured with an Evolve512 EMCCD camera ( Photometrics , Tucson , AZ ) . The SDCM was operated using SlideBook software ( version 5 . 0 . 031; Intelligent Imaging Innovations ) . A fixed specimen exposure time and gain setting were selected such that individual actin filaments could be observed , but actin filament bundles were not saturated . Maximum-intensity projections of z-series stacks were analyzed in ImageJ using algorithms described previously [31]–[33] . Gaussian blur and high-band pass filters were applied to projections prior to density analysis . No image processing was applied to maximum-intensity projections that were analyzed for skewness . At least 90 images were analyzed per time-point per treatment , from at least 30 individual seedlings for each measurement . Statistical comparisons and ANOVA test with Tukey HSD post-hoc analysis were carried out using KaleidaGraph ( version 4 . 1 . 3b1; Synergy Software , Reading , PA ) .
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The cytoskeleton is a dynamic platform for sensing and responding to a diverse array of biotic and abiotic stresses . The nature and timing of the changes in actin organization range from excessive bundling , to massive depolymerization , to new filament assembly , depending on the particular signal and the responding cell type . Here , we use the Arabidopsis–Pseudomonas pathosystem to dissect pathogen-derived cues that elicit changes in the plant host-cell cytoskeleton . Overall , we provide the first evidence that the actin cytoskeleton rearranges in response to a phytopathogenic bacterium and we quantified the temporal response of epidermal cells to Pseudomonas syringae pv . tomato DC3000 strains and susceptible Arabidopsis mutants , using a robust set of tools for measuring changes in actin organization . An immediate but transient increase in actin filament abundance was associated with pattern-triggered immunity . This response could be mimicked with microbe-associated molecular pattern peptide treatments . Second , we observed a late increase in actin filament bundling that appears to be part of effector-triggered susceptibility . We dissected the initial steps involved in the host-cell signaling pathway and demonstrated that FLS2 , BAK1 , and BIK1 were required for the actin response . Collectively , these findings demonstrate that rapid changes in host-cell cytoskeleton organization occur in response to receptor-mediated signaling during plant innate immunity .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology"
] |
2013
|
The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns
|
The Alternative Lengthening of Telomeres ( ALT ) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines . ALT is thought to involve templated extension of telomeres through homologous recombination , but the genetic or epigenetic changes that unleash ALT are not known . Recently , mutations in the ATRX/DAXX chromatin remodeling complex and histone H3 . 3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers , pediatric glioblastomas , and other tumors of the central nervous system , suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers . We have taken a comprehensive approach to deciphering ALT by applying genomic , molecular biological , and cell biological approaches to a panel of 22 ALT cell lines , including cell lines derived in vitro . Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT–immortalized cell lines . In addition , ALT is associated with extensive genome rearrangements , marked micronucleation , defects in the G2/M checkpoint , and altered double-strand break ( DSB ) repair . These attributes will facilitate the diagnosis and treatment of ALT positive human cancers .
In the absence of telomerase activity , telomeres shorten with cell division , ultimately leading to senescence . Hence , the development of human cancer is associated with an active telomere maintenance system that provides an infinite source of telomeric DNA to potentiate immortality . Although telomerase reactivation is the most common mechanism of telomeric repeat addition in cancers , a significant subset of human tumors employs a telomerase-independent telomere maintenance pathway , referred to as ALT [1] . The emerging view is that ALT maintains telomeres through homology-directed recombination ( HDR ) [2] . Supporting this view , ALT cells show an elevated frequency of sequence exchanges between telomeres [2]–[4] , contain extrachromosomal linear and circular telomeric DNA [5]–[8] , and often exhibit heterogeneously-sized telomeres [1] , features consistent with hyperactive HDR . The extrachromosomal telomeric DNA has been proposed to serve as a template for the extension of telomeres by a recombination mechanism akin to Break Induced Replication ( BIR ) [9] , [10] . In addition , ALT cells often carry altered PML bodies ( ALT-associated PML bodies , or APBs ) that contain telomeric DNA as well as numerous recombination factors [11] . Several proteins involved in recombination are known to be required for ALT , including the Mre11 complex , Mus81 , and the SMC5/6 sumoylation pathway [6] , [12]–[15] . ALT was discovered in a subset of immortalized cell lines that emerge at low frequency from human cell cultures in telomere crisis , but this pathway has increasingly been identified in human cancer . Approximately 10–15% of human cancers are suspected to use the ALT pathway based on their lack of telomerase activity in combination with certain hallmarks of ALT , such as the presence of extrachromosomal telomeric circles , APBs , and/or long and heterogeneous telomeres . Based on these criteria , ALT is relatively common in many sarcomas ( osteosarcoma and some types of soft tissue sarcomas ) , certain endocrine tumors ( pancreatic neuroendocrine tumors , paraganglioma ) , a subset of nervous system tumors ( e . g . glioblastoma , medulloblastoma , oligodendroglioma , astrocytoma , ganglioneuroblastoma ) , bladder small cell carcinoma , and nonseminomatous germ cell tumors [16]–[18] . The molecular basis for the activation of the ALT pathway is not known . In vitro , the frequency of conversion to ALT is low , typically requiring many months of culturing of virally-transformed ( p53/Rb deficient ) human cells that have entered telomere crisis [11] . The low frequency of conversion to ALT suggests that mutations and/or infrequent epigenetic alterations are required to unleash this pathway . Indeed , in telomerase-positive mammalian cells with fully functional telomeres , telomeric recombination is stringently repressed by the TRF2 , Rap1 , and POT1 components of shelterin as well as by the Ku70/80 heterodimer [6] , [19]–[23] . Mutations in shelterin and Ku might therefore be anticipated in ALT , a notion that is tested here . Recently , a subset of pancreatic neuroendocrine tumors ( PanNETs ) were found to have ultrabright telomeric FISH signals , suggesting the presence of ALT-like long telomeres [24] , [25] . These tumors also exhibited alterations in ATRX or its binding partner DAXX . Twenty-five PanNETs with ultrabright telomeric foci had ATRX/DAXX mutations or lacked nuclear ATRX/DAXX protein . In contrast , 16 PanNETs with apparently normal ATRX/DAXX lacked the aberrant telomeric staining patterns . The same correlation between inactivation of the ATRX pathway and the ALT-like phenotype was observed in pediatric glioblastoma and several other cancers [16] , [26] . ATRX and DAXX act together in a replication-independent chromatin assembly pathway that deposits the histone variant H3 . 3 at telomeres and perhaps at other G-rich repetitive elements [27]–[31] . The function of ATRX/DAXX and histone H3 . 3 at telomeres is not yet clear , but diminished ATRX function has been documented to increase TTAGGG repeat containing telomeric transcripts ( referred to as TERRA [32] ) , reduce telomeric loading of HP1α , and cause modest levels of telomere dysfunction in mouse ES ( but not NIH3T3 ) cells as gleaned from the localization of γ-H2AX at chromosome ends [29] , . ATRX deficiency also leads to a defect in sister chromatid cohesion and aberrant mitoses , culminating in the formation of micronuclei , lobulated nuclei , and chromatin bridges [33]–[35] . Because functional assessment of ALT is not possible in tumor specimens , further tests for the association of ALT with ATRX/DAXX deficiency requires analysis of in vitro immortalized cell lines . Here we describe the results of a comprehensive effort to characterize the genetic alterations and associated phenotypes of 22 human ALT cell lines . We found loss of ATRX in 90% of in vitro immortalized ALT lines , suggesting that inactivation of ATRX is a major step in generating the ALT phenotype . We also document that ALT cell lines are characterized by extensive genomic instability , formation of micronuclei , an aberrant G2/M checkpoint , and abnormal DSB repair kinetics .
We assembled a panel of 22 human ALT cell lines ( Table 1 ) , nineteen of which originated from in vitro immortalization experiments and four of which were derived from human tumors ( three osteosarcomas and one lung adenocarcinoma ) . Many of the in vitro immortalized lines expressed SV40- or HPV-derived oncoproteins . Seven cell lines originated from a single individual with cystic fibrosis ( JFCF-6 series ) ; an additional five cell lines were established from a Li-Fraumeni patient ( IIICF series ) . We confirmed the absence of telomerase activity by TRAP assay ( data not shown ) and the presence of extra-chromosomal telomeric C-circles , supporting the interpretation that these were ALT cells [8] , [17] ( Table 1; Figure S1 ) . The ALT cell lines and non-ALT controls ( hTERT-immortalized SV40-transformed BJ fibroblasts and the telomerase-positive HPV-E6/E7 expressing HeLa cervical tumor cell line ) were tested for the expression of ATRX and DAXX ( Figure 1A–1C and data not shown ) . Repeated immunoblots on independently harvested whole cell extracts indicated that among the 22 ALT cell lines , only six ( G292 , IIICF-E6E7/C4 , IVG-BF . LXN , LFS-05F-24 , SK-LU-1 , and MeT-4A ) , contained detectable full length ATRX protein and one of these ( IVG-BF . LXN ) appeared to lack DAXX . Of the six cell lines expressing ATRX , three ( G292 , IIICF-E6E7/C4 , and IVG-BF . LXN ) showed very low immunofluorescence signals for ATRX and/or DAXX ( Figure 1B ) , whereas the other three lines displayed the punctate nuclear staining pattern for ATRX and DAXX expected for this PML body component [36] , [37] . Thus , 19 out of 22 ALT lines had an alteration in the expression of ATRX and/or DAXX . As each of the seven JFCF-6-derived ALT lines lacked detectable ATRX , we considered the possibility that the parental JFCF-6 culture might have been deficient in ATRX expression . However , a non-ALT JFCF-6 line contained ATRX ( Figure 1C ) , indicating that the lack of ATRX in the other JFCF-6 lines was potentially correlated with the ALT phenotype . All ALT cell lines were tested for genetic changes in ATRX , DAXX , and H3 . 3 ( H3F3A ) using primers designed to PCR-amplify all exons from these genes for Sanger sequencing ( Table 1; Table S1 ) . Failure to amplify multiple consecutive exons was interpreted as a deletion event only when flanking exons did amplify . By that criterion , 6 cell lines ( JFCF-6/T . 1D , JFCF-6/T . 1L , JFCF-6/T . 1R , JFCF-6/T . 1J/5H , JFCF-6/T . 1J/1-3C , and IIICF-T/B3 ) harbored large deletions in the ATRX gene that ranged from 4 to 26 exons in size ( Table 1; Table S1 ) . Sequencing of genes that did not harbor detectable deletions revealed no potentially inactivating genetic changes . Nine of those cell lines lacked detectable ATRX protein suggesting that genetic alterations not detectable by Sanger sequencing , such as translocations , promoter or splicing mutations , or epigenetic changes may underlie their lack of expression . No genetic alterations were detected in DAXX or the H3F3A gene encoding H3 . 3 ( Table 1 ) . These data provide strong evidence that loss of ATRX expression is involved in either the initiation or maintenance of the ALT pathway in human cells . To test whether deficiency in ATRX was sufficient to induce the telomere-telomere recombination typical of ALT cells , we used an shRNA to deplete ATRX from HeLa cells and used Chromosome-Orientation FISH ( CO-FISH ) to measure sequence exchanges between sister telomeres ( Telomere Sister Chromatid Exchanges or T-SCEs ) . Despite repression of ATRX by more than 95% , the frequency of T-SCEs was not significantly altered ( Figure 1D and 1E ) . In addition , repression of ATRX or DAXX in SV40-transformed BJ fibroblasts failed to induce escape from crisis ( Figure 1F and 1G; Figure S2 ) , whereas expression of hTERT readily immortalized the cells ( Figure 1G; Figure S2 ) . After a prolonged period in crisis ( 6 weeks ) , immortal clones emerged from the culture in which ATRX expression was suppressed with ATRX sh590 . However , a parallel culture in which ATRX was not suppressed efficiently ( ATRX sh589 ) also yielded an immortal subpopulation ( Figure 1F and 1G ) and in both cases , the immortal cells expressed telomerase and lacked telomeric C-circles ( Figure S2A and S2B ) , indicating that they had not activated ALT . These observations argue that this level of ATRX/DAXX suppression is not sufficient to activate the ALT pathway . The lack of induction of ALT by shRNA-mediated suppression of ATRX and DAXX prompted us to inspect the ALT cell line panel for changes in the status of genes , pathways , and proteins with possible relevance to the activation of ALT . Because HDR is normally repressed at telomeres by shelterin , we examined the expression of all six shelterin components in the ALT lines by repeated immunoblotting ( Figure S3 ) . Using this approach on all 22 ALT lines failed to reveal a consistent change in the abundance or appearance of the six shelterin proteins . In addition , exome sequencing ( see below ) failed to reveal mutations in the genes encoding TRF1 , TRF2 , Rap1 , POT1 , TIN2 , and TPP1 . A second entity associated with telomeres is the telomeric RNA , TERRA [32] . As mouse ATRX deficient cells were reported to have elevated TERRA levels and selected ALT cells were previously found to have higher levels of TERRA [31] , [38] , we examined the expression of this RNA in the ALT lines . Quantitative Northern analysis confirmed that the ALT lines generally have higher TERRA levels than telomerase-positive cells , but some ALT lines ( e . g . JFCF-6/T . 1D and IIICF-E6E7/C4 ) approximated the levels of TERRA found in BJ/hTERT/SV40 and HeLa cells ( Figure S4 ) . There was no clear correlation between increased TERRA and ATRX status since the cell lines with apparently normal ATRX/DAXX ( LFS-05F-24 , SK-LU-1 , and MeT-4A ) had TERRA levels comparable to , or higher than , cell lines lacking ATRX ( e . g . JFCF-6/T . 1D , JFCF-6/T . 1R , and JFCF-6/T . 1L ) ( Figure S4 ) . In addition to shelterin and TERRA , we analyzed 299 genes with potential relevance to telomeres and the ALT pathway . This gene set included genes encoding proteins involved in telomere maintenance , DNA damage repair and signaling , chromosome duplication and segregation , and cell cycle regulation ( Table S2 ) . The coding regions of these genes were analyzed by massively parallel 454 sequencing on genomic DNA derived from 14 of the 22 ALT lines . We focused on ALT lines generated in vitro as opposed to tumor-derived ALT lines to exclude genetic changes selected for during tumorigenesis . The JFCF-6 and IIICF ALT lines are derived from two individuals , allowing identification of SNPs specific to the individual as well as potential ALT-correlated changes . After excluding known SNPs , the remaining sequence data were filtered to exclude apparent SNPs present in less than 25% of the reads . The rare SNPs that eluded the threshold filter affected 20 genes ( Table S3 ) . Within the JFCF-6 and IIICF cell line sets , a nucleotide change in multiple members of either set was interpreted as being present prior to the acquisition of the ALT phenotype , and thus not relevant . The variable occurrence of these SNPs in the multiple ALT lines derived from one individual ( e . g . only two of the five IIICF derived lines have the BLM K323R ) is likely due to chromosome losses occurring during or after telomere crisis . The florid chromosome instability exhibited by these cell lines ( see below ) makes this an appealing and parsimonious explanation . Some of these SNPs were also found in other cell lines ( e . g . MDC1 C1559G in LSF-05F-24 and IVG-BF . LXSN ) and deemed irrelevant based on their presence in the IIICF and/or JFCF-6 lines prior to the induction of ALT . Based on these criteria , no ALT specific change was observed in the five IIICF-derived ALT lines or in IVG-BF . LXN . Three of the JFCF-6 derived ALT lines had a specific change that could not be attributed to a pre-existing SNP ( A436S in Suv420H2 , S553F/S470F in PRMT5 , and D2579V in Ki67; Table S3 ) . Since these genes were not affected in the other 13 ALT lines analyzed , they are unlikely to be critical to ALT . Similarly , LSF-05F-24 contained changes in five genes that are not affected in the other 13 cell lines . Since none of these alterations were found in a substantive fraction of the ALT cell lines , it is unlikely that these genes represent clear contributors to the ALT phenotype . As ALT-relevant genetic alterations might be identified from recurrent regions of gene copy number changes , we analyzed the ALT cell line panel using Affymetrix 6 . 0 single nucleotide polymorphism ( SNP ) arrays to identify regions of recurrent copy number alteration . When we examined either individual cells ( Figure 2A and 2B ) or the entire set as a group ( GISTIC analysis , Figure S5 ) , we found that the number and complexity of these gains and losses were at least as extensive as the alterations in human cancers with genome instability [39] . When we considered all of the ALT cell lines as a group , we identified 18 regions of significant focal copy number gain , and many more regions of copy number loss ( Figure S5 ) . The genome rearrangements in the ALT lines are a likely consequence of the telomere dysfunction these cells experienced prior to their immortalization . Consistent with this idea , SKY analysis of five ALT lines ( JFCF-6/T . 1R , JFCF-6/T . 1Q , IVG-BF . LXN , IIICF/c , and LFS-05F-24 ) showed frequent non-reciprocal translocations , deletions , complex rearrangements , and hyper-triploid chromosome numbers ( Figure 3 ) , all of which can be caused by telomere dysfunction [40] , [41] . Previous cytogenetic analysis of two ALT cell lines ( Saos-2 and ZK-58 ) also indicated frequent translocations , deletions , and complex rearrangements [42] . In cells that have been immortalized by telomerase activation , the telomere-driven genome instability is largely dampened , resulting in a rearranged but now relatively stable karyotype [43] , [44] . We asked whether ALT similarly stabilizes the genome by examining two generations of clonal descendants of the JFCF-6/T . 1R ALT line . Although the clones exhibited features in common with the parental cell line , each of the clones showed new regions of copy number gain or loss ( Figure 2B ) . For instance , a new gain of a segment on chromosome 2q was observed in one single cell clone of the parental line ( and all the clones derived from it ) , and in another clone ( and its derivatives ) an overlapping segment showed a new deletion . Many other changes , including examples on chromosomes 6 , 12 , 14 , 15 , 16 and 21 , showed obvious new gains or losses as compared to the parental clone , with independent clones showing different alterations . In contrast , we found only rare clonal alterations in single cell clones derived from the telomerase-expressing HCT116 cell line . These observations suggest that the JFCF-6/T . 1R and perhaps other ALT lines have ongoing genome instability as well as highly rearranged genomes . We considered that ongoing genome instability in the ALT lines could be due to defects in mitosis and/or dicentric chromosomes resulting from telomere dysfunction and ATRX deficiency . Defects in mitosis and dicentric chromosomes can give rise to lagging chromosomes that can form micronuclei , which were recently shown to engender massive genomic alterations [45] . We therefore examined the ALT lines for the spontaneous occurrence of micronuclei . Fifteen of the 22 ALT lines showed a high frequency ( 10–30% ) of cells with micronuclei ( Table 1; Figure 4A and 4B ) . As expected , SV40-transformed hTERT-expressing BJ fibroblasts and HeLa cells showed the low level of spontaneous micronucleation ( <8% ) previously noted in other transformed human cells [46] . Micronucleation frequencies of up to 15% were previously only observed in cells experiencing high levels of DNA damage ensuing from γ-irradiation , in genetic contexts associated with high levels of spontaneous DNA damage , and in genomically unstable tumor cell lines ( e . g . [47] , [48]; Neill Ganem and David Pellman , pers . comm . ) . Thus , many ALT cell lines show an unusually high frequency of micronucleation , indicative of ongoing genome instability . The micronucleation phenotype of ALT lines is likely to be in part due to the absence of ATRX since depletion of ATRX with two shRNAs induced the formation of micronuclei in BJ/hTERT/SV40 and HeLa cells ( Figure 4C and 4D ) , consistent with a previous report on the induction of lobulated nuclei after ATRX depletion [35] . The signs of ongoing genome instability in ALT cells led us to examine the functionality of DNA damage checkpoints and DSB repair . As the ALT lines were expected to have an impaired G1/S DNA damage checkpoint due to their p53 deficiency , we focused our efforts on the G2/M checkpoint . Entry into mitosis is blocked upon activation of the ATM and/or ATR signaling pathways . ATM signaling is primarily responsible for the initiation of the checkpoint , whereas ATR signaling ensures the maintenance of the arrest [49] . To determine the efficiency of initiation and maintenance of the G2/M checkpoint , subconfluent cells were subjected to ionizing radiation , and the mitotic index was determined by flow cytometric quantitation of histone H3 ( S10 ) phosphorylation . One hour after treatment with 10 Gy ionizing radiation ( IR ) , the mitotic index of control hTERT-immortalized Retinal Pigment Epithelial cells ( RPE/hTERT ) was reduced approximately ten-fold ( from 2 . 7% to 0 . 3% ) , whereas reduction in the mitotic index of ATM-deficient GM5849 cells was less than two-fold ( from 4 . 1% to 2 . 3% ) ( Table 1; Figure 5A ) . Of fourteen ALT lines that had a sufficiently high mitotic index to yield interpretable results , half showed a severe defect in G2/M checkpoint initiation ( Figure 5A; Table 1 ) . In six ALT lines , the checkpoint defect was more severe than that of ATM-deficient cells . Qualitatively similar results were observed in response to low dose ( 1 Gy ) IR treatment , with the same cell lines exhibiting defects as at the high dose ( data not shown ) . Similarly , 10 of 11 ALT lines tested displayed a deficiency in the maintenance of the G2/M checkpoint , monitored at 16 hr after γ-irradiation with 4 . 5 Gy ( Table 1; Figure 5B ) . Some of the ALT lines were proficient in the initiation of the checkpoint but failed to maintain it ( e . g . , IIICF/c ) . Collectively , we observed impaired G2/M checkpoint function in 11 out of 17 ALT lines tested . This defect in the G2/M checkpoint is not seen in established non-ALT human cell lines . For example , normal G2/M arrest following γ-irradiation is observed in HT1080 , HCT116 , IMR90 ( JHJP , unpubl . data ) , 293T [50] , MCF7 , and genetically complemented HCC1937 cells [51] . The decrement in the G2/M checkpoint did not reflect a global impairment of DNA damage response ( DDR ) signaling in the ALT cells , since phosphorylation of CHK2 , an outcome of ATM signaling , was readily apparent upon IR treatment in all cell lines , but undetectable in GM5849 ( ATM-deficient ) cells ( Figure S6 ) . Indeed , CHK2 phosphorylation was evident in several of the ALT cell lines prior to irradiation , consistent with the finding that the ALT phenotype is associated with chronic genotoxic stress ( Figure S6 ) . In addition to an abnormal G2/M checkpoint , many of the ALT cell lines showed unusually slow DSB repair kinetics . To assay this aspect of the DDR , cells were γ-irradiated with 0 . 5 Gy and the fraction of cells with 10 or more 53BP1 foci was scored after 1 and 24 hr as well as in non-irradiated controls ( Figure 6A; Figure S7; Table 1 ) . As expected , non-ALT cell lines ( HeLa and hTERT immortalized SV40-transformed BJ ) showed a nearly complete disappearance of the IR-induced DNA damage foci at 24 hr . In contrast , 11 out of 12 ALT cell lines retained a significant fraction of the induced 53BP1 DNA damage foci after a day ( Table 1; Figure 6A ) . This result was corroborated with a pulsed field gel electrophoresis ( PFGE ) -based gel assay for the disappearance of IR-induced DNA fragmentation after 24 hr ( Figure 6B; Table 1 ) . We also noted that approximately half of the ALT cell lines had a high basal level of cells containing 10 or more 53BP1 foci , further confirming that ALT cells experience ongoing genome damage ( Table 1 ) . These spontaneous 53BP1 foci could be the result of both genome-wide DNA breaks and the presence of dysfunctional telomeres [52] . Their deficiency in the G2/M checkpoint , combined with the absence of a functional p53 pathway likely explains why these cells proliferate despite severe genome damage .
Here we report that ATRX is either undetectable or severely depleted from PML bodies in ∼90% of human ALT cell lines tested , including ALT lines derived in vitro . This result establishes a strong correlation between the initiation or maintenance of ALT and deficiency in the ATRX/DAXX pathway . In a subset of cases , the loss of ATRX is due to large deletions within the ATRX gene , further underscoring the relevance of ATRX status to ALT . We expect that alterations in ATRX/DAXX in cells and tumors will be a useful indicator of the presence of the ALT pathway . However , manipulating ATRX/DAXX expression failed to unleash ALT , suggesting that deficiency of these proteins is not sufficient for the ALT phenotype and pointing to the need to identify the cooperating ( epi- ) genetic changes . In addition , a key issue for further investigation is the mechanism by which the loss of ATRX/DAXX and concomitant lack of deposition of H3 . 3 into telomeric chromatin might allow or promote telomere recombination . We note that it remains possible that the ATRX/DAXX pathway has an indirect effect , for instance by repressing genes that promote telomere recombination . Our data have uncovered a hitherto unappreciated level of genome instability in ALT cells . All ALT lines , including cell lines that arose from in vitro immortalization experiments , harbor scrambled genomes and have signs of ongoing genome instability . The ALT lines have frequent micronuclei , a high basal level of DNA damage foci , and elevated checkpoint signaling in absence of exogenous damage . Furthermore , in one ALT cell line , repeated subcloning established frequent copy number alterations . These features of ALT raise the possibility that tumors employing this pathway will have unique vulnerabilities that could offer clinical utility , such as has been shown for cells lacking BRCA1 that are sensitive to PARP inhibition [53] . This possibility underscores the importance of establishing a signature for the ALT phenotype . In this context , it is notable that G2/M checkpoint deficiency emerged as a prominent attribute of many ALT cells in this study . It is likely that the diminished G2/M checkpoint response allows these cells to proliferate , despite a considerable burden of DNA damage both at telomeres and elsewhere in the genome . G2/M checkpoint deficiency is an example of a potential vulnerability unique to ALT cells . Indeed , G2/M checkpoint inhibitors to enhance efficacy of clastogenic therapies are currently being developed and evaluated in clinical trials [54] . The altered G2/M response could not be ascribed to mutations in known components of the ATM or ATR signaling pathways . Furthermore , DNA damage signaling was demonstrably intact since CHK2 phosphorylation could be induced by exogenous DNA damage . The implication of ATRX and DAXX in the ALT phenotype may have some relevance to the checkpoint defects observed , as the involvement of chromatin remodelers in the DDR becomes increasingly clear [55] . Nevertheless , identification of the mechanisms underlying G2/M checkpoint dysfunction in ALT cells is warranted given the data presented here . Our results would appear to exclude the possibility that the ALT cells arise from a single dominant mutation in addition to p53/Rb loss . Loss of ATRX/DAXX function appears to be required for ALT but it seems likely that a defect in the G2/M checkpoint is also needed in order for the cells to proliferate , given their high level of spontaneous DNA damage . The view that multiple steps are required to allow ALT-mediated immortalization is consistent with the low frequency by which it arises , even in SV40- or HPV-transformed cells .
ATL cell lines are described in Table 1 . CellBank Australia provided cell line quality control . All cell lines were verified by 16-locus STR profiling and confirmed to be free of Mycoplasma species , or were analyzed within 10 population doublings of being obtained from ATCC . Non-ALT cell lines were obtained from the ATCC . The HeLa cell line ( subclone HeLa1 . 3 ) was described previously [56] . Primers were synthesized by Invitrogen ( San Diego , CA ) to cover all exons of ATRX , DAXX , and H3F3A1 ( H3 . 3 ) . PCR was performed in 5 µl reactions containing 1× PCR Buffer ( 67 mM Tris-HCl , pH 8 . 8 , 6 . 7 mM MgCl2 , 16 . 6 mM NH4SO4 , 10 mM 2-mercaptoethanol ) , 1 mM dNTPs ( Invitrogen , San Diego , CA ) , 1 µM forward and 1 µM reverse primers , 6% DMSO , 2 mM ATP , 0 . 25 U Platinum Taq ( Invitrogen , San Diego , CA ) and 3 ng DNA . Reactions were carried out in 384-well ABI 9700 thermocyclers ( Applied Biosystems , Foster City , CA ) using a touchdown PCR protocol: 1 cycle of 96°C for 2 min; 3 cycles of 96°C for 10 sec , 64°C for 10 sec , 70°C for 30 sec; 3 cycles of 96°C for 10 sec , 61°C for 10 sec , 70°C for 30 sec; 3 cycles of 96°C for 10 sec , 58°C for 10 sec , 70°C for 30 sec; 41 cycles of 96°C for 10 sec , 57°C for 10 sec , 70°C for 30 sec; 1cycle of 70°C for 5 min . The PCR products were evaluated for presence and size by electrophoresis on 2% agarose gels and sequenced as described ( refs . 25 and 57 [25] , [57] ) . All failed PCRs were repeated at least two more times . Coding regions of 299 genes were amplified from genomic DNA of 15 samples . Following PCR amplification , products were normalized and pooled prior to emulsion PCR amplification of single stranded DNA and 454 pyrosequencing . Sequencing was performed using a FLX Titanium machine at Agencourt Bioscience Corp . ( Beverly MA ) , to a depth of 20× coverage per sample per amplicon ( attempted ) . Assembly and mapping of reads was performed using AVA software . Immunoblotting was performed using standard procedures on whole cell extracts . ATRX was detected primarily with A301-045 ( Bethyl Labs ) or with HPA001906 ( Sigma Aldrich ) . DAXX was detected with A301-353A ( Bethyl Labs ) . Immunoblotting for CHK2-P was done with CHK2-T69 ( Cell Signaling ) and total CHK2 was detected with clone 7 Ab ( Millipore ) . Shelterin components were analyzed as described previously [56] . Cells grown on glass coverslips were fixed with 3% paraformaldehyde for 10 min at room temperature , washed three times with PBS , and permeabilized with 0 . 5% Triton X-100 in PBS for 10 min on ice . Cells were then washed four times with PBS and blocked for 30 min at room temperature with PBG ( 0 . 2% cold water fish gelatin ( Sigma ) , 0 . 5% BSA , in PBS ) . Primary antibodies recognizing ATRX and DAXX ( Bethyl Laboratories , Inc . , see above ) were diluted in PBG and incubated on cells for 1 . 5 h at 37°C . Following three washes with PBG , cells were incubated with FITC-conjugated donkey anti-rabbit secondary antibody ( Life Technologies ) , diluted in PBG , for 30 min at 37°C . Cells were washed three times with PBS and counterstained with 4′ , 6′-diamidino-2-phenylindole ( DAPI ) . RNA was extracted from cell lines using the RNeasy kit ( Qiagen ) . 0 . 5 µg of RNA was reverse transcribed using RT-Advantage kit ( Clontech ) in a volume of 20 µl according to the manufactures instructions . Following reverse transcription 180 µl of water was added and 5 µl was used for qPCR with specific primers and SYBR green mix ( Applied Biosystems ) . Primers: DAXX 5′: AGACGGTTTCTGAGCATCATC; DAXX 3′: AGAGGAGCTAGGGGCTTCTG; TERT 5′: GCCTTCAAGAGCCACGTC; TERT 3′: CCACGAACTGTCGCATGT . Affymetrix 6 . 0 SNP data were generated at the Broad Institute for 22 ALT cell lines and 6 single cell ALT ( JFCF-6/T . 1R ) and non-ALT ( HCT116 ) cell line clones . Single cell clones were generated by fluorescence activated cell sorting ( FACS ) sorting into 96 well plates . Normalized copy number estimates ( log2 ratios ) were made and segmented by the Circular Binary Segmentation algorithm as previously described [58] . The GISTIC 2 . 0 algorithm was performed as previously described on the resulting segmented copy number data from the 22 ALT lines [39] , [59] . The boundaries of the peak amplified and deleted regions identified by GISTIC 2 . 0 were set with at least 95% confidence to include the target gene ( s ) . SV40-transformed BJ fibroblasts at PD 38 were infected with either ATRX- or DAXX-specific shRNAs ( Sigma ) with the following target sequences: ATRXsh589: GCAGATTGATATGAGAGGAAT; ATRXsh590: CGACAGAAACTAACCCTGTAA; ATRXsh592: CCGGTGGTGAACATAAGAAAT; DAXXsh800 : GAAGGGATGGACTAAGCTAAT; DAXXsh801: TCACCATCGTTACTGTCAGAA; DAXXsh802: GCCACACAATGCGATCCAGAA . LacZ-specific shRNA was used as negative control , while infection with hTERT was used as a positive control . Cells were selected with puromycin ( 0 . 5 µg/ml ) for 2 days , and at least 106 cells of each sub-line were maintained in culture for 110 days . Cultures were split at <80% confluence , and growth medium was replaced every 3 days . Cells were counted at each passage and cumulative PDs were recorded . The suppression of ATRX in HeLa1 . 3 cells was achieved with ATRXsh592 . Cells were grown in 10 µM BrdU∶BrdC ( 3∶1 ) for 16 hr with the addition of 0 . 1 µg/ml colcemid ( Roche ) for the final 2 h . Slides were treated with 0 . 5 mg/ml RNAse A for 10 min at 37°C , stained with 0 . 5 µg/ml Hoechst 33258 in 2× SSC for 15 min at room temperature , and exposed to 365-nm UV light ( Stratalinker 1800 UV irradiator , 5400 J/m2 ) . Following digestion with Exonuclease III ( Promega , 10 U/µl , 2×10 min ) at room temperature , slides were dehydrated through an ethanol series ( 70% , 90% , 100% ) and incubated sequentially with TAMRA-TelG 5′-[TTAGGG]3-3′ and FITC-TelC 5′-[CCCTAA]3-3′ probes at room temperature . The percentage of chromosome ends displaying telomeric sister chromatid exchanges ( T-SCEs ) was calculated from four independent experiments . Cells were treated with colcemid for 1 hr , harvested , pelleted , re-suspended in a hypotonic solution of 0 . 075 M KCl for 18 min , fixed in Carnoy's fixative ( 3∶1 methanol∶glacial acetic acid ) , and washed four times with Carnoy's fixative . All fixed samples were spread on slides for staining or hybridization . Chromosomes were stained with Giemsa or Hoechst to visualize chromosomal abnormalities . Spectral karyotyping ( SKY ) was performed on mitotic samples according to the SkyPaint DNA kit H-5 for human chromosomes procedure ( Applied Spectral Imaging , SKY000029 ) and imaged on a Nikon Eclipse E6000 microscope equipped with the SD300 Spectracube and Spectral Imaging acquisition software . To assess G2/M checkpoint initiation , subconfluent cells were exposed to γ-irradiation and harvested at 1 hr with non-irradiated cells for mitotic index measurement by flow cytometry . Briefly , cell pellets were fixed in 70% ethanol , washed twice with cold PBS , and permeabilized with 0 . 25% Triton X-100 in PBS for 15 min on ice . Cells were then rinsed with PBS containing 1% BSA and stained with anti-phospho-histone H3 S10 antibody ( Cell Signaling ) for 1 . 5 hr at room temperature . Following two washes with PBS containing 1% BSA , cells were stained with a FITC-conjugated donkey anti-mouse secondary antibody ( Life Technologies ) for 30 min at room temperature . Cells were washed twice with PBS and then stained with propidium iodide ( 25 µg/ml ) in the presence of 0 . 1 mg/ml RNase . Flow cytometry was performed on a BD Biosciences FACSCalibur , and the percentage of mitotic cells was determined as those that were FITC-positive and contained 4N DNA content . To assess maintenance of the G2/M checkpoint , nocodazole ( 1 µg/ml ) was added 1 hr after irradiation , and cells were incubated at 37°C for 16 hr . Cells were then harvested and processed as described above . To account for differences in the rate of cell cycle progression between cell lines , the mitotic index of irradiated cells was normalized to that of non-irradiated cells treated with nocodazole for 16 hr . Asynchronously growing cells were fixed and examined for the presence of micronuclei after DAPI staining for DNA . Data were obtained from three independent experiments with >100 nuclei examined in each . For the C-circle assay , DNA from cell lines was isolated using the DNeasy Blood and Tissue Qiagen kit , digested with HinfI and RsaI , and quantified using Hoechst fluorimetric analysis . DNAs were diluted to approximately 3 ng/µl , quantified again , and the dilutions were adjusted to exactly 3 ng/µl . The assay was performed using 30 ng of quantified DNA as described by Henson et al . [8] using dot-blotting with an end-labeled [AACCCT]4 telomeric oligonucleotide probe . C-circle values in Table 1 were derived from three independent DNA preparations for each cell line . Cells grown on glass coverslips were irradiated with 0 . 5 Gy IR and incubated at 37°C for 1 hr or 24 hr . Irradiated and non-irradiated cells were fixed with 3% paraformaldehyde for 10 min at room temperature , washed three times with PBS , and permeabilized with 0 . 5% Triton X-100 for 10 min on ice . Cells were then washed four times with PBS and blocked for 30 min at room temperature with PBG ( 0 . 2% cold water fish gelatin ( Sigma ) , 0 . 5% BSA , in PBS ) . Primary 53BP1 ( Novus ) antibody was diluted in PBG and incubated on cells for 1 hr at 37°C . Following three washes with PBG , cells were incubated with FITC-conjugated donkey anti-rabbit secondary antibodies ( Life Technologies ) , diluted in PBG for 30 min at 37°C . Cells were washed three times with PBS and counterstained with 4′ , 6′-diamidino-2-phenylindole ( DAPI ) . The percentage of cells containing >10 53BP1 foci per nucleus was calculated from three independent experiments . For the PFGE assay , subconfluent cells were irradiated with 100 Gy IR and incubated at 37°C for 24 hr . Irradiated and non-irradiated cells were washed once with PBS , trypsinized , and counted . One million cells per sample ( in duplicate ) were centrifuged and washed a second time with PBS . Cell pellets were resuspended in 50 µl 10 mM Tris pH 7 . 2 , 20 mM NaCl , 50 mM EDTA and pre-warmed at 55°C for 10 min . The cell suspension was then mixed with 50 µl of 2% agarose/PBS at 55°C and solidified in plastic molds ( BioRad ) . Agarose plugs were incubated with 1 mg/ml Proteinase K , 0 . 2% sodium deoxycholate , 1% N-lauroylsarcosine-sodium salt , 100 mM EDTA , pH 8 . 0 for 20 hr at 50°C and then washed four times for 1 hr with 20 mM Tris/HCl , 50 mM EDTA , pH 8 . 0 . Duplicate plugs were irradiated with 600 Gy IR , and all samples were equilibrated in 0 . 5× TBE for 30 min . Agarose plugs were inserted into the wells of a 1% agarose gel and sealed with 1% agarose . PFGE was carried out using a CHEF-II apparatus in 0 . 5× TBE buffer at 6 V/cm with pulse times of 70 sec ( 15 hr ) and 120 sec ( 11 hr ) . Following electrophoresis , the gel was stained with 0 . 5 µg/ml ethidium bromide in TBE for 30 min .
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Telomeres , the protective elements at the ends of chromosomes , need to be maintained for cells to proliferate indefinitely . In many human cancers , the telomeric DNA is replenished by telomerase . However , a second pathway for telomere maintenance , referred to as the ALT pathway , has increasingly been recognized in human cancers . The genetic basis for activation of ALT is not known , but recent data have implicated a chromatin remodeling complex ( ATRX/DAXX ) and the histone variant H3 . 3 as players in the repression of ALT . We have examined a large panel of ALT cell lines for their genetic and cell biological features and found that loss of ATRX is a common event in the genesis of ALT lines . In addition , we document that ALT cell lines frequently undergo chromosomal changes and are impaired in their ability to detect and repair damage in their DNA . These hallmarks of ALT are expected to facilitate the detection of ALT–type tumors in the clinic and may lead to ALT–specific treatments .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"medicine",
"biology"
] |
2012
|
Loss of ATRX, Genome Instability, and an Altered DNA Damage Response Are Hallmarks of the Alternative Lengthening of Telomeres Pathway
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Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals . Meiotic recombination is initiated by programmed double-strand break ( DSB ) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation . This histone modification is recognized by Spp1 , a PHD finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex . During meiosis , Spp1 binds H3K4me3 and interacts with a DSB protein , Mer2 , to promote DSB formation close to gene promoters . How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear . Here , combining genome-wide localization analyses , biochemical approaches and the use of separation of function mutants , we show that Spp1 is present within two distinct complexes in meiotic cells , the Set1 and the Mer2 complexes . Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation . Conversely , the Spp1-Mer2 interaction is required for normal meiotic recombination initiation , but dispensable for Set1 complex-mediated histone H3K4 methylation . Finally , we provide evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex . We propose a model where Spp1 works in three ways to promote recombination initiation: first by depositing histone H3K4 methylation ( Set1 complex ) , next by “reading” and protecting histone H3K4 methylation , and finally by making the link with the chromosome axis ( Mer2-Spp1 complex ) . This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots .
In sexually reproducing organisms , recombination between homologous chromosomes at meiosis plays an important role to form gametes with a normal chromosome content . Meiotic recombination is initiated by the programmed formation of DNA double-strand breaks ( DSBs ) catalyzed by the conserved Spo11 protein together with largely conserved accessory DSB proteins [1–3] and is required to promote genetic diversity and accurate homolog segregation [4] . Histone modifications are key players of the chromatin organization . Among these , histone H3K4 trimethylation ( H3K4me3 ) is able to recruit downstream effectors such as chromatin remodelers [5] . In meiosis , Set1 , the subunit of the Set1 complex that catalyzes histone H3K4 methylation , is required for normal levels and distribution of meiotic DSBs [6 , 7] . In budding yeast , the vast majority of meiotic DSBs occur in the ~ 140 bp nucleosome depleted regions ( NDR ) at gene promoters , close to nucleosomes bearing the histone H3K4me3 modification [6 , 8] . The tendency of meiotic recombination to occur in gene promoters and H3K4me3 regions is conserved among many organisms such as dogs , plants and birds [9–11] . In mice and humans , meiotic recombination also occurs at H3K4me3 sites , but only those created by the meiotic-specific histone H3K4 methyltransferase , PRDM9 [12–14] . PRDM9 recognizes specific DNA sequences with its array of zinc finger motifs , where it methylates H3K4 and promotes recombination , away from gene promoters [15] . Remarkably , the mutation of Prdm9 in mice redirects meiotic recombination events towards gene promoters and H3K4me3 [16] as if PRDM9 had a dominant role over the default promoter/histone H3K4me3 conserved pathway . The link between histone H3K4 methylation and meiotic DSB formation has recently been explained in budding yeast by the role of the PHD finger protein , Spp1 , in spatially linking DSB sites to the recombination initiation machinery [17 , 18] . During meiotic prophase , chromosomes adopt a specific three-dimensional structure formed of chromatin loops anchored at their basis to a chromosome axis [19] . DSBs are formed into loop DNA sequences , whereas the DSB proteins are located on the chromosome axis , implying a spatial contact between these two physically distant chromosomal regions during DSB formation [3 , 20–23] . In meiosis , Spp1 , a member of the Set1 complex , is , like Set1 , required for normal DSB levels [17 , 18] . Spp1 is specifically important for H3K4 trimethylation , and in its absence , H3K4me3 levels are reduced to about 20% of wild type [18] . This has been attributed to Spp1 being important for opening the catalytic site of Set1 and allowing trimethylation [24] . Spp1 also interacts with Mer2 , one of the axis-associated DSB proteins required for DSB formation , and is preferentially located on the chromosome axis [1 , 17 , 18 , 23] . The PHD finger of Spp1 interacts also with H3K4me2/me3 at +1 nucleosomes and is required for normal DSB formation , and thus Spp1 makes the physical link between gene promoters close to H3K4me2/3 sites and the DSB formation machinery [17 , 18] . Thus , Spp1 may facilitate or stabilize the interaction between these distant regions , which triggers DSB formation by Spo11 , the protein that bears the catalytic DSB forming activity [17 , 18] . In vegetative cells , Spp1 belongs to the Set1 complex and its distribution mirrors that of RNA pol II [18] . By contrast , in meiosis , the chromosomal distribution of Spp1 shows no spatial correlation with that of RNA pol II [18] , raising the question whether Spp1 is still part of the Set1 complex in meiosis , and if so , how it distributes between the Set1 complex and the DSB proteins . In addition , given that Spp1 is required for normal levels of H3K4me3 , known to recruit downstream chromatin remodelers , it is not clear as well if the functional role of Spp1 within the Set1 complex for H3K4 methylation can be separated from its implication in DSB formation through its interaction with Mer2 . In this paper , we show that Spp1 interacts both with the Set1 complex and Mer2 in meiotic cells . However , Set1 complex does not associate with chromosome axes in meiosis , and its subunits do not interact with Mer2 , revealing that Spp1 is present in two distinct complexes . Next , we show that surprisingly , the presence of Spp1 in the Set1 complex is not important for maintaining H3K4 trimethylation levels and that Spp1 acts independently of the Set1 complex to promote meiotic DSB formation . Finally , we show that a mutant of MER2 that no longer interacts with Spp1 but binds normally to chromosome axes is impaired for DSB formation . This demonstrates that solely affecting Spp1 interaction with Mer2 is sufficient to impair DSB formation , independently of any H3K4 methylation-related change in chromatin . Finally this work is relevant for understanding meiotic DSB formation in mammals and other organisms , for which a mechanism linking H3K4me3 and the DSB machinery likely exists but has not yet been elucidated .
During vegetative growth , Spp1 is a member of the eight-unit Set1 complex , and is predominantly located at highly transcribed genes , consistent with the Set1 complex being associated with elongating RNA pol II [18 , 25] . However , in meiosis , Spp1 interacts with the Mer2 protein and is predominantly located on chromosomes axes [18] . We thus investigated how Spp1 distributes between the Set1 complex and its interaction with the axis-associated Mer2 DSB protein in meiotic cells . For this , we affinity-purified Spp1-TAP from cells at 3 . 5 hr in meiotic prophase , the expected time of DSB formation [26] . As shown before for Myc-tagged Spp1 , the Spp1-TAP fusion protein associated with chromosome axis sites during meiosis ( Fig 1A ) [18] . Spp1-TAP interacting proteins were purified and identified by label-free mass-spectrometry-based quantitative proteomics . Analyses revealed that Spp1-TAP interacts with the whole Set1 complex ( composed of Bre2 , Sdc1 , Spp1 , Set1 , Swd1 , Swd2 , Swd3 , Shg1 ) and with Mer2 in meiotic cells ( Fig 1B and 1C and S1 Table ) . No other proteins significantly purified with Spp1 . Our finding that Spp1 is associated with the Set1 complex in meiosis is in apparent contradiction with the fact that Spp1 appears mainly associated with regions of axis attachment , largely co-localizing with Mer2 , as assessed by genome wide mapping [18] . The contribution of Mer2 to the localization of Spp1 was assessed by mapping genome-wide Spp1 binding sites in mer2∆ meiotic cells . Whereas in wild type , Spp1 binding was not correlated with that of RNA pol II , in the absence of MER2 , Spp1 location became positively correlated with that of RNA pol II ( Pcorr = 0 . 57 ) ( Fig 2A ) . Indeed , in the absence of Mer2 , the strongest Spp1 peaks were now at highly transcribed genes , similar to the distribution of Spp1 in vegetative cells , likely reflecting its association with the Set1 complex ( Fig 2B ) . The localization of Spp1 was confirmed by ChIP-qPCR in WT ( wild-type ) and mer2∆ cells ( Fig 2D ) . In WT cells , Spp1 was present at two highly transcribed genes , ACS1 and CIT2 , like in mer2∆ , in addition to its association with chromosome axes ( Fig 2D ) . By contrast , Spp1-Myc fully associated with meiotic chromosomes axes in the absence of SET1 , as shown by its positive correlation with that of the Rec8 protein ( Pcorr = 0 . 66 ) , similar to the correlation between Spp1 and Rec8 in SET1 cells ( Pcorr = 0 . 69 [18] ) ( Fig 2C and S1 Fig ) . However , the signal at the two highly transcribed genes ACS1 and CIT2 was lost ( Fig 2D ) . We propose that in wild-type cells , the association of Spp1 with Mer2 occurs at the highly localized axis association sites , resulting in strong peaks that mask its association with chromatin through the Set1 complex , which may be more diffuse along chromatin , and less concentrated in strongly defined peaks . Our findings are consistent with the existence of two classes of Spp1 binding sites , ones with axis sites , dependent on MER2 , and ones with transcription sites , dependent on SET1 . We next asked if when Spp1 is associated with Mer2 on chromosomes axes , it is within the Set1 complex . To answer this , we mapped the sites of chromatin association of Set1 and Swd1 , another subunit of the Set1 complex , in meiosis . Set1 and Swd1 bound the two highly transcribed genes , ACS1 and CIT2 , during meiosis , but only weakly a chromosome axis-associated site ( Fig 3A and S2 Fig ) . This was confirmed genome-wide , where the location of Swd1 in meiotic cells was positively correlated with that of RNA pol II and Swd1 bound only weakly to chromosome axis sites ( Fig 3B and 3C , respectively ) . Consistent with these findings , the strongest Swd1 peaks in the genome were at highly transcribed regions ( Fig 3D ) . In agreement with our genome-wide localization results , Swd1 immunoprecipitated Spp1 from meiotic cells , but not Mer2 , and reciprocally , Mer2 pulled down Spp1 , but not Swd1 ( Fig 3E and 3F , respectively ) . The latter result was confirmed by mass spectrometry-based analysis of Mer2-TAP interactome ( S1 Table ) . Together , our experiments point toward Spp1 being located in two physically distinct complexes . We next set out to determine if the presence of Spp1 in the Set1 complex is required for its DSB formation function . For this , we designed a mutation to disrupt the Set1-Spp1 interaction without affecting Spp1’s PHD finger or the interaction of Spp1 with Mer2 . The domain of Spp1 that interacts with Set1 has not been determined , and we failed to identify a Spp1 mutant that would disrupt its interaction with Set1 . On the Set1 side , a short region ( amino acids 762–794 ) close to the regulatory nSet domain of Set1 is sufficient for the interaction with Spp1 in a two-hybrid test [27] . We searched for conserved amino acids in this region that could potentially be involved in protein-protein interactions [28] . We identified a conserved negatively charged acidic motif ( AIKDEEDM ) that we mutated into a neutral motif ( ASKSSSSM ) ( set1_sid mutant for Spp1 interaction-deficient ) ( Fig 4A and S3 Fig ) . Remarkably , the mutated Set1 protein lost all interaction with Spp1 in a two-hybrid assay , while keeping its interaction with its two other known direct binding partners , Shg1 and Swd2 ( Fig 4B ) [29] . Furthermore , the set1_sid mutation induced the loss of detectable interaction of Spp1 with the Set1 complex in vivo , as assessed by the loss of interaction between Spp1 and the Swd1 subunit , both in vegetative and in meiotic cells ( Fig 4C ) and by mass spectrometry analysis of Spp1-TAP co-purified proteins from meiotic set1_sid cells ( S4 Fig and S1 Table ) . Thus , no other subunit of the Set1 complex seems able to retain Spp1 on its own . In addition , in vegetative or in meiotic set1_sid cells , Spp1-Myc was no longer detected at the ADH1 , ACS1 and CIT2 highly transcribed genes , whereas it still associated with chromosome axis in meiotic cells ( Fig 4D ) . This set1_sid mutant allowed us to assess if DSB formation occurs normally even if Spp1 is not associated with the Set1 complex . Meiotic DSB frequency at two Spo11 DSB hotspots , CYS3 and DEP1 ( 8 ) , in the set1_sid mutant was indistinguishable from wild-type ( Fig 4E , upper panel ) . In situations where H3K4 methylation or Spp1 is absent , such as in set1∆ or spp1∆ mutants , respectively , DSB frequencies are generally reduced , but DSB formation is increased at a few sites , including the PES4 gene promoter [6 , 18] . Importantly , in the set1_sid mutant , no DSBs were induced at the PES4 promoter ( Fig 4E , lower panel ) . These results clearly indicate that the presence of Spp1 in the Set1 complex is dispensable for DSB formation . In agreement with these results , the transient interaction of Spp1 with two DSB hotspots during meiosis was still detected in the set1_sid mutant ( S5 Fig ) . This indicates a different behavior of Spp1 than that at the ACS1 and CIT2 genes , which are not DSB hotspots [8] , where Spp1 interaction is lost in the set1_sid mutant ( Fig 4D ) . Since Spp1 is believed to facilitate the Set1 catalysis of H3K4 trimethylation , we would expect that in the set1_sid mutant where Spp1 is no longer associated with the Set1 complex , H3K4me3 levels should decrease similarly to spp1∆ mutant , to about 20% of WT levels ( [18] and Fig 5A and 5B and S4 Table ) . Surprisingly , the set1_sid mutant still showed high levels of H3K4me3 , as assessed by Western blot and chromatin immunoprecipitation at the ADH1 locus ( Fig 5A and 5B and S4 Table ) . Furthermore , H3K4me3 levels were significantly reduced in the double set1_sid spp1∆ compared to the single set1_sid mutant , indicating that Spp1 still promotes H3K4me3 levels outside of the Set1 complex ( Fig 5A and 5B and S4 Table ) . One possibility is that its binding to H3K4me3 stabilizes this mark and/or protects it from active demethylation . We asked if this function could be through its PHD finger , known to recognize H3K4 methylation in vitro [30] . A deletion of the PHD finger or the point mutation spp1W45A of the PHD finger mildly affects global levels of H3K4me3 ( [17] and Fig 5A ) and H3K4me3 levels at the ADH1 locus ( Fig 5B ) . However , a set1_sid spp1W45A double mutant showed a reduction of H3K4me3 similar to that of the set1_sid spp1∆ mutant ( Fig 5A and 5B ) . These results uncover a previously unknown role of the PHD finger of Spp1 in the maintenance of H3K4me3 levels that takes place even without stable interaction with the Set1 complex . Likewise , we asked if the spp1∆ phenotype for DSB formation is solely due to absence of Spp1 within the Mer2 complex , and not to indirect effects on Set1 complex function . For this , we designed a mutant that would disrupt the Mer2-Spp1 interaction but preserves the Spp1-Set1 interaction . The domain of Spp1 interacting with Mer2 lies in the last 131 amino acids of Spp1 , and the deletion of four amino acids C263 to C266 in this domain was proposed to abolish Mer2-Spp1 interaction in vivo and be important for new DSB targeting by a Gal4BD-Spp1 fusion [17] . However , this Spp1 mutant retained two-hybrid interaction with Mer2 , and when inserted behind its endogenous promoter , was largely proficient in DSB formation at the tested hotspots ( S6 Fig ) . We thus looked for altering the region of Mer2 that interacts with Spp1 , with as little as possible alteration of Mer2’s other functions . This region has been mapped to amino acids 165 to 232 [17] , which corresponds to one of the two major coiled coils of Mer2 predicted structure ( Fig 6A ) . Combining analyses of structure prediction and conservation of amino acids in this region , we identified several conserved amino acids predicted to be surface exposed ( Fig 6A and S7 Fig ) . The mutation of one of these , V195 to D , totally abolished the two-hybrid interaction of Mer2 with Spp1 ( mer2_sid mutant , Fig 6B ) . When tested in vivo , the mutant Mer2_sid protein was still recruited to the chromosome axis like the non-mutated protein ( Fig 6C ) . However , the Mer2_sid mutant protein was totally deficient for interaction with Spp1 in meiotic cells , in agreement with the two-hybrid results ( Fig 6D ) . Furthermore , like in mer2∆ cells , Spp1 interaction with the chromosome axis was lost in the mer2_sid mutant ( Fig 6E ) . We examined the meiotic phenotypes of this Spp1 interaction-defective mer2_sid mutant . The mer2_sid mutant had reduced DSB formation at the two Spo11 hotspots , CYS3 and DEP1 , like spp1∆ ( Fig 6F , upper panel ) . In addition , DSB formation at the PES4 site was detected at an increased level in the mer2_sid mutant , similar to spp1∆ ( Fig 6F , lower panel ) . We conclude that the mer2_sid mutation recapitulates all the meiotic DSB phenotypes of spp1∆ . Next , we checked meiotic progression , since spp1∆ was reported to have a meiotic delay [18] . The mer2_sid mutant had wild-type meiotic progression ( Fig 6G and S8 Fig ) . The meiotic delay of spp1∆ may thus be unrelated to a recombination phenotype of spp1∆ but to defects related to the Set1 complex . To answer this question , we examined meiotic progression in a spo11Y135F DSB deficient strain . Indeed , spp1∆ still showed a meiotic delay in this context , which is thus DSB-independent ( Fig 6G and S8 Fig ) . Finally , mer2_sid showed a wild-type level of spore viability ( 97% among 100 tetrads ) , contrary to mer2∆ , but similar to spp1∆ [18] . Since Mer2 is an essential DSB protein , this indicates that Mer2-sid keeps its core meiotic functions , apart from its interaction with Spp1 . Altogether , these results highlight that all meiotic DSB formation defects of spp1∆ can be attributed to its lack of interaction with Mer2 , and are unrelated to any change in chromatin opening or accessibility that might be due to affecting the Set1 complex .
Mer2 is one of the ten DSB proteins identified in S . cerevisiae , which are all connected by physical interactions , and was proposed to form a complex with two other DSB proteins , Mei4 and Rec114 [31 , 32] . However , we did not identify any other candidate than Mer2 and the Set1 complex in the meiotic Spp1 purifications . Likewise , we previously found that Spp1 does not interact with Mei4 in co-immunoprecipitation experiments [18] . Furthermore , in a meiotic Mer2-TAP pulldown , we retrieved Spp1 as the top candidate , but no peptide of Mei4 or Rec114 ( S1 Table ) . This indicates that the strength or the frequency of interaction between Mer2 and Spp1 is higher than that of Mer2 with Mei4 and Rec114 . Spp1 interacts strongly with Mer2 in two-hybrid assays , and thus this interaction does not need meiosis-specific modification of Mer2 [17] . By contrast , the co-immunoprecipitation of Mei4 and Rec114 with Mer2 and the recruitment of Mei4 and Rec114 to the chromosome axis require Mer2 to be phosphorylated , upon DNA replication , by CDK and DDK [23 , 33–35] . Thus , the interaction of Mer2 with Mei4 and Rec114 is perhaps more transient than the Mer2-Spp1 interaction and too weak to be detected in our analysis . Finally , our mer2_sid mutant , despite losing interaction with Spp1 , still likely keeps interaction with Mei4 and Rec114 , which are essential DSB proteins [1] . We thus propose two separable functions of Mer2: one in recruiting the essential DSB proteins Mei4 and Rec114 , and the other in increasing the tethering of DSB sites to the axis , through Spp1 , in order to favor cleavage by Spo11 . The first function is regulated by replication and Mer2 phosphorylation , whereas the latter is a “constitutive” function of Mer2 . Histone H3K4me3 levels are slightly decreased in the set1_sid mutant , consistent with the hypothesis that the presence of Spp1 in the Set1 complex is important for normal H3K4me3 levels to stimulate Set1 catalytic activity for H3K4me3 deposition as proposed before [24] . However , it is surprising that histone H3K4me3 levels were much less affected in the set1-sid mutant than in the spp1∆ mutant , revealing an additional function of Spp1 for histone H3K4me3 levels in the absence of detectable interaction with the Set1 complex . The in vivo function of Spp1 recognition of H3K4me3 by its PHD finger has been poorly studied . Within the Set1 complex , it may restrict Set1 activity to the +1 nucleosomes of genes , which harbor the combination of H3K4me2/3 and H3R2 not asymmetrically methylated that is specifically recognized by Spp1 PHD finger [36] . However , this explanation does not hold for the effect we saw in the set1_sid mutant . We thus propose that both in wild type SET1 cells and in the set1_sid mutant , the PHD finger module of Spp1 binds and protects H3K4me3 from demethylation by the Jhd2/JARID1 enzyme , which specifically demethylates H3K4me3 in vivo [37 , 38] ( Fig 7 ) . This binding may involve a small proportion of the total Spp1 population or occur only transiently during the cell cycle , for instance in S phase , when Jhd2 was proposed to act [39] . This would explain why we failed to detect it at the tested loci in the set1_sid mutant . An alternative explanation might be that Spp1 directly interacts with Jhd2 and inhibits its histone H3K4 demethylation activity , in a way that requires Spp1 PHD finger . Further studies will be required to test these hypotheses and the crosstalk between Spp1 and Jhd2 in regulating H3K4me3 . The closest homolog of Spp1 in Mammals , CXXC1 ( CFP1 ) has a N-terminal PHD finger that is able to interact with histone H3K4 methylated peptide in vitro , and seems important to allow Set1 complex binding to chromatin in vitro [40] . However , the in vivo function of this PHD finger has not been determined . In addition , CXXC1 contains a motif that binds unmethylated CpG islands , allowing CXXC1 to restrict H3K4 methylation by the Set1 complex to these sites at promoters [41 , 42] . It is not known if CXXC1 may associate with these regions without the associated Set1 complex . Finally , CXXC1 was reported to interact with the DNMT1 methyltransferase , through a region distinct from its Set1 interacting region [43] . However , it was not investigated if in vivo this interaction occurs when CXXC1 is in the Set1 complex . To conclude , so far , no Set1 complex independent function in H3K4me3 levels have been described for the homolog of Spp1 in mammals . Although Spp1 is required both for deposition , reading of H3K4me3 and tethering to DSB proteins , we show here that surprisingly , DSB formation is as proficient when Spp1 is physically separated from the Set1 complex as in the wild-type conditions . One could thus wonder why Spp1 divided its tasks in meiosis to both participate to H3K4me3 deposition and work as a reader and tether of this mark . There are 18 other PHD finger proteins in S . cerevisiae , among which 8 are able to bind H3K4me2/3 in vitro [30] . Our findings indicate that any PHD-finger motif that could interact with Mer2 would be able to promote DSB formation as Spp1 . Maybe it is just by chance that evolution selected the same protein for fulfilling its complementary functions . However , the fact that this configuration may also be conserved in mammals makes it unlikely ( see below ) . Since we revealed here that Spp1 is able to bind H3K4me3 independently of the Set1 complex , it is possible that in wild-type cells , Spp1 “persists” on H3K4me3 after the passage of the Set1 complex , and this constitutes a first step in the tethering of DSB sites to the chromosome axis ( Fig 7 ) . There may thus be an evolutionary advantage to the same protein having three separate but sequential activities , without all occurring in the same physical complex . It could be argued that H3K4me3 could only be a sign of open chromatin regions , and if it had any role , it could just be to make chromatin more accessible for DSB formation . Our present data argue against this . Indeed , in our mer2_sid mutant , the Set1 complex is intact , and thus chromatin modifications resulting from its activity are expected to be unaltered compared to wild-type . Nevertheless , the DSB phenotype recapitulates the one seen in the absence of H3K4 methylation or Spp1 , suggesting that impaired tethering of DSB sites to the chromosome axis is the only cause of the DSB reduction and redistribution and that open chromatin structure is not sufficient for normal DSB formation . Outside of budding yeast , histone H3K4me3 is emerging as being widely used for meiotic recombination . Two main pathways for choosing sites of meiotic recombination have been identified [44] . The first one , which occurs in mice , humans , cattle and a certain number of other vertebrates involves PRDM9 , which promotes both H3K4me3 and meiotic DSB formation at its binding sites [12–15] . The other one uses open chromatin at promoters , enriched in H3K4me3 and/or CpG islands , to direct meiotic recombination [6 , 9–11 , 45] . Although the role of H3K4me3 in meiotic recombination has not been proven apart from in budding yeast , interesting findings showed recently that CXXC1 , the homolog of Spp1 in mammals , is able to interact in two-hybrid tests both with PRDM9 and IHO1 , the proposed functional homolog of Mer2 [3 , 46 , 47] . Like Spp1 , CXXC1 is a member of the Set1 complex , and has a PHD finger that may recognize H3K4me3 . In addition , it also binds unmethylated CpG at promoters . A model was proposed in which PRDM9 , by interacting with CXXC1 , would direct it away from promoters , and would tether the PRDM9-bound sites to the axis for Spo11 cleavage . In Prdm9-/- mice or in organisms that do not have PRDM9 , CXXC1 would bind H3K4me3 and/or CpG islands and tether these sites to chromosome axis-bound IHO1 , exactly as in budding yeast . Since we have shown that in budding yeast , Spp1 is able to bind H3K4me3 outside of the Set1 complex , CXXC1 may similarly recognize H3K4me3 deposited by PRDM9 outside of the Set1 complex with which it is normally associated . This likely conservation is fascinating , and indicates that H3K4me3 and/or CpG islands could be an evolutionary conserved tether for meiotic recombination sites .
All yeast strains are derivatives of the SK1 background and are listed in S2 Table . For synchronous meiosis , cells were grown in SPS presporulation medium and transferred to 1% potassium acetate with vigorous shaking at 30°C as described [48] . For strain constructions and spore viability measurements , sporulation was performed on solid sporulation medium for two days at 30°C . Yeast strains were obtained by direct transformation or crossing to get the desired genotype . All transformants were confirmed using PCR ( Polymerase Chain Reaction ) discriminating between correct and incorrect integrations and sequencing for epitope tag insertion or mutagenesis . Spp1 and Mer2 were fused at their C-terminus with a TAP-tag at their endogenous loci using the pBS1539 plasmid [49] . Set1 was tagged with 6 copies of HA at its N-terminus at its endogenous locus by using plasmid pOM10 [50] and Cre-Lox excision of the marker between the SET1 promoter and the tag . Swd1 was tagged at its C-terminus at its endogenous locus by 3 copies of HA [51] . Site directed mutagenesis was performed using PCR . The mutagenic PCR was performed on the region of interest where the gene was flanked by a selectable marker and transformed into yeast . For making the spp1W45A and spp1∆263–266 mutants without tag , we first introduced an hphMX drug resistant cassette behind the 3’UTR of SPP1 . This construct was fully functional for meiotic DSB formation ( S6 Fig ) . Next , we used this construct to introduce the desired mutation by PCR and transformation of the fragment containing the mutated gene and its 3’UTR hphMX cassette . For mer2_sid-Flag we used genomic DNA from a strain containing the MER2-FLAG-kanMX allele for PCR mutagenesis . For introducing the set1_sid and the mer2_sid mutations without an associated tag or marker , we first deleted the SET1 or MER2 gene with kanMX cassette by yeast transformation . We next used CRISPR-Cas9 mediated cleavage , using a plasmid encoding Cas9 and expressing a guide RNA targeted to the kanMX cassette ( plasmid generously provided by G . Zhao and B . Futcher ) , co-transformed together with a healing set1 or mer2 fragment containing the desired mutation . Yeast two-hybrid assays were performed exactly as described [52] . SET1 , SHG1 , SPP1 and SWD2 ORFs were PCR-amplified from SK1 genomic DNA . MER2 cDNA sequence was amplified from pCA5-MER2 containing MER2 cDNA , given by Scott Keeney [31] . Briefly , PCR products were cloned in plasmids derived from the 2 hybrid vectors pGADT7 ( GAL4-activating domain ) and pGBKT7 ( GAL4-binding domain ) creating N terminal fusions and transformed in yeast haploid strains Y187 and AH109 ( Clontech ) , respectively . Interactions were scored , after mating and diploid selection on dropout medium without leucine and tryptophan , as growth on dropout medium without leucine , tryptophan and histidine . Cells bearing the dmc1∆ mutation to accumulate DSBs were harvested from meiotic time courses at each time point . Genomic DNA was prepared in low melting temperature agarose plugs and digested with the AflII restriction enzyme as described [48] . Southern blotting and signal quantification was performed as described [18] . Probes used were from nt 123046 to 124295 chr1 for CYS3 and DEP1 DSBs , and from nt 194846 to 196285 , chr6 for PES4 DSB ( R64-2-1 version , Nov . 2014 S . cerevisiae genome coordinates ) . Spp1-TAP or Mer2-TAP purification ( strain VBD1266 for Spp1-TAP , VBD1877 for Spp1-TAP set1_sid , VBD1402 for Mer2-TAP or ORD7339 for the untagged control ) was performed from 1L ( 2 . 1010 cells ) of a synchronous meiotic culture at t = 3 . 5 hr in meiosis . Each purification was performed in parallel with a control untagged strain , ORD7339 . The protocol was essentially as described in [49] with the following modifications: PMSF ( phenylmethylsulfonyl fluoride ) to a final concentration of 1 mM was added to the culture prior harvesting the cells . Cells were washed with TAP lysis buffer ( 50 mM Tris/HCl pH 7 . 5; 1 mM EDTA; 0 . 5% NP-40; 10% glycerol; 300 mM NaCl; 1 mM PMSF; 10 mM NEM; 1X Complete Mini EDTA-Free ( Roche ) ; 1X PhosSTOP ( Roche ) ) , resuspended in about 2 ml of the same buffer and frozen as noodles in liquid nitrogen . For lysis , cells were ground in a mortar in liquid nitrogen , and lysis was performed in TAP Lysis buffer plus 1 mM PMSF and 1X Complete EDTA-free protease inhibitor cocktail ( Roche ) . Protein preparation for mass spectrometry-based proteomic analyses was as described [53] . Briefly , extracted proteins were stacked in the top of a SDS-PAGE gel ( NuPAGE 4–12% , Invitrogen ) before in-gel digestion using trypsin ( Promega , sequencing grade ) . Resulting peptides were analysed by online nanoLC-MS/MS ( UltiMate 3000 coupled to LTQ-Orbitrap Velos Pro and Ultimate 3000 RSLCnano coupled to Q-Exactive Plus , Thermo Scientific , for Spp1 and Mer2 interactomes analyses , respectively ) using a 120-min gradient . Peptides and proteins were identified and quantified using MaxQuant ( version 1 . 5 . 8 . 3 [54] ) and SwissProt database ( June 2017 version , Saccharomyces cerevisiae S288c taxonomy ) . Only proteins identified with a minimum of two unique + razor peptides were taken into account for further analyses . Statistical analyses were performed using ProStaR [55] . Proteins identified in the reverse and contaminant databases , proteins only identified by site and proteins exhibiting less than 3 intensity values in one condition were discarded from the list . After log2 transformation , intensity values were normalized by median centering before missing value imputation ( replacing missing values by the 2 . 5 percentile value of each column ) ; statistical testing was conducted using limma t-test . Differentially recovered proteins were sorted out using a log2 ( fold change ) cut-off of 7 and a FDR threshold on remaining p-values of 1% using the Benjamini-Hochberg method . 6 x 108 cells were washed with PBS ( Phosphate Buffered Saline ) , and lysed in 1 . 5 ml lysis buffer ( 20 mM HEPES/KOH pH7 . 5; 150 mM NaCl; 0 . 5% Triton X-100; 10% Glycerol; 1 mM MgCl2; 2 mM EDTA; 1 mM PMSF; 1X Complete Mini EDTA‐Free ( Roche ) ; 1X PhosSTOP ( Roche ) ) and 125 U/mL benzonase ( Sigma ) and glass beads three times for 30 s in a Fastprep instrument ( MP Biomedicals ) . The lysate was incubated 1 hr at 4°C with 125 U/ml benzonase , and cleared by centrifugation at 13 , 000 g for 5 minutes . For Spp1-TAP purifications , magnetic PanMouse IgG beads ( Life Technologies ) were added and incubated overnight at 4°C . Beads were washed 4 times with lysis buffer and precipitated proteins were eluted by Tev cleavage ( 2 μg in 20 mM Tris pH8; 150 mM NaCl; 0 . 1% NP-40; 5% glycerol; 1 mM MgCl2; 0 . 5 mM EDTA; 1 mM DTT for 1 hr at room temperature ) . One volume of 2 x SDS protein sample buffer was added and sample were denatured 10 min at 95°C before electrophoresis . For Swd1-HA IP , 25 μl of Protein G magnetic beads ( New England Biolabs ) and 5 μg of mouse HA monoclonal antibody 16B12 ( Covance ) ) were added . After overnight incubation at 4°C , beads were washed 4 times with lysis buffer and resuspended in 30 μl of 2 x SDS protein sample buffer . The beads were heated at 95 °C for 10 min . For Mer2-Flag IP , Sigma Anti-FLAG M2 magnetic beads were added to the lysate and incubated overnight at 4°C . Beads were washed twice with lysis buffer , and eluted for 2 hrs at 4°C with 12 . 5 μg of Flag peptide in elution buffer ( 20 mM Tris pH8; 150 mM NaCl; 0 . 1% Tween; 10% Glycerol; 5mM MgCl2; 0 . 5 mM EDTA ) . One volume of 2 x SDS protein sample buffer was added and sample were denatured 10 min at 95°C before electrophoresis . Protein eluates were loaded onto a 4–12% SDS-polyacrylamide gel and blotted to PVDF ( Polyvinylidene difluoride ) membrane . Antibodies used were as follows: anti-H3K4me3 ( MC315 , Millipore , 1/5000 ) ; anti-H3K4me2 ( 07–030 , Millipore , 1/5000 ) ; anti-PGK1 ( Invitrogen , 1/20000 ) ; anti-HA ( Roche , 12CA5 , 1/750 ) ; anti-Myc ( Santa Cruz , 9E10 , 1/500 ) ; anti-Flag ( Sigma , 1/1000 ) ; anti-TAP ( Invitrogen , 1/2000 ) ; anti-Spp1 ( rabbit polyclonal , 1/2000 ) . The Spp1 polyclonal antibody was made in rabbit against the full-length Spp1 fused at its N-terminus with a 6His-MBP tag . Signal was detected using the SuperSignal West Pico Chemiluminescent Substrate ( ThermoFisher ) and a Chemidoc touch system ( Biorad ) . Signal was quantified using Image J software . For each meiotic time point , 2 x 108 cells were processed as described [52] . For HA-Set1 , we used 1 μg of monoclonal 16B12 anti-HA antibody ( Covance ) and 50 μL PanMouse IgG magnetic beads ( Life Technologies ) . For H3K4me3 , we used 2 μl of MC315 H3K4me3 monoclonal antibody ( Millipore ) and 30 μL Protein G magnetic beads ( New England Biolabs ) . For Spp1-Myc , in ChIPchip experiment we used 0 . 8 μg of c-Myc monoclonal antibody ( 9E10 , Santa Cruz ) and 30 μL Protein G magnetic beads , and for ChIP qPCR , we used 1 . 6 μg of c‐Myc monoclonal antibody ( 9E10 , Santa Cruz ) and 50 μL PanMouse IgG magnetic beads . Quantitative PCR was performed from the immunoprecipitated DNA or the whole‐cell extract using a 7900HT Fast Real‐Time PCR System ( Applied Biosystems ) and SYBR Green PCR master mix ( Applied Biosystems ) as described [6] . Primers for ADH1 , BUD23 , GAT1 , axis ( chr3 , nt 232942 to 233010 ) and control site ( NFT1 , chr11 nt 654468 to 654549 ) have been described [18 , 56] . Primers for ACS1 and CIT2 genes amplified fragments with the coordinates: chr1 , nt 44778–44843 and chr3 , nt 122043–122105 , respectively . Results were expressed as % of DNA in the total input present in the immunoprecipitated sample and normalized to the negative control site in the middle of NFT1 , a 3 . 5 kb long gene ( indicated “control site” in the figures ) . For microarray hybridizations , whole-cell extract or immunoprecipitated DNA was amplified and labeled with either Cy3 ( whole-cell extract ) or Cy5 ( immunoprecipitated sample ) and hybridized on an Agilent 44K yeast whole-genome oligonucleotide array as described [6] . Microarray images were read using an Axon 4000B scanner and analyzed using GenePix Pro 6 . 0 software ( Axon Instruments , Inc . ) . Files were converted to text files and analyzed using the R software . The signal was normalized , denoised , smoothed and peak calling was done exactly as described before [18] . The R code has been deposited to the github depository at https://github . com/ValerieBorde/ChIPchip-analysis . In all figures , ChIP ratio is the decile-normalized ratios after denoising and smoothing using a 1 kb ( Figs 2A , 2C and 3B ) or 2 kb ( Fig 3C ) window . To each probe of the array , we attributed the mRNA level of the corresponding gene determined from vegetative cells [57] or cells at 4 hr in meiosis [58] . For probes lying in a promoter , the mRNA level of the downstream gene was attributed , and for probes in divergent promoters , the mean value of the two divergent genes was attributed . For generating the graphs in Figs 2B and 3D , the average signal of the probes at the summit of the top 100 peaks ( 100 values ) was calculated , and compared to the signal for the entire microarray ( 41479 values ) . For generating the boxplot graphs in Fig 3C , we considered the ChIP signal at the probes corresponding to the summit of the top 200 Red1 ( for axis ) or DSB peaks . The definition of Red1 and DSB peaks from previously published datasets [23 , 59] has been described previously [56] . The ChIP-chip data generated in this study have been deposited in the Gene Expression Omnibus database , accession number GSE102790 . Processed data for all chromosomes are provided in S3 Table . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [60] with the dataset identifier PXD007590 .
|
Meiotic recombination is a conserved pathway of sexual reproduction that is required to faithfully segregate homologous chromosomes and produce viable gametes . Recombination events between homologous chromosomes are triggered by the programmed formation of DNA breaks , which occur preferentially at places called hotspots . In many organisms , these hotspots are located close to a particular chromatin modification , the methylation of lysine 4 of histone H3 ( H3K4me3 ) . It was previously shown in the budding yeast model that one protein , Spp1 , plays an important function in this process . We further explored the functional link between Spp1 and its interacting partners , and show that Spp1 shows genetically separable functions , by depositing the H3K4me3 mark on the chromatin , “reading” and protecting it , and linking it to the recombination proteins . We provide evidence that Spp1 is in distinct complexes to perform these functions . This work opens perspectives for understanding the process in other eukaryotes such as mammals , where most of the proteins involved are conserved .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
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] |
2018
|
The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
|
Buruli ulcer ( BU ) is a skin disease caused by Mycobacterium ulcerans , with endemicity predominantly in sub-Saharan Africa and south-eastern Australia . The mode of transmission and the environmental reservoir ( s ) of the bacterium and remain elusive . Real-time PCR investigations have detected M . ulcerans DNA in a variety of Australian environmental samples , including the faeces of native possums with and without clinical evidence of infection . This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria , Australia . Twenty-seven clinical cases of M . ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998–2011 . Common ringtail possums ( Pseudocheirus peregrinus ) , a common brushtail possum ( Trichosurus vulpecula ) and a mountain brushtail possum ( Trichosurus cunninghami ) were included in the clinically affected cohort . Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions , generally confined to the face , limbs and/or tail . The disease was minor and self-limiting in the case of both Trichosurus spp . possums . In contrast , many of the common ringtail possums had cutaneous disease involving disparate anatomical sites , and in four cases there was evidence of systemic disease at post mortem examination . Where tested using real-time PCR targeted at IS2404 , animals typically had significant levels of M . ulcerans DNA throughout the gut and/or faeces . A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces ( subclinical cases ) , and in one of these the organism was cultured from liver tissue . Comparisons were made between clinically and subclinically affected possums , and 61 PCR-negative , non-affected individuals , with regards to disease category and the categorical variables of species ( common ringtail possums v others ) and sex . Animals with clinical lesions were significantly more likely to be male common ringtail possums . There is significant disease burden in common ringtail possums ( especially males ) in some areas of Victoria endemic for M . ulcerans disease . The natural history of the disease generally remains unknown , however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection , whereas some severely affected animals , especially common ringtail possums , may become systemically , and potentially fatally affected . Subclinical gut carriage of M . ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient . Further work is required to determine whether M . ulcerans infection poses a potential threat to possum populations , and whether these animals are acting as environmental reservoirs in certain geographical areas .
Mycobacterium ulcerans is an environmental organism that causes distinctive dermal lesions in people and animals , via the elaboration of the cytotoxic and immunosuppressive polyketide toxin , mycolactone [1] . The disease is known internationally as Buruli Ulcer ( BU ) . It has a worldwide but highly focal distribution , with endemicity recorded in 33 countries to date , predominately in sub-Saharan Africa and Australia . Sporadic cases and a number of localised outbreaks have been documented in people [2]–[13] and a variety of wild and domestic mammals [14]–[22] in southeastern Victoria , Australia , since the first reports of the disease over 60 years ago [3] , [23] . Shortly after the initial reports of infection in people , it was shown that the common brushtail ( CBT ) possum ( Trichosurus vulpecula , Kerr 1792 ) was experimentally susceptible to the disease [24] , [25] . These investigations were undertaken because this species was noted to be highly susceptible to infection caused by members of the M . tuberculosis complex [26] . In brief , ulcerative lesions could be induced by local subcutaneous inoculation of organisms , and two animals inoculated via the intraperitoneal route developed lesions containing acid-fast bacilli ( AFB ) at peripheral sites ( tail or tail base , scrotum , inguinal lymph node , stifle and front paw ) . Despite this apparent susceptibility , there was no evidence of the infection in the wild CBT possum population in an endemic area of Victoria in the decades immediately following the initial outbreak , despite ongoing sporadic disease in the human population [3] . However , following an outbreak of the disease in people on Phillip Island , Victoria in the mid 1990s , two local wild common ringtail ( CRT ) possums ( Pseudocheirus peregrinus , Boddaert 1785 ) were confirmed with M . ulcerans infection [18] . Since 2000 , there has been an ongoing outbreak of BU in residents and visitors to the Bellarine Peninsula of Victoria [19] . Analysis of environmental samples using a highly sensitive real-time polymerase chain reaction ( PCR ) technique targeting sequences within IS2404 , IS2606 and KR [19] , [27] , [28] has confirmed low levels of M . ulcerans DNA in plant biofilms , vegetation , soil samples and local mosquitoes [29] , and much higher levels ( in some instances greater than 106 organisms/gram ) , in the faeces of local wild CRT and CBT possums [19] . Despite this , the culture of viable organisms from these samples has remained elusive [19] . Variable-number of tandem repeats ( VNTR ) profiles of the DNA extracted from Point Lonsdale possum faeces was indistinguishable from that of the Victorian human outbreak strain of M . ulcerans [19] , [28] . This report expands on the previous work performed by the authors' investigative group , by describing in detail the clinical , microbiological and pathological features of M . ulcerans infection of 27 clinical and 12 subclinical possum cases domiciled in three endemic areas of southern Victoria , Australia: Cowes ( Phillip Island ) , Bellbird Creek ( East Gippsland ) and Point Lonsdale ( Bellarine peninsula ) . This report represents the largest case series of naturally occurring M . ulcerans infection in any non-human species to date . Possible links between the disease in people and the resident possum population , and future avenues of investigation are discussed .
‘Clinical’ cases were defined as any possum with ulcerative skin lesions from which M . ulcerans was cultured , or was detected by IS2404 PCR from clinical material , including swabs and necropsy tissues . ‘Subclinical’ cases were defined as any possum from which M . ulcerans was cultured or was detected by PCR from clinical material , including gut contents and/or faeces , where there was no gross or histopathological evidence of disease . During the course of the investigation , any possum in which no M . ulcerans DNA could be detected via PCR in any clinical sample was classified as ‘non-affected’ . Cases were identified by a review of the record database of the Mycobacterium Reference Laboratory of the Victorian Infectious Diseases Reference Laboratory ( VIDRL ) from 1998 to 2011 , and from prospective capture and sampling of possums indigenous to some known M . ulcerans-endemic areas of Victoria; Bellbird Creek , between 2007–2012 , as part of a routine surveillance program conducted by the Department of Environment and Primary Industries , and Point Lonsdale between 2008–2010 , as part of an epidemiological study of M . ulcerans disease in these species . The methods of the latter study have been published elsewhere [19] . Clinical and/or autopsy data were also retrieved from case records of Newhaven Veterinary Clinic , Phillip Island , Victoria , the veterinary department of Melbourne Zoo , Parkville , Victoria , the laboratory records of the University of Melbourne Veterinary Hospital , Werribee , Victoria and the Department of Environment and Primary Industries , Veterinary Diagnostic Services , Victoria . Where available , data such as species , sex , estimated age at diagnosis ( juvenile/adult ) , year of diagnosis , geographical location of domicile , anatomical location and nature of lesions , and results of autopsy examination ( including histopathology ) were recorded . Fresh faeces from mountain brushtail ( MBT ) possums ( Trichosurus cunnighami , Lindenmayer 1990 ) trapped at Bellbird Creek were collected from within or underneath traps . For some possums trapped in Point Lonsdale , one to two millilitres of blood was collected from the cephalic , medial saphenous , or tail vein and placed in a plain tube . The blood sample was centrifuged at 16 , 100 g ( Eppendorf 5415D , Hamburg , Germany ) for 2 minutes before the serum was separated and stored at −80°C , the pellet was used for IS2404 PCR analysis . Urine was collected via percutaneous cystocentesis where possible . Buccal and nasal swabs were obtained from some individuals . Fresh faeces were collected from underneath traps or from holding bags; a cloacal swab was collected from individuals if no faeces were obtained . Details of the methods utilised in the capture and sampling of possums has been described previously [19] . The project was approved by the University of Melbourne Faculty of Veterinary Science Animal Experimentation Ethics Committee ( project no . 0706769 ) and was carried out under a permit from the Victorian Department of Sustainability and Environment ( DSE permit no . 10004406 ) . Apart from case 28 , which was found deceased , animals that underwent autopsy examination were euthanased with an overdose of sodium pentobarbitone , and tissues collected were either fixed in 10% buffered formalin for histopathology or were submitted fresh for microbiology . Formalin-fixed tissues were then embedded into paraffin blocks and processed routinely prior to staining with haematoxylin-eosin and/or acid-fast staining with either Ziehl-Neelsen ( ZN ) stain or Wade's modification of the ZN stain [30] . Initially , IS2404 PCR was performed by the Microbiological Research Unit , Royal Children's Hospital ( prior to 2002 ) and thence at VIDRL , utilising the methods published by Ross et al [31] . Since 2003 , a multiplex real-time PCR assay has been used to test clinical and environmental samples for the presence of M . ulcerans DNA [27] . Culture of M . ulcerans from lesions and necropsy tissue was carried out after decontamination via incubating at room temperature for 15 minutes with either 2 or 4% sodium hydroxide , followed by neutralisation with 10% orthophosphoric acid . The samples were then centrifuged at 3082 g ( Beckman Coulter Allegra X-22 , Pasadena , California , USA ) for 20 minutes and the resultant pellet was re-suspended in 2 ml of Ringer's solution . 400 µl was then used to inoculate Brown and Buckle slopes , and Mycobacterium Growth Indicator Tubes ( BD , Franklin Lakes , N . J . ) admixed with 0 . 8 ml of manufacturer-supplied antibiotic mixture and incubated for up to 12 weeks at 31°C , with weekly or fortnightly monitoring by either visual inspection or IS2404 real-time PCR . Isolates were typed by either restriction fragment length polymorphism ( RFLP ) typing [32] , variable-number of tandem repeats ( VNTR ) typing [33] , [34] and in two instances , whole genome sequencing [35] . The two-tailed Fischer exact test was used to test for associations between categorical variables . The t-test was performed when comparing the mean of continuous variable data sets . P values<0 . 05 were considered significant .
Twenty-seven clinically affected animals were identified for inclusion in the study ( Table 1 ) . There were 23 CRT , one CBT and one MBT possum . For two possums , species was not recorded . Of the 25 animals for which sex was recorded 17 ( 69% ) were male . Of the 24 animals in which an age estimate was performed , one was a juvenile and 23 were estimated to be adults ( although more objective data such as dental examination were not utilised ) . Thirteen of the animals ( 11 CRT possums and two species not recorded ) were from the Cowes region of Phillip Island , Victoria ( Figure 1 ) . These animals had been surrendered to the local veterinary hospital ( Newhaven Veterinary Clinic ) because of illness or trauma between 1998 and 2011 . The MBT possum was captured in 2005 at Bellbird Creek ( Figure 1 ) . This animal was transferred to Melbourne Zoo , Parkville , Victoria , for management . Thirteen animals ( 12 CRT and one CBT ) were from Point Lonsdale , Victoria ( Figure 1 ) ; all were trapped between 2008–2010 as part of the prospective epidemiological study . The nature and anatomical location of lesions were recorded for 25 possums ( Table 1 ) . Twelve animals had single skin lesions , five had two skin lesions and eight animals had multiple skin lesions , involving three or more anatomical sites . Lesions recorded on CRT possums were located on the face and/or head ( n = 13 ) , feet and/or limbs ( n = 11 ) and tail and/or tail base ( n = 14 ) ( Figure 2 ) . One animal also had scrotal ulcers ( as well as multiple lesions at other sites ) . One CRT fitted with a radio-tracking collar ( case 26 ) , noted to initially have a solitary tail lesion , was found deceased several months later with a large IS2404 PCR-positive lesion on the ventrolateral thorax . The CBT possum had a small ulcer on a front toe ( Figure 3a ) and the MBT had a single ulceration on the left hind foot ( Figure 3b ) . Both of these animals were noted to have spontaneous healing of lesions without specific treatment over a 3–6 month period . Only CRT possums were recorded to have two or more lesions , with six disparate anatomical sites in two instances . Eight animals ( cases 5 , 8 , 14–16 , 24 , 25 , and 27 ) underwent partial or complete autopsy examination . The necrotic skin ulcers appeared grossly and histopathologically similar to those described in other species [16] , [22] , [36] . The lesions were characterised by extensive loss of the epidermis . A superficial crust composed of serous exudate and degenerate leukocytes was present , overlying a necrotic base ( Figure 4a ) . The margins of the lesions were characterised by proliferative epidermis overlying fibrotic and sometimes , oedematous dermal tissue admixed with pyogranulomatous and/or lymphoplasmacytic inflammation . In ZN-stained sections , abundant numbers of AFB were present in the dermis , either extra-cellularly , or within macrophages ( Figure 4b ) . Four animals with extensive cutaneous disease ( cases 16 , 24 , 25 and 27 ) also had evidence of systemic involvement , with patchy granulomatous , neutrophilic and/or lymphoplasmacytic lesions in the liver and lung . Rare AFB were present in macrophages in some sections of liver ( Figure 4c ) . The results of the microbiological investigations for clinically affected animals performed on either lesion swabs and/or necropsy material and , in most cases , gut contents or faeces are presented in Tables 2 and 3 . M . ulcerans was cultured from skin lesions only in 19 animals , the liver , spleen and a mandibular lymph node in case 19 , and skin lesions , liver , lung and small intestinal contents in case 27 . The organism was also cultured from the abdominal cavity of the decomposing carcass of case 26 . RFLP typing of isolates was performed in four cases and found to be “V1” type [32] . VNTR typing was performed in 15 cases and was found to be indistinguishable from M . ulcerans strains causing human and animal disease in Victoria . Analyses of the whole genome sequences of the isolates from cases 10 and 16 , which have been described previously [35] , confirmed a close genetic relationship with M . ulcerans isolates from Victorian human patients . The duodenum of case 16 also contained a large tapeworm ( species not identified ) , which was PCR-negative , despite the moderate real-time PCR-positivity of the small-intestinal contents of this animal . PCR-positive fly larvae ( species not identified ) were also retrieved from the carcass of case 26 , however M . ulcerans , could not be cultured successfully from these larvae . Twelve animals met the criteria for classification as a subclinical case ( Table 4 ) . These were all adult animals , comprising one male and three female CBT possums , four female CRT possums , and one male and three female MBT possums . The animals were domiciled in Point Lonsdale ( CBT and CRT possums ) , and Bellbird Creek ( MBT possums ) . One animal ( case 28 ) was identified by a Point Lonsdale resident , while the remainder were trapped as part of the aforementioned studies ( cases 29–39 ) . Some CBT and MBT possums were trapped multiple times; the time-line of results from PCR analysis on faecal samples from these animals is presented in Figure 5 . [30] → Two subclinical cases ( cases 28 and 32 ) underwent autopsy examination . Case 28 had been seen at ground level during the late afternoon and had appeared unusually docile . The animal was found deceased the following day and its body was submitted to the veterinary pathology service of The University of Melbourne a few days later . Although there was significant tissue autolysis , and cause of death could not be determined , there were no cutaneous lesions consistent with M . ulcerans infection . Low levels of M . ulcerans DNA were detected by real-time PCR in tissue from the liver , spleen , lung and gut wall , as well as small and large bowel contents and faecal pellets retrieved from the rectum . M . ulcerans as also cultured from the liver tissue . The carcass also contained fly larvae ( species not identified ) , which were also weakly IS2404 PCR-positive . Case 32 was found to have IS2404 PCR-positive faeces and was euthanased and underwent autopsy as part of the epidemiological study . The animal had no gross or histopathological lesions . A moderate level of M . ulcerans DNA was detected in all gut compartments ( Table 3 ) , and salivary gland tissue was also weakly PCR-positive [35] . Of the 69 animals trapped in Point Lonsdale , 50 ( 72% ) had no detectable M . ulcerans DNA ( via IS2404 real-time PCR ) in any clinical sample . This cohort comprised 13 male and 6 female CBT possums , and 13 male and 18 female CRT possums . Eleven ( 73% ) MBT possums trapped at Bellbird Creek had PCR-negative faecal samples on all trapping occasions . This group of animals comprised 7 males and 4 females . Categorical variables of ( i ) case type ( clinical , subclinical and non-affected ) ( ii ) species ( CRT possum and non-CRT possum ) , and ( iii ) sex ( male and female ) were compared using 2×2 contingency tables ( raw data presented in Table 5 ) . A comparison of animals with ( clinical ) and without cutaneous lesions ( subclinical and non-affected possums ) was made; the former animals were more likely to be CRT possums , or male , although the latter variable did not reach significance . When the variables of sex and species were combined , male CRT possums were more likely to have clinical lesions than other animals ( odds ratios , including 95% confidence intervals and P values are presented in Table 6 ) . When comparisons were made between animals with a PCR-positive sample ( both clinical and subclinical cases ) and those in which all clinical samples were PCR-negative ( non-affected cases ) ( results presented in Table 7 ) , individuals in the former group were more likely to be CRT possums . There was no sex predilection for PCR-positivity . The mean IS2404 real-time PCR signal strength ( CT ) of approximately 100 mg of faeces collected from clinically ( n = 10 ) and sub-clinically affected ( n = 12 ) animals was calculated as 29 . 32 ( SD ±5 . 43 ) and 33 . 54 ( SD ±4 . 82 ) , respectively ( raw data not shown ) ( Figure S1 ) . When the values were compared , the difference was not statistically significant ( P = 0 . 075 ) .
Although there are previous reports of M . ulcerans infection in possums [18] , [19] , this is the first study to examine the clinical , pathological and microbiological aspects of the disease in detail , and also represents the largest and most comprehensive case series of the disease in any non-human species . Affected possums have only been recorded from areas of known M . ulcerans endemicity in Victoria , Australia . This is true for all reports of the infection in non-human mammalian species , despite the fact that the disease has been reported in many disparate areas of the world . The reasons for this epidemiological discrepancy are currently unknown , but may be due to specific host or environmental factors ( for example , the presence of particular insect vectors in a temperate climate ) , or genomic differences related to virulence or host specificity of the ‘Victorian’ strain of M . ulcerans , compared to strains found elsewhere . There has been some attempt to identify animal cases in endemic areas of Africa and other parts of Australia , however this research has not yet yielded any positive results ( J . Fyfe , C . Lavender , PDR . Johnson unpublished observations ) [37] , [38] . It has been reported that some species of marsupials appear prone to particular mycobacterial diseases [39] ( for example , CBT possums and M . bovis infection , macropods and infections caused by the M . avium complex ) and it is noteworthy that the next animal species most commonly reported with M . ulcerans infection is another Australian arboreal marsupial , the koala [15]–[17] . The prevalence of M . ulcerans infection in the possum populations in this study cannot be accurately estimated due to the inherent difficulties in sampling from a wild population; however , it appears that CRT possums are significantly more susceptible to clinical M . ulcerans infection than other possum species . Male CRT possums appeared to be particularly predisposed to clinical lesions , perhaps due to behaviours such as fighting ( which was touted as a possible reason why male koalas appeared over-represented in an earlier study [15] ) or as a result of stress associated with competition for territories . More extensive trapping and sampling and/or captive studies would be informative in revealing whether CRT possums have higher susceptibility to infection and disease across all BU-endemic areas in Victoria . Further , it is noteworthy that only CRT possums were observed with advanced clinical disease , with a number of animals displaying multiple lesions at disparate anatomical sites , and that some cases progressed to systemic disease involving the liver and lungs . It is unclear whether the apparent susceptibility of the CRT possum is a function of impaired immunity or epidemiological factors such as high rates of environmental exposure , perhaps via communal sharing of contaminated dens ( CRT possums are more social than most other possum species [40] ) , inoculation of the bacterium via penetrative wounds or insect vectors , or possibly via the practice of auto and/or allocoprophagy [41] . Due to the economic impact of CBT possums as reservoirs for M . bovis in New Zealand , much effort has been expended in assessing the immunobiology of this species . No such data exist for the CRT possum and testing of the immunological competence of non-infected controls is needed to establish whether there are any inherent immunological deficiencies in these populations , and would be helpful in differentiating any local or systemic immunosuppressive effects of mycolactone in infected individuals . The significance of variable levels of M . ulcerans DNA in the gastrointestinal tract of both clinically and subclinically affected possums in endemic areas is unknown . There were no lesions consistent with established gastrointestinal infection in any animals where histopathological examination of the gut was performed . The question as to whether these are viable organisms ( and therefore represent a significant source of potentially infective organisms to other animals and people ) remains unanswered , despite concerted attempts to culture M . ulcerans from fresh and aged PCR-positive faecal material collected from the environment , and fresh gut contents collected at autopsy . A single isolate was recovered from the small intestinal contents of a systemically affected CRT possum from Phillip Island , however the possibility of cross-contamination from lesions cannot be ruled out in this instance . Alternative methods to demonstrate metabolic activity of M . ulcerans within possum gut contents/faeces , for example , via rRNA or mycolactone detection remain a possibility , although negative findings may theoretically result if the organisms have become metabolically dormant . Based on real-time PCR signal strength , there is no evidence that the organism is amplified in any particular region of the gut of affected animals . Nor does it appear that clinically affected cases have significantly greater amounts of M . ulcerans DNA in the gut compared to subclinical cases . There is also no sex predilection for PCR-positivity in the gut contents or faeces , perhaps suggesting a common environmental source of oral inoculation in all animals ( even though there may be separate risk factors for clinical disease between the sexes ) . If the organisms are viable within the gut , the daily practice of autocoprophagy in CRT possums [42] provides a possible route for re-inoculation and amplification of organisms in the gut of an individual ( and thus ongoing colonisation/infection ) . It is not known whether this represents a source of horizontal transmission to other individuals ( thus maintaining colonisation/infection in the population ) via allocoprophagy . This seems most likely , perhaps , in juveniles approaching weaning . In the koala , which like the CRT is also a specialist folivore , establishment of normal gut flora in dependent juveniles occurs during weaning via allocoprophagy [43] . An alternative significant environmental source of oral inoculation ( for example , a contaminated food source ) remains elusive , although interrogation of a variety of plant species ( potentially foraged by CRT possums in some endemic areas ) via IS2404 real-time PCR demonstrated low levels of M . ulcerans DNA ( data not shown ) . Except in one instance , only animals with cutaneous lesions were found to have PCR-positive buccal swabs , which might suggest that the positivity of these samples may be due to contamination of the oral cavity via licking of ulcers . However , it cannot be conclusively shown that these findings are not due to ingestion of M . ulcerans DNA from the environment or contaminated caecotrophs . The fact that the non-coprophagous CBT has also been noted to have similar levels of M . ulcerans DNA in the faeces [19] also argues for a potential oral environmental source . It is unknown at this stage whether the finding of M . ulcerans DNA in the gut of possums represents transient or persistent gut contamination , colonisation or infection , although in two CBT and four MBT possums trapped repeatedly over the course of the study low level faecal PCR-positivity was observed to be transient . The possibility that the M . ulcerans DNA was derived from soil or environmental detritus contamination of the external surface of the faeces was considered unlikely , given that the samples were mostly collected directly from within traps or collection boxes , rather than the ground . No record of whether gut PCR-positivity was transient or permanent in CRT possums was obtained due to the difficulties of repeat trapping of this species , even with the aide of radio-tracking collars ( A . Legione , K Handasyde unpublished observations ) . It is also not known whether gut colonisation/infection is directly linked with prior or eventual clinical disease . The only clinically affected individual without PCR-positive faeces ( case 13 ) had numerous superficial cutaneous lesions on the tail that were different in appearance from the typically deep , undermined ulcers observed in other animals in the study . Whilst these lesions were weakly PCR-positive , it cannot be excluded that they were actually caused by a different disease entity ( such as pox-virus infection [44] ) that had become contaminated with M . ulcerans DNA from the environment . Based on real-time PCR results of urine samples , it does not appear that M . ulcerans is shed via the urinary tract , nor is there evidence of mycobacteraemia in the animals from which blood samples were obtained . Thus , it is unlikely that possums are a significant source of potentially infective blood meals for vectors such as mosquitoes or flies , although mechanical transmission from the wounds of infected possums to humans via contaminated insect vectors cannot be ruled out . While the natural history of this disease in possums is generally unknown , lesions were observed to undergo spontaneous remission in a CBT and a MBT possum in this study . It is not known if either of these two individuals eventually suffered disease relapse ( one individual was kept in captivity but has been lost to follow-up , and one was released back into the wild ) . Due to the aforementioned difficulties in repeated trapping of CRT possums it is unknown whether clinical lesions are able to undergo spontaneous remission in this species . Given the state of ill-health of some of the CRT possums in this study , ( and possibly the CBT possum , case 28 ) it seems that the disease may become progressive , possibly leading to death either directly due to the effects of the M . ulcerans infection , or due to secondary illness ( although no co-morbidities were identified in severely affected animals that underwent autopsy ) . Unfortunately , in the two animals found deceased ( cases 26 and 28 ) the cause of death could not be determined due to the state of decomposition , thus it is impossible to say what role , if any , M . ulcerans played in their demise . The systemically affected CRT possums had histopathological evidence of disease in the lungs and liver . Lower respiratory tract infections have been previously reported in koalas with extensive nasal cavity disease , presumably due to inhalation of the organisms [17] . The authors speculated that due to the organism's strict temperature requirements ( 27–33°C ) the infection was possibly maintained in this anatomical site due to a sub-normal core body temperature that could occur in a moribund animal . Unfortunately , the core body temperature of the severely affected possums in this study was not measured , however the normal core body temperature of the CRT possum is 35–36°C [45] , thus it is conceivable that these individuals may become sufficiently hypothermic to allow growth of M . ulcerans within the body [39] . Circulating mycolactone has been detected in human patients [46] , and may also play a role in producing systemic immunosuppression in animals with extensive disease . Our initial study of M . ulcerans infection in possums has highlighted a number of areas that warrant further investigation . More detailed studies are necessary to document the natural history of the disease and the level of M . ulcerans DNA shed in the faeces over time , in both naturally and experimentally infected animals ( especially CRT possums ) . One difficulty is that the stress of bringing wild possums into captivity is likely to confound results from any such investigations , and although ideally such aims could be achieved via radio-tracking of wild animals , the heavy attrition of CRT possums in their natural habitat [47] , makes this work difficult , as a pilot study by our group has confirmed ( A . Legione , K . Handasyde Unpublished observations ) . Research into the potential for horizontal and vertical transmission between possums is also needed , as well as ongoing attempts to determine the viability of M . ulcerans within gut contents/faeces . In conclusion , the disease burden in CRT possums ( especially males ) in some areas of Victoria endemic for M . ulcerans disease appears significant . Whilst it appears that CBT and MBT possums with solitary cutaneous lesions have the ability to overcome the infection , the natural history of the disease generally remains unknown . In some instances , severely affected animals , especially CRT possums , may become systemically , and potentially fatally affected . As previous work has shown , subclinical gut carriage of M . ulcerans DNA in possums is quite common [19] , and this study has shown that in some CBT and MBT possums it is transient . It is unknown whether this is also the case for CRT possums . Further work is required to establish whether this disease poses a potential threat to possum populations , and whether these animals are contributing to the high incidence of M . ulcerans infection in people in certain geographical areas by acting as environmental reservoirs .
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Mycobacterium ulcerans causes skin disease predominantly in sub-Saharan Africa and southeastern Australia . The mode of transmission and the environmental reservoir ( s ) of the bacterium are unknown . Investigations have detected M . ulcerans DNA in a variety of Australian environmental samples , including the faeces of native possums . This report expands on these studies by detailing the clinical , pathological and microbiological findings in affected wild possum species in endemic areas . Twenty-seven clinically and 12 subclinically affected individuals were identified . Most clinical cases were adults with skin ulcers of the face , limbs and/or tail . The disease was mild and self-limiting in both Trichosurus spp . possums . In contrast , many of the common ringtail possums had multiple skin ulcers and in some there was evidence of internal disease . There were also significant levels of M . ulcerans DNA throughout the gut . Comparisons were made with regards to disease category , species and sex; with clinical cases more likely to be male common ringtail possums . Asymptomatic gut carriage of M . ulcerans DNA is quite common and may be transient in some individuals . Further work is needed to determine whether M . ulcerans infection poses a potential threat to possum populations , and whether these animals are acting as reservoirs in some areas .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"bacterial",
"diseases",
"infectious",
"diseases",
"buruli",
"ulcer",
"animal",
"types",
"wildlife",
"emerging",
"infectious",
"diseases",
"veterinary",
"microbiology",
"neglected",
"tropical",
"diseases",
"biology",
"microbiology",
"veterinary",
"science"
] |
2014
|
Clinical, Microbiological and Pathological Findings of Mycobacterium ulcerans Infection in Three Australian Possum Species
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Type III secretion systems ( T3SS ) are essential for virulence in dozens of pathogens , but are not required for growth outside the host . Therefore , the T3SS of many bacterial species are under tight regulatory control . To increase our understanding of the molecular mechanisms behind T3SS regulation , we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis . We identified two unique transposon insertions in YPTB2860 , a gene that displays 79% identity with the E . coli iron-sulfur cluster regulator , IscR . A Y . pseudotuberculosis iscR in-frame deletion mutant ( ΔiscR ) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS . To determine the mechanism behind IscR control of the Ysc T3SS , we carried out transcriptome and bioinformatic analysis to identify Y . pseudotuberculosis genes regulated by IscR . We discovered a putative IscR binding motif upstream of the Y . pseudotuberculosis yscW-lcrF operon . As LcrF controls transcription of a number of critical T3SS genes in Yersinia , we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF . Indeed , purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y . pseudotuberculosis in the absence of IscR . Importantly , mice orally infected with the Y . pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches , mesenteric lymph nodes , spleens , and livers , indicating an essential role for IscR in Y . pseudotuberculosis virulence . This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator , LcrF .
Type III secretion systems ( T3SS ) are important components in the progression of disease for a number of clinically relevant human pathogens , including those in the genera Shigella , Salmonella , Escherichia , Chlamydia , Vibrio , Pseudomonas , and Yersinia [1] , [2] . The T3SS functions as an injectisome that delivers bacterial effector proteins directly into the host cell cytoplasm [2] . While the T3SS apparatus itself is structurally conserved , the repertoire of T3SS effector proteins used by each group of pathogens is distinct [2] . Thus , the effect of the T3SS on the host is unique to the needs of the pathogen [2] . While the T3SS is generally essential for a T3SS-expressing pathogen to cause disease , several aspects of the T3SS may be detrimental to bacterial growth [2] . For example , T3SS components are recognized by the host immune system [3] , [4] . In addition , expression of the T3SS is energetically costly and , in some organisms , T3SS induction correlates with growth arrest [5] . Therefore , regulation is essential for proper T3SS function in order to ensure that it occurs only during host cell contact in the appropriate host tissue [2] , [6] . Members of the genus Yersinia that utilize a T3SS are important human pathogens: Y . pestis , the causative agent of plague , and the enteropathogens Y . enterocolitica and Y . pseudotuberculosis . The Y . pseudotuberculosis Ysc T3SS is encoded on a 70-kb plasmid termed pYV [7]–[9] and is made up of approximately 25 known proteins comprising three main structures: the basal body , the needle apparatus , and the translocon [10] , [11] . The basal body , which displays a high degree of similarity to the flagellar basal body , is made up of rings that span the inner and outer membranes and a rod that traverses the periplasmic space [12] . Basal body associated proteins include YscN , an ATPase that aids in the secretion and translocation of effector proteins [13] . The needle complex , which is thought to act as a molecular channel for effector protein translocation , is a straight hollow appendage approximately 60 nm in length and is made up of helical polymerized subunits of YscF [12] . The translocon is comprised of three proteins: YopD , YopB and LcrV , which are essential for pore formation in the target host membrane and proper translocation of effector proteins YopHEMOJTK to the host cytoplasm [12] , [14] . Also encoded on pYV are chaperones important for efficient translocation of a subset of effector proteins [15] . Lastly , several transcriptional and post-transcriptional regulators of the T3SS are found on pYV , including the AraC-like transcriptional regulator LcrF . LcrF is responsible for expression of a number of T3SS structural genes and Yop effectors , specifically the virC and lcrGVH-yopBD operons as well as genes encoding effector Yops , the adhesin YadA , and the lipoprotein YlpA [16]-[22] . LcrF itself is thermoregulated at both the transcriptional and translational levels through the action of the histone-like protein YmoA and a cis-acting RNA thermosensor located on the lcrF transcript , respectively [23] , [24] . This enables Yersinia to express T3SS genes at 37°C within the mammalian host , but not at lower temperatures [23] , [24] . Importantly , proper LcrF-mediated control of T3SS expression is important for Y . pseudotuberculosis virulence [24] . IscR belongs to the Rrf2 family of winged helix-turn-helix transcription factors [25] , [26] and has been studied extensively in E . coli , where its DNA-binding activity is dependent on coordination of an iron-sulfur [2Fe-2S] cluster through three conserved cysteines and a histidine [27]–[30] . E . coli IscR recognizes two distinct DNA motifs , type 1 and type 2 , depending on the Fe-S status of the protein [31] . Holo-IscR coordinating an Fe-S cluster binds both type 1 and type 2 motifs , while clusterless apo-IscR recognizes only the type 2 DNA-binding motif [27] , [32] , [33] . As iron starvation , oxidative stress , and oxygen limitation affect the holo-IscR/apo-IscR ratio , these environmental cues are thought to have a direct effect on gene expression through IscR in E . coli [28]–[30] . For example , holo-IscR represses transcription of the housekeeping iscRSUA-hscBA-fdx Fe-S cluster biogenesis operon [32] , [34] , while either holo- or apo-IscR promotes transcription of the inducible sufABCDSE Fe-S cluster biogenesis operon [33] , [35] . Both pathways function to insert Fe-S clusters onto proteins involved in a range of metabolic processes including electron transfer , substrate binding/activation , iron/sulfur storage , regulation , and enzyme activity [36] . In addition , E . coli IscR is also known to regulate transcription of other Fe-S cluster assembly genes such as erpA ( yadR ) as well as genes integral to oxidative stress resistance , biofilm formation , and anaerobic respiration [28]–[30] , [34] . IscR is widely conserved among bacteria [25] and its regulatory activity is integral to the infectious process of the plant pathogen Erwinia chrysanthemi [37] . Furthermore , IscR plays an important role in the virulence of the human pathogens Pseudomonas aeruginosa through modulation of the catalase katA [38] , Burkholderia mallei through resistance to reactive nitrogen species [39] , and Vibrio vulnificus through induction of several virulence-associated pathways [39] , [40] . While the iron-dependent transcriptional repressor Fur has been shown to control T3SS expression in Salmonella and Shigella [41] , [42] , IscR has never been linked to regulation of the T3SS in any organism and has not been studied in Yersinia . In this study , we isolated two independent IscR transposon insertion mutants in a novel screen for Y . pseudotuberculosis genes important for T3SS function . We assessed the impact of iscR deletion on Y . pseudotuberculosis in vitro and in vivo growth , type III secretion , and global gene expression . We found IscR to be essential for full T3SS function and virulence in a mouse model of infection . In addition , we provide evidence that IscR control of the T3SS stems from direct transcriptional regulation of the T3SS master regulator LcrF .
To identify regulators of the Y . pseudotuberculosis T3SS , we utilized a novel screen to isolate transposon mutants with defects in T3SS function . We previously showed that Y . pseudotuberculosis expressing a functional T3SS induces NFκB activation in HEK293T cells [43] , enabling us to use host cell NFκB activation as a readout for T3SS function in Y . pseudotuberculosis transposon mutants . As some T3SS effector proteins inhibit NFκB signaling [44] , we performed the screen using a Y . pseudotuberculosis transposon mutant library in a genetic background that lacked the known T3SS effector proteins YopHEMOJT ( Δyop6; [43] ) . We identified several transposon mutants with defects in triggering activation of NFκB in HEK293T cells ( L . Kwuan , N . Herrera , H . Ramirez , V . Auerbuch , data not shown ) , suggesting defective T3SS function . Among these were two strains with unique transposon insertions in YPTB2860 ( Figure 1A ) , encoding a protein with 79% identity to the E . coli iron-sulfur cluster regulator IscR , part of the iscRSUA-hscBA-fdx operon involved in Fe-S cluster biogenesis ( Figure 1B ) . Importantly , the helix-turn-helix DNA binding domain as well as the three cysteines and histidine known to coordinate an iron-sulfur ( Fe-S ) cluster in E . coli IscR are conserved in all three Yersinia species ( Figure 1B ) . These data indicate that Yersinia IscR may coordinate an Fe-S cluster and , as in E . coli , may regulate gene transcription . To validate that loss of IscR in Y . pseudotuberculosis leads to T3SS defects , we isolated the two iscR transposon mutants ( iscR::Tn1 and iscR::Tn2 ) from our library and again measured their ability to trigger NFκB activation in HEK293T cells compared to the Δyop6 parental strain and a ΔyscNU T3SS-null mutant [43] . In addition , we constructed an in-frame iscR deletion mutant in the Δyop6 genetic background ( Δyop6/ΔiscR ) and tested it in this assay . We found that disruption of iscR led to ∼2-fold less NFκB activation relative to the Δyop6 T3SS+ parental strain , although NFκB activation levels were still ∼5-fold higher than a strain with complete lack of T3SS function ( ΔyscNU; Figure 2A ) , suggesting that loss of iscR leads to partial T3SS loss . To verify further that deletion of iscR leads to alterations in T3SS function , we assessed the ability of the Δyop6/ΔiscR mutant to insert YopBD pores in target host cell membranes by measuring entry of ethidium bromide ( EtBr ) inside Y . pseudotuberculosis-infected bone marrow derived macrophages [45] , [46] . Pore formation by the Δyop6/ΔiscR mutant was decreased by 7-fold ( p<0 . 05 ) relative to the Δyop6 parental strain , which could be restored upon complementation with plasmid-encoded iscR ( Figure 2B ) . To determine whether loss of iscR affects T3SS function in a wild type genetic background , we constructed an in-frame iscR deletion ( ΔiscR ) in the wild type Y . pseudotuberculosis IP2666 strain expressing six of the seven known T3SS effector proteins YopHEMOJK [47] . We then visualized the secretome of the ΔiscR mutant relative to wild type . Deletion of iscR led to a dramatic decrease in secretion of T3SS cargo relative to the wild type background , which can be restored upon complementation with plasmid-encoded iscR ( Figure 2C ) . Importantly , this lack of type III secretion did not result from a defect in growth of the mutant , as the ΔiscR mutant actually grew better than wild type bacteria under T3SS-inducing conditions ( Figure S1A ) . This is consistent with a T3SS defect in this strain , as wild type Yersinia display a characteristic growth arrest upon T3SS expression [5] , [48] , [49] . Collectively , these data demonstrate that Y . pseudotuberculosis IscR is required for proper T3SS function . Based on the knowledge that the T3SS plays an important role in the virulence of human pathogenic Yersinia , we sought to investigate whether the diminished type III secretion observed in the Y . pseudotuberculosis ΔiscR strain would lead to a reduction in the infectious capacity of this mutant . Mice were orogastrically infected with 2×108 CFU of either the Y . pseudotuberculosis wild type or isogenic ΔiscR mutant strains . At 5 days post-inoculation , mice infected with the ΔiscR mutant displayed significantly decreased colonization of Peyer's patches and mesenteric lymph nodes ( MLN ) as well as diminished systemic colonization ( Figure 3 ) . Specifically , we noted 10- and 130-fold reductions in CFU recovered from the Peyer's patches and MLNs , respectively , in mice infected with the ΔiscR mutant strain relative to wild type . Notably , we observed a 1000- to 10 , 000-fold decrease in bacterial burden in the spleen and liver respectively . The diminished ability of the ΔiscR mutant strain to colonize deep tissue sites is underscored by the fact that bacteria were not detected in seven of the nine livers analyzed . These findings suggest that IscR is essential for Y . pseudotuberculosis virulence in an oral infection model . To begin to understand the mechanistic contribution of IscR to Y . pseudotuberculosis pathogenesis , we performed high throughput transcriptome sequencing ( RNAseq ) analysis to determine the Y . pseudotuberculosis genes directly and indirectly controlled by IscR under iron replete , T3SS-inducing conditions . Total RNA was collected from wild type Y . pseudotuberculosis as well as the ΔiscR mutant strain grown in M9 at 37°C for 3 h , a point at which the ΔiscR and wild type strains display comparable growth rates ( Figure S1A ) . For the ΔiscR mutant relative to the wild type , a total of 226 genes demonstrated a statistically significant fold change of ≥2 ( Table S1 ) . Of these , 134 genes were up-regulated in the ΔiscR mutant relative to the wild type ( Table 1 & Figure 4A ) , while 92 were down-regulated ( Table 2 & Figure 4B ) . Genes found to be up-regulated in the ΔiscR mutant include key elements of Fe-S cluster biosynthesis , cellular detoxification , metabolism , and protein fate ( Figure 4A ) . The most notable increases in transcription were observed for genes encoding Fe-S cluster biosynthesis proteins including those encoded in the isc operon , iscS ( 18 . 7-fold ) , iscU ( 21 . 7-fold ) and iscA ( 13-fold ) ( Table 1 & Figure S2A ) . Additional genes encoding proteins involved in Fe-S cluster assembly and their respective fold increases include iscX/yfhJ ( 10 . 8 ) , fdx ( 10 . 9 ) , hscB ( 10 ) , hscA ( 9 . 3 ) , yadR/erpA ( 6 . 8 ) , pepB ( 10 . 1 ) and nfuA ( 7 . 0 ) . To validate these findings , we performed qRT-PCR analysis on the second gene encoded in the iscRSUA operon , iscS , as well as on the gene encoding the Fe-S biosynthesis protein ErpA . Transcription of iscS was increased by 30-fold , while erpA expression was increased 5-fold ( p<0 . 05; Figure 5A ) . Bioinformatic analysis identified two IscR type 1 motifs upstream of the iscRSUA-hscBA-fdx operon ( Figure S2B ) as well as one site each located upstream of both erpA and nfuA ( data not shown ) . Based on this data , we propose that Y . pseudotuberculosis IscR modulates Fe-S cluster biosynthesis expression in a manner akin to that of E . coli IscR . In total , 92 genes were significantly down-regulated in the ΔiscR mutant relative to wild type Y . pseudotuberculosis ( Table 2 ) . These data demonstrate that the majority of pYV-encoded genes are decreased in the ΔiscR mutant relative to the wild type strain , including genes essential for proper T3SS expression and function . The virC and lcrGVH-yopBD operons as well as genes encoding the T3SS cargo YopHEMOJTK were the most affected upon deletion of iscR: the effector proteins YopJ ( −3 . 4-fold ) , YopM ( −5 . 3-fold ) and YopT ( −5 . 5-fold ) , the effector protein and translocation regulator YopK ( −9 . 3-fold ) , as well as a number of genes encoding T3SS structural proteins . Genes encoding regulators that control T3SS expression and function were decreased in the mutant including lcrQ ( −2 . 1-fold ) , lcrF ( −3 . 3-fold ) , lcrG ( −2 . 8-fold ) and lcrH ( −3 . 9-fold ) . To verify that T3SS gene expression was indeed decreased in the ΔiscR mutant , we measured the transcript levels of the genes encoding T3SS structural proteins YscN , YscF , and the T3SS transcriptional regulator LcrF via qRT-PCR . As detailed in Figure 5B , we observed fold decreases of 2 . 8-fold ( p<0 . 05 ) , 6 . 9-fold ( p<0 . 001 ) , and 5 . 4-fold ( p<0 . 0001 ) for yscN , yscF , and lcrF , respectively . These data support our RNAseq analysis and confirm that IscR is required for robust transcription of Y . pseudotuberculosis T3SS genes . In addition to T3SS genes , 25 other pYV-encoded genes were decreased in the mutant , but these are annotated as hypothetical proteins , transposases , and pseudogenes . Analysis of the relative abundance of pYV in the Y . pseudotuberculosis wild type and ΔiscR strains was performed in order to verify that the decreases in pYV-encoded genes were not a result of plasmid loss ( Figure S3 ) . The concentration of plasmid isolated from the wild type and ΔiscR mutant was comparable , suggesting that the decreased transcription of pYV-encoded genes , including those encoding the T3SS , are not a result of decreased stability of the pYV plasmid . To assess the contribution of Fe-S cluster ligation to IscR control of the T3SS , we constructed an IscR mutant strain in which the three conserved cysteines were substituted with alanines ( C92A , C98A , C104A; apo-locked IscR ) . Identical mutations in E . coli IscR render the protein unable to coordinate an iron-sulfur cluster , yet able to bind type 2 DNA binding motifs and to regulate target gene transcription [28]–[30] . We analyzed the secretome of the Y . pseudotuberculosis apo-locked IscR strain under T3SS-inducing conditions and found that the mutant was just as defective as the ΔiscR strain in Yop secretion ( Figure 2C ) . This defect could be complemented with plasmid-encoded wild type IscR . As apo-locked IscR is insufficient to promote type III secretion , holo-IscR-mediated regulation of gene expression through one or more type 1 motifs may be specifically involved in regulating T3SS gene expression . Alternatively , forcing all IscR expression within the cell to the clusterless form , which leads to IscR overexpression , may lead to alterations of bacterial pathways that indirectly affect type III secretion . Consistent with this latter explanation , the apo-locked IscR mutant exhibited decreased colony size on LB agar , slower growth in rich media ( Figure S1 ) , and decreased motility ( Figure 6A ) . The flagellar basal body is a T3SS itself , indicating that the defect in the Ysc T3SS for this strain may be a result of gross abnormalities in secretion systems . Based on these findings , we set out to examine whether the apo-locked IscR mutant demonstrated alterations in membrane potential , as this has been shown to be important for both motility and Ysc T3S in Y . enterocolitica [50] . To this end , we examined bacterial membrane potential under T3SS inducing conditions . As demonstrated in Figure 6B , there is a notable decrease in membrane potential in the apo-locked IscR mutant relative to the wild type strain , which can be complemented upon addition of wild type iscR on a plasmid . Furthermore , the membrane potential of the ΔiscR mutant strain is comparable to that of the wild type . Collectively , these data suggest that the apo-locked IscR mutant has a proton motive force defect , leading to decreased type III secretion and motility . These findings highlight the importance for Yersinia to maintain appropriate levels of holo-IscR relative to apo-IscR in order maintain normal membrane potential . To begin to understand the nature of the T3SS defect in the presence of only apo-IscR , we carried out RNAseq analysis on the Y . pseudotuberculosis apo-locked IscR mutant grown under T3SS-inducing conditions and compared the results with data from the wild type and ΔiscR strains . Curiously , the apo-locked IscR mutant displayed aberrant expression of genes involved in stress response , transport , cell envelope , as well as electron transport ( data not shown ) . Of note , the Fe-S cluster biosynthesis proteins encoded in the iscRSUA-hscBA-fdx-iscX-pepB-sseB , yadR/erpA and nfuA operons are significantly increased in this background , similar to that of the ΔiscR mutant strain ( Figure S2A ) , indicating that holo-IscR represses expression of these genes under the aerobic , iron-replete conditions used . In contrast , increases in the sufABCDS Fe-S cluster biogenesis operon were observed for the apo-locked IscR strain when compared to both the wild type and ΔiscR strains ( Figure S4 ) . As IscR is overexpressed by 30-fold ( p<0 . 05 ) in the apo-locked iscR mutant compared to wild type ( Figure S2A ) , we speculate that the suf operon is positively regulated by IscR in Yersinia as in E . coli . In contrast , the extensively studied E . coli IscR target , hyaABCDEF , is not encoded in the Y . pseudotuberculosis genome . Importantly , our RNAseq analysis demonstrated that transcription of genes within the virA , virB , virC , yscW-lcrF , and lcrGVH-yopBD operons was restored in the apo-locked IscR mutant compared to the ΔiscR mutant ( Figure 7 and Table S2 ) . However , we observed a decrease in transcription of genes encoding the T3SS effector proteins YopH ( −4 . 4-fold ) , YopM ( −3 . 0-fold ) , YopK ( −7 . 1-fold ) , and YopE ( −2 . 1-fold ) in the apo-locked IscR mutant compared to wild type . Transcription of yopE has been shown to be regulated by Yop secretion through a positive feedback loop [51] , [52] , suggesting that the defect in YopHEMK transcription observed in the apo-locked IscR mutant may be caused by the lack of Yop secretion we observed in this strain . Together , these data suggest that both holo- and apo-IscR can promote T3SS gene transcription , possibly through binding to one or more type 2 DNA motifs . To determine whether IscR might directly regulate T3SS gene expression , we carried out bioinformatic analysis to search pYV for sequences resembling the E . coli IscR type 2 motif ( xxWWWWCCxYAxxxxxxxTRxGGWWWWxx ) [30] , [31] , [33] , as the DNA-binding domain of Yersinia IscR is 100% identical to that of E . coli IscR ( Figure 1A ) . We searched within the 150 nucleotides upstream of the 99 genes encoded on the pYV plasmid and obtained a ranked list of putative type 2 motifs ( data not shown ) . Among these was a site located within the yscW-lcrF promoter region ( Figure 8A ) [24] . To test whether IscR bound specifically to this site , we performed equilibrium DNA competition assays utilizing purified E . coli IscR-C92A ( apo-locked IscR ) [33] , with a fluorescently-labeled E . coli hya type 2 site previously identified by Nesbit et al . [33] . Purified E . coli IscR was utilized in this assay , as complementation of the Y . pseudotuberculosis ΔiscR mutant strain with IscR of E . coli encoded on a plasmid fully restored secretion of T3SS cargo ( Figure 8B ) . Competitor DNA included unlabeled E . coli hya as a positive control , the identified site within the Yersinia yscW-lcrF promoter region , a mutated version of this sequence ( mlcrF ) , where nucleotides previously demonstrated in E . coli to be important for type 2 motif binding were altered [33] , as well as one of the Y . pseudotuberculosis isc type 1 motif sites we identified as a negative control ( Figure S2B & Figure 8C ) . We found that unlabeled lcrF DNA competed as well as unlabeled hya DNA ( IC50 27 nm and 61 nm , respectively ) , suggesting that IscR can indeed bind to the identified type 2 motif upstream of lcrF ( Figure 8D ) . Furthermore , mutation of key nucleotides in the lcrF promoter sequence led to alleviation of competition and increased the IC50 to greater than 1000 nM , a level comparable to that of the isc negative control type 1 motif site ( Figure 8D ) . These findings suggest that IscR may regulate transcription of the T3SS through a type 2 motif within the yscW-lcrF promoter region .
In this study , we present the first characterization of the iron-sulfur cluster regulator , IscR , of Yersinia . Initially identified through a genetic screen for modulators of Ysc T3SS function , iscR-deficient Y . pseudotuberculosis had a dramatic defect in secretion of T3SS effector proteins and in targeting macrophages through their T3SS , yet displayed normal growth in broth culture and wild type flagellar motility . Bioinformatic and DNA binding analysis revealed an IscR binding site upstream of the operon encoding the T3SS master regulator LcrF , indicating that IscR controls expression of the Ysc T3SS . Collectively , these findings indicated that IscR is a central component of the Y . pseudotuberculosis T3SS regulatory cascade . Both E . coli holo- and apo-IscR are active transcription factors with distinct DNA binding targets . Holo-IscR can bind both type 1 and 2 motifs whereas apo-IscR can only bind type 2 motifs . IscR of E . coli autoregulates the isc operon , iscRSUA-hscBA-fdx , through binding to type 1 motifs within the isc promoter region [34] . In addition , Giel et al . described increased transcription of the genes located immediately downstream of the isc operon , yfhJ-pepB-sseB , in an iscR mutant , suggesting a negative regulatory effect on these genes as well [30] . We observed derepression of the iscRSUA-hscBA-fdx operon and the yfhJ-pepB-sseB locus in the Y . pseudotuberculosis ΔiscR mutant as well as the mutant expressing apo-locked IscR . Furthermore , we identified two sites within the Y . pseudotuberculosis isc promoter that closely match the E . coli IscR motif I consensus sequence . These data indicate that the iscRSUA-hscBA-fdx operon , and possibly the yfhJ-pepB-sseB locus , are negatively regulated by holo-IscR in Yersinia as they are in E . coli ( Figure 9A ) . IscR in E . coli is known to activate transcription of the sufABCDSE operon through binding to a type 2 motif [29] . Our analysis revealed that the Y . pseudotuberculosis apo-locked IscR mutant overexpresses the sufABCDS operon compared to the wild type and ΔiscR strains , which we predict results from the overexpression of IscR observed in the apo-locked mutant as found in E . coli [32] , [33] . We identified a site within the Y . pseudotuberculosis suf promoter region that closely resembles an E . coli IscR type 2 motif ( data not shown ) . Together , these data indicate that the suf operon is positively regulated by IscR in Yersinia as in E . coli . Thus , we propose that IscR of Y . pseudotuberculosis modulates transcription of both the isc and suf Fe-S cluster biosynthesis pathways via mechanisms established for its E . coli ortholog . In addition to control of Fe-S cluster biogenesis pathway expression , we present evidence that IscR controls expression and function of the Y . pseudotuberculosis T3SS . Bioinformatic analysis revealed a type 2 motif within the promoter of the T3SS master regulator LcrF that contained all nine bases previously found to be important for IscR binding ( Figure 8A ) [33] . Indeed , DNA binding assays demonstrated that IscR is able to specifically recognize this type 2 motif , suggesting that IscR may be acting directly to promote transcription of lcrF ( Figure 9B ) . In support of this , we observed a marked decrease in transcription of numerous T3SS genes in the ΔiscR mutant strain . These include the gene that encodes LcrF , as well as a number of LcrF-regulated genes including the virC operon , yopK , yopT , yopM , yopH , yopJ , and lcrGVH-yopBD [17] , [20] , [22] , [53] , [54] . The lcrF type 2 motif is further upstream of the -10/-35 region previously identified by Böhme et al . [24] than other IscR binding sites that promote transcription [33] , as we propose this site does . However , there may be an alternative −10/−35 region closer to the identified motif 2 site that might be used under specific growth conditions . Together , these data suggest that IscR is required for full expression of lcrF and LcrF-regulated genes through binding to a type 2 motif in the yscW-lcrF promoter ( Figure 9B ) . Based on these findings , an IscR mutant unable to coordinate an Fe-S cluster ( apo-locked IscR ) should lead to restoration of T3SS expression . Indeed , transcription of the yscW-lcrF and virC operons , as well as the majority of genes in the lcrGVH-yopBD operon , were no longer significantly decreased in the apo-locked IscR mutant compared to the ΔiscR strain . However , decreased transcription of yopE , yopK , yopM , and yopH as well as a severe defect in secretion of Yops was still observed . This could be explained by a deficiency in the apo-locked mutant's membrane potential , but not in the ΔiscR strain ( Figure 9B ) . Wilharm et al . , demonstrated that Y . enterocolitica motility and type III secretion requires the proton motive force [50] . Indeed , the apo-locked Y . pseudotuberculosis strain displayed a significant motility defect while the ΔiscR mutant was fully motile . Therefore , the type III secretion defect of the Y . pseudotuberculosis apo-locked IscR mutant can be explained by a deficiency in the proton motive force . Furthermore , the defect in YopHEMK transcription in the apo-locked IscR mutant may be explained by the fact that Yop secretion has a positive regulatory effect on Yop transcription [51] , [52] . Together , these data suggest that apo-IscR can promote LcrF transcription , but that locking iscR is the apo form causes a proton motive force defect that prevents effector Yop transcription and secretion ( Figure 9B ) . It is unclear why locking IscR in the apo-locked form leads to a proton motive force defect . We observed ∼9-fold more suf transcript in the apo-locked IscR mutant compared to the ΔiscR strain that does not have a proton motive force defect , whereas the isc operon was expressed to the same degree in both mutants . Ezraty et al . recently showed that expression of the suf , but not the isc , operon in E . coli leads to a proton motive force defect , possibly as a result of impaired loading of Fe-S clusters into aerobic respiratory complexes [55] . Although the isc operon is expressed in the apo-locked Y . pseudotuberculosis mutant , perhaps overexpression of the suf pathway leads to misassembly of the Fe-S complexes of the electron transport chain that drive the proton motive force . Both holo- and apo-IscR are predicted to bind to the type 2 motif within the yscW-lcrF promoter [33] . Based on previous data on E . coli IscR [28]–[30] , [34] , [56] , low iron , aerobic growth , or high oxidative stress conditions are predicted to result in high expression of IscR through derepression of the isc operon , which in turn should increase T3SS gene expression . Likewise , high iron , anaerobic , or low oxidative stress conditions should lead to decreased IscR levels and therefore lower T3SS expression . Under normal aerobic culture conditions , we do not observe a change in wild type Y . pseudotuberculosis type III secretion when iron levels are altered ( data not shown ) . However , in vivo , bacteria may be present in microaerophilic or anaerobic niches , where changes in iron bioavailability and reactive oxygen species production may impact iscR and T3SS gene expression . Upon ingestion by a host animal , Y . pseudotuberculosis enters the lumen of the intestine , which receives approximately 15 mg of iron per day [57] , [58] . In the small intestine , Y . pseudotuberculosis can cross the gut barrier and enter the bloodstream and deeper tissues , which have very low iron bioavailability ( ∼10−24 M free serum iron ) [59]–[61] . Sequestration of iron by iron carriers in mammalian tissues is an important host defense mechanism to prevent growth of bacterial pathogens , the majority of which require iron for growth [62] . The Ysc T3SS has been shown to be required for Y . pseudotuberculosis pathogenesis in these deep tissue sites that are low in iron bioavailability [44] . Perhaps Y . pseudotuberculosis uses IscR to sense iron , O2 , and/or ROS concentration in order to optimally control T3SS expression in vivo . Consistent with the severe T3SS expression defect displayed by the Y . pseudotuberculosis ΔiscR strain , this mutant was deficient in colonization of the Peyer's patches , spleen , and liver . Interestingly , the ΔiscR mutant was also defective in colonization of the mesenteric lymph nodes ( MLN ) , yet T3SS mutants were previously shown to persist in the MLN and chromosomally-encoded factors were found to be important for Y . pseudotuberculosis survival in this tissue [24] , [63] , [64] . These results indicate that the virulence defect of the Y . pseudotuberculosis ΔiscR strain may not be due solely to misregulation of the T3SS , suggesting the existence of other IscR gene targets important for virulence . IscR of Pseudomonas aeruginosa has been shown to be important for full virulence through its ability to upregulate KatA , encoding a catalase that protects against oxidative stress [38] , [65]–[67] . In Vibrio vulnificus , IscR upregulates two genes encoding the antioxidants peroxiredoxin and glutaredoxin 2 , and is essential for survival during exposure to reactive oxygen species [40] . Interestingly , our analysis suggests that Y . pseudotuberculosis IscR plays an opposite regulatory role , as IscR negatively affects expression of the genes encoding cellular detoxification proteins KatY , Tpx , SodC and SodB . Furthermore , hydrogen peroxide sensitivity assays showed comparable levels of survival between the Y . pseudotuberculosis wild type and ΔiscR strains ( Figure S5 ) . This suggests that the virulence defect observed for the ΔiscR Y . pseudotuberculosis mutant is not due to increased susceptibility to oxidative stresses encountered during infection . Pathways other than the T3SS , such as the hmu hemin uptake system , were found to be misregulated in the Y . pseudotuberculosis ΔiscR strain ( Table 2 & Figure 4B ) . While the hmu operon was shown to not affect Y . pestis virulence , it is possible that IscR control of the Y . pseudotuberculosis hmu pathway is important for virulence . In summary , we present the first characterization for the iron-sulfur cluster regulator , IscR , of Yersinia . We reveal that IscR regulates genes involved in Fe-S cluster assembly in a manner akin to that of E . coli . Most notably , we demonstrate that mutation of IscR leads to decreased function of the Y . pseudotuberculosis T3SS and that this is due to a decrease in transcription of genes encoding structural , regulatory , and effector proteins . Furthermore , we present evidence showing that IscR is essential for the virulence of Y . pseudotuberculosis and that this attenuation is likely due , in part , to direct regulation of the T3SS by IscR . Collectively , this study argues for the important and novel role of IscR in the virulence of Y . pseudotuberculosis as well as regulation of the Ysc T3SS , and identifies IscR as a potential target for novel antimicrobial agents .
All strains used in this study are listed in Table 3 . Y . pseudotuberculosis strains were grown in either 2xYT or M9 minimal media supplemented with casamino acids [68] , referred to here as M9 , at 26°C with shaking at 250 rpm , unless otherwise indicated . Where stated , Yop synthesis was induced via back-dilution of cultures into either M9 or low calcium media ( 2xYT plus 20 mM sodium oxalate and 20 mM MgCl2 ) to an OD600 of 0 . 2 and grown for 1 . 5 h at 26°C/shaking followed by 2 h at 37°C/shaking as previously described [69] . The iscR deletion mutant ( ΔiscR ) was generated via splicing by overlap extension PCR [70] . Primer pairs F5'iscR/R5'iscR and F3'iscR/R3'iscR ( Table S3 ) , designed using MacVector and Primer 3 software ( http://fokker . wi . mit . edu/primer3/input . htm ) , were used to amplify ∼500 bp 5′ and 3′ of the iscR coding region , respectively . Amplified PCR fragments served as templates in an overlap extension PCR using the outside primers F5'iscR and R3'iscR . The IscR-C92A/C98A/C104A mutant ( apo-IscR ) was generated via splicing by overlap extension PCR [70] . Primer pairs F5'apo-IscR/R5'apo-IscR and F3'apo-IscR/R3'apo-IscR ( Table S3 ) , designed using MacVector and Primer 3 software ( http://fokker . wi . mit . edu/primer3/input . htm ) , were used to amplify ∼500 bp 5′ and 3′ within the iscR coding region , respectively . Amplified PCR fragments served as templates in an overlap extension PCR using the outside primers F5'apo-IscR and R3'apo-IscR . Nucleotide changes within the internal primers R5'apo-IscR and F3'apo-IscR allowed for amplification of iscR target containing sequences coding for alanine substitutions of the three conserved cysteines that coordinate an Fe-S cluster . The resulting ∼1 kb fragments were cloned into the TOPO TA cloning vector ( Invitrogen ) and further subcloned into a BamHI- and NotI-digested pSR47s suicide plasmid ( λpir-dependent replicon , kanamycinR ( KanR ) , sacB gene conferring sucrose sensitivity ) [71] , [72] . Recombinant plasmids were transformed into E . coli S17-1 λpir competent cells and later introduced into Y . pseudotuberculosis IP2666 via conjugation . The resulting KanR , irgansanR ( Yersinia selective antibiotic ) integrants were grown in the absence of antibiotics and plated on sucrose-containing media to select for clones that had lost sacB ( and by inference , the linked plasmid DNA ) . KanS , sucroseR , congo red-positive colonies were screened by PCR and subsequently sequenced to verify loss of the intended iscR coding region . The iscR complement construct was generated by insertion of a fragment containing the iscR coding region as well as 530 bp of 5′ upstream sequence . This was PCR amplified using primer pair FiscRC and RiscRC , and cloned into the vector pACYC184 via BamHI/SalI restriction sites [73] , [74] . Recombinant plasmids were transformed into E . coli S17-1 λpir competent cells and later introduced into Y . pseudotuberculosis IP2666 ΔiscR via a modified transformation method [75] . Briefly , recipient Yersinia strains were grown overnight in LB containing 2% glucose at 26°C . Cultures were centrifuged at 3 , 500 rpm for 3 min then washed with 750 µl of ice-cold sterile diH2O and repeated for a total of three washes . Washed pellets were resuspended in 100 µl of sterile diH2O , combined with 3 µl of plasmid and electroporated at EC2 . Cells were allowed to recover in 1 mL SOC media for 1 h at 26°C followed by plating on LB containing carbenicillin to select for Yersinia bearing the plasmid of interest . Clones were confirmed by PCR analysis , using a combination of gene- and vector-specific primers , to construct both the ΔiscR complemented strain ( ΔiscR pIscR ) and the apo-IscR complemented strain ( apo-IscR pIscR ) . The nonmotile Δyop6/flhDCY . pestis mutant was generated by crossing in the Y . pestis flhDC gene into Y . pseudotuberculosis . Y . pestis flhD has a frameshift mutation , resulting in suppression of flagellin production [76] . The suicide plasmid pSB890 encoding a partial flhC gene and the full flhD gene from the Y . pestis KIM strain , generously provided by Dr . Brad Cookson , was conjugated into Y . pseudotuberculosis Δyop6 and nonmotile , recombinant mutants isolated as previously described [46] . Transposon mutagenesis was preformed similarly to Crimmins et al . [64] . Briefly , E . coli SM10λpir harboring pSC189 , which encodes Himar1 [77] , was mated with Y . pseudotuberculosis Δyop6 . Mating culture was then pelleted , resuspended , spread out evenly among six 150 mm×15 mm petri plates containing LB supplemented with 2 µg mL−1 irgasan and 30 µg mL−1 Kan , and incubated for 3 days at room temperature . Colonies were patched onto LB supplemented with 100 µg mL−1 carbenicillin to ensure insertion of the transposon . Colony patches were used to grow 2xYT overnight cultures in 96-well plates , which were then frozen down to preserve the library . HEK293T cells were plated in 96-well white clear bottom plates ( Corning ) and transfected with a plasmid encoding a luciferase reporter gene fused to an NFκB-dependent promoter ( Stratagene ) . Mutants from the transposon library were grown overnight in 96 well plates in M9 at 26°C and used to infect the transfected HEK293T cell monolayers . After 4 h incubation at 37°C , 100 µl of 1∶1 NeoLite:PBS solution was added to each well of the 96-well clear-bottom white plate ( Corning ) , and luminescence was measured using a Victor3 plate reader ( PerkinElmer ) . Each transposon mutant was assayed in duplicate . The positions of the transposons in the iscR::Tn1 and iscR::Tn2 mutants were determined by plasmid rescue , as previously described [78] , except BamHI was used for digestion of genomic DNA . Validation of the transposon screen was performed through the use of an NFΚB activity assay , which is based on our previous work showing that Y . pseudotuberculosis induces NFκB activation in HEK293T cells dependent on expression of a functional T3SS [43] . Briefly , HEK293T cells were transfected with a plasmid encoding a luciferase reporter gene fused to an NFκB-dependent promoter ( Stratagene ) . Bacterial strains were grown overnight in 2xYT and subcultured to an OD600 of 0 . 2 into low calcium media and grown at 26°C for 1 . 5 h followed by a shift to 37°C for an additional 1 . 5 h to induce the T3SS . Bacterial cultures were resuspended in prewarmed ( 37°C ) DMEM and 200 µl aliquots were then used to infect the HEK293T cells containing the luciferase reporter plasmid at an MOI of 10 . After 4 h incubation at 37°C , 100 µl of 1∶1 NeoLite:PBS solution was added to each well of the 96-well clear-bottom white plate ( Corning ) , and luminescence was measured using a Victor3 plate reader ( PerkinElmer ) . Data from three separate wells were averaged for each independent experiment . Visualization of T3SS cargo secreted in broth culture was performed as previously described [46] . Briefly , Y . pseudotuberculosis in M9 low calcium media ( M9 plus 20 mM sodium oxalate and 20 mM MgCl2 ) was grown for 1 . 5 h at 26°C followed by growth at 37°C for 2 h . Cultures were normalized by OD600 and pelleted at 13 , 200 rpm for 10 min at room temperature . Supernatants were removed and proteins precipitated by addition of trichloroacetic acid ( TCA ) at a final concentration of 10% . Samples were incubated on ice for 20 min and pelleted at 13 , 200 rpm for 15 min at 4°C . Resulting pellets were washed twice with ice-cold 100% EtOH and subsequently resuspended in final sample buffer ( FSB ) containing 20% dithiothreitol ( DTT ) . Samples were boiled for 5 min prior to running on a 12 . 5% SDS PAGE gel . Evaluation of pore formation was performed via the ethidium bromide ( EtBr ) entry assay as previously described [46] . Briefly , 2×104 immortalized C57Bl/6 BMDMs were plated in a 96 well clear bottom black plate ( Corning ) in 100 uL DMEM +10% FBS . Infection was performed in triplicate at an MOI of 25 . Plates were centrifuged at 750×g at 4°C for 5 min to facilitate contact . Infections were carried out at 37°C with 5% CO2 for 2 h , at which point media was aspirated and replaced with 30 µL of PBS containing 25 µgmL−1 ethidium bromide ( EtBr ) and 12 . 3 µg mL−1 Hoechst dye . The cell monolayer was visualized using an ImageXpressMICRO automated microscope and MetaXpress analysis software ( Molecular Devices ) . The percent of EtBr-positive cells was calculated by dividing the number of EtBr-stained cells by the number of Hoechst-stained cells . Data from three separate wells was averaged for each independent experiment . Y . pseudotuberculosis strains were cultured overnight in 2xYT or M9 at 26°C and sub-cultured to an OD600 of 0 . 2 in 25 mL of either 2xYT or M9 . Cultures were incubated at either 26°C or 37°C with shaking at 250 rpm and optical density measured at 600 nm every hour for 9 h . All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the UC Santa Cruz Institutional Animal Care and Use Committee . Eleven to twelve-week-old 129S6/SvEvTac mice from our breeding facilities were used for oral infections as previously described [79] . Briefly , mice were orogastrically inoculated with 2×108 CFU in a 200 µl volume using a feeding needle . Mice were given food and water ad libitum and were euthanized at 5 days post-inoculation . Peyer's patches , mesenteric lymph nodes , spleens , and livers were isolated and homogenized for 30 s in PBS followed by serial dilution and plating on LB supplemented with 1 µg mL−1 irgasan for CFU determination . RNA was isolated from the IP2666 wild type and isogenic ΔiscR and apo-IscR strains grown for 3 h at 37°C in M9 , using the RNeasy Mini Kit ( Qiagen ) as per the manufacturer's protocol . We chose M9 media for our RNASeq analysis because this condition enables expression of T3SS genes and secretion of T3SS cargo at 37°C [68] . Contaminating DNA was removed from the RNA samples using a DNA-free kit ( Life Sciences ) . Samples were subjected to removal of contaminating rRNA via the Ribo-Zero Magnetic Kit for Gram-negative bacteria ( Epicentre ) . The cDNA library was created using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina ( NEB ) . These studies were performed with three biological replicates per condition . Six indexed samples were sequenced per single lane using the HiSeq2500 Illumina sequencing platform for 50 bp single reads ( UC Davis Genome Center ) and subsequently analyzed and visualized via the CLC Genomics Workbench version 5 . 5 . 1 ( CLC bio ) . Samples were normalized for both sequence depth and gene size by determining RPKM ( Reads Per Kilo base per Million reads ) and mapped to the Y . pseudotuberculosis genome ( IP32953 ) . Differentially regulated genes were identified as those displaying a fold change with an absolute value of 2 or greater . Statistical significance was determined by baySeq test with a corrected FDR post hoc test where p<0 . 05 was deemed significant [80] . Total RNA generated from our RNAseq analysis at a concentration of 2 µg was used to make cDNA as previously described [81] . SYBR Green PCR master mix ( Applied Biosystems ) was used for qPCR reactions according to the manufacturer's instructions and a 60°C annealing temperature . Primers used are listed in Table S4 . Control primers were for the 16S rRNA as described previously [82] . Results were analyzed using the Bio-Rad CFX software . Average RPKM values generated from RNAseq analysis for the wild type , ΔiscR and apo-IscR mutants were converted to log2-ratios ( log2 ( RPKMmutant/RPKMwt ) for each gene encoded on the virulence plasmid , pYV . These values were converted to a Circos heatmap [83] and plotted against the respective pYV base coordinate positions from Y . pseudotuberculosis IP32953 . Position specific scoring matrix ( PSSM ) was generated by the alignment of the known E . coli IscR type 2 motifs ( Table S4 ) ( Maverix Biomics , Inc ) [31] . PSSM of type 2 was used to scan against the 150-nt upstream of 99 genes encoded on the Y . pseudotuberculosis pYV plasmid and obtained a ranked list of putative type 2 motifs . Fluorescence anisotropy was measured similar to Nesbit et al . , [33] . E . coli apo-IscR lacking the [2Fe-2S] cluster ( IscR-C92A ) was isolated anaerobically following the protocol described previously for wild type IscR [30] . Competition assays were performed using 5nM of 30-mer dsDNA of the known E . coli hyaA type 2 motif containing a 5′ TAMRA fluorophore ( IDT ) on the top strand and unlabeled competitor dsDNA concentrations ranged from 8 to 1000 nM ( IDT , Table S4 ) . DNA was annealed by heating equimolar concentrations of complementary DNA strands in annealing buffer ( 40 mM Tris ( pH 7 . 9 ) , 30 mM KCl ) to 95°C for 5 min followed by slow cooling to room temperature over 2 hours . Annealed DNA was incubated with 90 nM apo-IscR in anisotropy buffer ( 40 mM Tris pH 7 . 9 , 150 mM KCl , 100 ng ul−1 Salmon Sperm DNA ) for 12 min at room temperature and anisotropy was measured using an EnVision 2103 Multilabel Reader ( Perkin Elmer ) with Wallac EnVision Manager software . Data is representative of experiments performed on three separate days . Motility was analyzed by spotting 1 µl aliquots of either a nonmotile strain bearing an inactive , Y . pestis allele of flhD ( Δyop6/flhDY . pestis ) , WT , ΔiscR , or apo-IscR strains onto motility agar plates ( 1% tryptone , 0 . 25% agar ) from overnight cultures standardized to an OD600 of 2 . 5 . Plates were incubated at room temperature for 1 day , at which point the diameters of the colonies were determined and used to calculate percent motility relative to WT , which was set at 100% . The electrical potential was measured similar to the JC-1 red/green dye assay previously described for E . coli [84] . JC-1 is a membrane-permeable dye that emits green fluorescence ( ∼530 nm ) upon excitation when the dye is in the monomeric form . Due to the membrane potential of the bacterial cell , JC-1 dye will form J aggregates which emit red fluorescence ( ∼590 nm ) . If the membrane potential decreases , there will be a decrease in J aggregate formation and subsequently a decrease in red fluorescence . As such , membrane potential can be displayed as a ratio of red/green fluorescence . Briefly , Y . pseudotuberculosis wild type and isogenic ΔiscR , ΔiscR complemented ( ΔiscR pIscR ) , apo-IscR and apo-IscR complemented ( apo-IscR pIscR ) strains were grown overnight in M9 at 26°C . Strains were subcultured to an OD600 of 0 . 2 in M9 and grown at 37°C for 3 hours . A negative control containing a sample of wild type Y . pseudotuberculosis treated with 40 µM of the protonophore , CCCP ( carbonyl cyanide m-chlorophenylhydrazone ) , during the last 30 min of growth was included in each experiment . After incubation at 37°C , 1 mL aliquots were harvested for each strain and pelleted at 4 , 500×g for 3 min followed by resuspension in 1 mL of permeabilisation buffer ( 10 mM Tris-HCl , pH 7 . 6 , 1 mM EDTA and 10 mM glucose ) . Post resuspension , 2 µl of the membrane-permeable JC-1 dye ( 5 mg/mL ) was added and the samples were incubated at room temperature for 30 min . Samples were then pelleted at 4 , 500×g for 3 min and resuspended in 500 µl of permeabilisation buffer . Slides were prepared by first coating with Poly-L-Lysine solution through addition of 100 µl aliquots of a 0 . 01% solution followed by a 5 min incubation at room temperature . Slides were washed a total of 3 times with sterile diH2O . Once dry , 10 µl of prepared sample was added to the slide and allowed to adhere for 5 min . Unattached bacteria were removed by washing with PBS and excess liquid removed via aspiration . A coverslip was applied and the cells were imaged using a LSM 5 PASCAL laser scanning microscope ( Zeiss ) fitted with a Plan-Apochromat 63x/1 . 4 Oil DIC objective and analyzed using the LSM 510 software ( Zeiss ) . Quantification of image intensities was performed using ImageJ [85] .
|
Bacterial pathogens use regulators that sense environmental cues to enhance their fitness . Here , we identify a transcriptional regulator in the human gut pathogen , Yersinia pseudotuberculosis , which controls a specialized secretion system essential for bacterial growth in mammalian tissues . This regulator was shown in other bacterial species to alter its activity in response to changes in iron concentration and oxidative stress , but has never been studied in Yersinia . Importantly , Y . pseudotuberculosis experiences large changes in iron bioavailability upon transit from the gut to deeper tissues and iron is a critical component in Yersinia virulence , as individuals with iron overload disorders have enhanced susceptibility to systemic Yersinia infections . Our work places this iron-modulated transcriptional regulator within the regulatory network that controls virulence gene expression in Y . pseudotuberculosis , identifying it as a potential new target for antimicrobial agents .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology",
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"life",
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2014
|
IscR Is Essential for Yersinia pseudotuberculosis Type III Secretion and Virulence
|
Circulating levels of both seasonal and pandemic influenza require constant surveillance to ensure the health and safety of the population . While up-to-date information is critical , traditional surveillance systems can have data availability lags of up to two weeks . We introduce a novel method of estimating , in near-real time , the level of influenza-like illness ( ILI ) in the United States ( US ) by monitoring the rate of particular Wikipedia article views on a daily basis . We calculated the number of times certain influenza- or health-related Wikipedia articles were accessed each day between December 2007 and August 2013 and compared these data to official ILI activity levels provided by the Centers for Disease Control and Prevention ( CDC ) . We developed a Poisson model that accurately estimates the level of ILI activity in the American population , up to two weeks ahead of the CDC , with an absolute average difference between the two estimates of just 0 . 27% over 294 weeks of data . Wikipedia-derived ILI models performed well through both abnormally high media coverage events ( such as during the 2009 H1N1 pandemic ) as well as unusually severe influenza seasons ( such as the 2012–2013 influenza season ) . Wikipedia usage accurately estimated the week of peak ILI activity 17% more often than Google Flu Trends data and was often more accurate in its measure of ILI intensity . With further study , this method could potentially be implemented for continuous monitoring of ILI activity in the US and to provide support for traditional influenza surveillance tools .
Each year , there are an estimated 250 , 000–500 , 000 deaths worldwide that are attributed to seasonal influenza [1] , with anywhere between 3 , 000–50 , 000 deaths occurring in the United States of America ( US ) [2] . In the US , the Centers for Disease Control and Prevention ( CDC ) continuously monitors the level of influenza-like illness ( ILI ) circulating in the population by gathering information from sentinel programs which include virologic data as well as clinical data , such as physicians who report on the percentage of patients seen who are exhibiting influenza-like illness [2] . While the CDC ILI data is considered to be a useful indicator of influenza activity , its availability has a known lag-time of between 7–14 days , meaning that by the time the data is available , the information is already 1–2 weeks old . To appropriately distribute vaccines , staff , and other healthcare commodities , it is critical to have up-to-date information about the prevalence of ILI in a population . There have been several attempts at gathering non-traditional , digital information to be used to predict the current or future levels of ILI , and other diseases , in a population [3]–[11] . The most notable of these attempts to date has been Google Flu Trends ( GFT ) , a proprietary system designed by Google , which uses Google search terms that are correlated with ILI activity in the US to make a estimation of the current level of ILI [12] . Google Flu Trends was initially quite successful in its estimation of ILI activity , but was shown to falter in the face of the 2009 H1N1 swine influenza pandemic ( pH1N1 ) due to much-increased levels of media attention surrounding the pandemic [13] . Similarly , GFT greatly over-estimated ILI activity in the 2012–2013 influenza season , again likely due to that fact that it was a more severe influenza season than normally observed and therefore garnered much media attention [14] . In the face of these obstacles , Google has continued to update and re-evaluate its models [15]–[17] . Although GFT has performed well in the past , with the exception of two high ILI activity time periods , new methods of estimating current ILI activity that are less susceptible to error in the face of media coverage should be sought . Additionally , as the global community continues to become increasingly in favor of open-access data and methods [18] , new methods of ILI estimation should be freely available for everyone to investigate and improve upon , unlike GFT , which does not share the search terms it uses in its algorithms ( though results are public ) . To this end , we have created a method of estimating current ILI activity in the US by gathering information on the number of times particular Wikipedia articles have been viewed . Wikipedia is a massive , user-regulated , online encyclopedia . Launched in 2001 , Wikipedia harnesses the power of the online community to create , edit , and modify encyclopedia-like articles that are then freely available to the entire world . Currently operating in 232 languages , Wikipedia has ∼30 million articles available , expanding at approximately 17 , 800 articles per day , with nearly 506 million visitors per month , representing 27 billion total page views since its launch , and has approximately 31 , 000 active Wikipedia editors ( http://stats . wikimedia . org ) [19] . With a wealth of detailed information on an almost limitless range of topics , Wikipedia is ideally suited as a platform that could potentially be of use for legitimate scientific investigation in many different areas . Not only is the information held within Wikipedia articles very useful on its own , but statistics and trends surrounding the amount of usage of particular articles , frequency of article edits , region specific statistics , and countless other factors make the Wikipedia environment an area of interest for researchers . It has previously been shown that Wikipedia can be a useful tool to monitor the emergence of breaking news stories , to track what topics are “trending” in the public sphere , and to develop tools for natural language processing [20]–[23] . Furthermore , Wikipedia makes all of this information public and freely available , greatly increasing and expediting any potential research studies that aim to make use of their data . The purpose of this study was to develop a statistical model to provide near real-time estimates of ILI activity in the US using freely available data gathered from the online encyclopedia , Wikipedia .
In an attempt to use Wikipedia data to estimate ILI activity in the US , we compiled a list of Wikipedia articles that were likely to be related to influenza , influenza-like activity , or to health in general . These articles were selected based on previous knowledge of the subject area , previously published materials , and expert opinion . In addition to articles that were potentially related to ILI activity , several articles were selected to act as markers for general background-level activity of normal usage of Wikipedia . For example , information was gathered on the number of times the Wikipedia main page ( www . en . wikipedia . org/wiki/Main_page ) was accessed per day , as a measure of normal website traffic . As well , the Wikipedia article for the European Centers for Disease Control was included in models in an attempt to control for non-American article views . Table 1 displays the Wikipedia articles that were considered for inclusion in our models . Wikipedia article view information is made freely available by Wikipedia , under a project called Wikimedia Statistics ( http://en . wikipedia . org/wiki/Wikipedia:Statistics ) , and is available as the number of article views per hour , which may include multiple views on the same article by the same user . A freely available , user-written tool was independently developed to more easily access the information that Wikipedia makes available ( http://stats . grok . se ) , which aggregates article view data to the day-level , and this tool was used to gather total daily article view information . Daily Wikipedia article view data was retrospectively collected beginning at the earliest available date , December 10 , 2007 , through to August 19th , 2013 , and then aggregated to the week level , with each week beginning on Sunday . The CDC compiles data on the weekly level of ILI activity in the United States by collecting information from sentinel sites across the country where physicians report on the number of patients with influenza-like illness . CDC ILI data is freely available through ILInet , via the online FluView tool ( www . cdc . gov/flu/weekly ) , and downloadable as week-level data . Google Flu Trends data is also freely available through the Google Flu Trends website ( http://www . google . org/flutrends ) and is provided weekly at the country and state level . GFT data is the result of Google's proprietary algorithm that uses Google search queries to estimate the level of ILI activity in a given region . We gathered Wikipedia article view data beginning from the week of December 10th , 2007 , the earliest records available , until August 19th , 2013 . Accordingly , retrospective CDC ILI data and GFT data was obtained for the same period as the Wikipedia article view information , although both the CDC and GFT data extends much further back in time . When aggregated to week-level , all data sources accounted for 296 weeks of retrospective information , capturing five full influenza seasons as well as partial 2007–2008 data . Due to a lapse in the Wikipedia database , article view information is not available between July 13th and July 31st , 2008 , inclusive . Therefore , the total set of data available accounts for 294 weeks . Models to estimate ILI activity using Wikipedia article view information were developed using a generalized linear model framework . The outcome variable , age-weighted CDC ILI activity , is a proportion and is therefore appropriately modeled using a Poisson distribution , and so the Poisson family was used in the GLM framework , with a log-link function . In an attempt to adjust for potential over-fitting , models were run using jackknife resampling . Two principle models were created , which include Mf , a Poisson model that used the full set of collected Wikipedia article page view data , and Ml , a Poisson model that used Lasso ( Least Absolute Shrinkage and Selection Operator ) regression analysis . Lasso regression dynamically and automatically selects predictor variables for inclusion or exclusion by penalizing the absolute size of the regression coefficients toward zero , thereby selecting a subset of predictor variables which best describe the outcome data [24] , [25] . To investigate the reliability of the models , we used a split-sample analysis on the Ml models to compare how well the Lasso selected predictors for a subset of the data ( including years 2007 , 2008 , 2009 , and 2010 ) accounted for the observed data in the remaining subset ( years 2011 , 2012 , and 2013 ) . Additionally , each of these aforementioned models were also run while excluding data at key time periods which reflect higher than normal ILI activity or Wikipedia article view traffic ( during the early weeks of the 2009 pandemic H1N1 swine influenza pandemic and the unusually severe influenza season of 2012–2013 ) as a means of investigating the models' ability to deal with large data spikes . By comparing the models with or without higher than normal Wikipedia usage , we can investigate what impact , if any , spikes in Wikipedia activity ( potentially caused by increased media reporting of influenza-related events ) have on the accuracy of the models , and whether or not these spikes in traffic need to be accounted for . In addition to a factor variable representing the year being included in the models , the month was also controlled for in an effort to adjust for the seasonal patterns that influenza outbreaks exhibit in the United States . All models were investigated for appropriate fit using the Pregibon's goodness-of-link test [26] and by examining Anscombe and deviance residuals . Models were compared to one another by comparing Akaike's Information Criteria , response statistics , and by performing likelihood-ratio tests on the maximum-likelihood values of each model . Goodness-of-fit ( GOF ) tests , both Pearson and deviance , were tested for; all presented models had GOFs≫0 . 05 . All statistics and models were performed using Stata 12 ( Statacorp . , College Station , Texas , US ) .
The Mf model , containing all 35 predictor variables ( including year , month , CDC page views , ECDC page views , and Wikipedia Main Page views ) and 294 weeks of data , resulted in a Poisson model with an AIC value of 2 . 795 . Deviance residuals for this model ranged from −0 . 971–1 . 062 ( mean: −0 . 006 ) and were approximately normally distributed . Although many of the dependent variables showed spikes in page view activity around the beginning of the 2009 pH1N1 event , the Mf model was able to accurately estimate the rate of ILI activity , with a mean response value ( difference between observed and estimated ILI values ) of 0 . 48% in 2009 between weeks 17–20 , inclusive . Overall , the absolute response values for the Mf model ranged from 0 . 00–2 . 38% ( mean: 0 . 27% , median: 0 . 16% ) . In comparison , the absolute response values between CDC ILI data and GFT data ranged from 0 . 00–6 . 04% ( mean: 0 . 42% , median: 0 . 21% ) . The Pearson correlation coefficient between the CDC ILI values and the estimated values from the Mf model was 0 . 946 ( p<0 . 001 ) . The actual observed range of ILI activity throughout the entire period for which data is available , as reported by the CDC , was from 0 . 47–7 . 72% , with a median value of 1 . 40% . In comparison , the Mf model estimated ILI activity for the same period ranged from 0 . 44–8 . 37% , with a median value of 1 . 50% , and the GFT ILI data ranged from 0 . 60–10 . 56% , with a median value of 1 . 72% . The Ml model , which contained 26 variables ( including year , month , and CDC page views ) that were chosen as significant by the Lasso regression method , resulted in a model with an AIC of 2 . 764 . Deviance residuals for this model ranged from −0 . 790 to 1 . 205 ( mean: −0 . 007 ) and were approximately normally distributed , though less so than in Mf . The absolute response values for this Ml model ranged from 0 . 00–2 . 53% ( mean: 0 . 29% , median: 0 . 18% ) . During weeks 17–20 of the 2009 pH1N1 event , the mean response value for this model was 0 . 45% , suggesting it was slightly less accurate over this unusually high article view activity time period than the Mf model for the same period . The Pearson correlation coefficient between CDC ILI data and the estimated mean value for the Ml model was 0 . 938 ( p<0 . 001 ) , and the range of estimated ILI values for this model was from 0 . 55–8 . 66% , with a median value of 1 . 48% . Split-sample analysis was used to investigate the reliability of the Ml model . A Lasso regression model that was trained on data from years 2007–2010 , inclusive , and the selected predictor variables were used to estimate the ILI activity for each week in the remainder of the dataset ( years 2011–2013 , inclusive ) . The cross-validation Pearson correlation between the actual observed CDC ILI data and the ILI estimates provided by the Ml model based on the first subset of data was 0 . 9854 ( p<0 . 001 ) . Figure 1 shows the time series for CDC ILI data , GFT data , and the estimated ILI values from both the Mf and Ml models . In the following models , data from the beginning weeks of the 2009 pH1N1 event ( weeks 17–20 , inclusive ) , which showed large spikes in Wikipedia article views due to increased media attention , were excluded from analyses . As well , because of the higher-than-normal influenza activity of the 2012–2013 influenza season , that data was also removed from analyses , beginning from week 40 of 2012 to week 13 of 2013 , inclusive . By running the Poisson models without these high volume time-sections , comparisons can be made to the full models in order to investigate the estimating ability of models in the face of higher-than-normal levels of influenza activity or Wikipedia article views . When removing the above-mentioned data , the Mf model produced an AIC value of 2 . 772 , only marginally smaller than that of the complete Mf model , and was comprised of 263 weeks of data . The range of deviance residuals from this model , −0 . 650 to 0 . 891 , is slightly narrower than the complete Mf model , suggesting a better fit . For the truncated Lasso model , the Poisson regression model was refit to only include the available data , and therefore produced a different set of 24 predictor variables . From this model , an AIC value of 2 . 727 was obtained , with a range of deviance residuals from −0 . 677 to 1 . 081 , a marginal narrowing over the original Ml model . Pearson correlation coefficient values between CDC ILI data and estimated values by the Mf and Ml models , for peak-truncated data , were 0 . 958 ( p<0 . 001 ) and 0 . 942 ( p<0 . 001 ) , respectively . In the United States , seasonal influenza activity usually peaks during January or February . Using the maximum value of the CDC ILI data in a single influenza season as the true peak time and value , we compared the peak value and week for influenza activity as estimated by our two models , Mf and Ml , as well as the Google Flu Trends data . Results are summarized by model and by year in Table 2 . The Mf model was able to accurately estimate the ILI activity peak in 3 of 6 influenza seasons for which data is available ( 2009–2010 , 2010–2011 and 2012–2013 seasons ) , and was within one week of an accurate estimation in another season ( 2007–2008 ) . The Ml model accurately estimated the ILI peak activity week in 2 of 6 seasons ( 2007–2008 and 2010–2011 ) , and estimated 2 others within a week ( 2009–2010 and 2012–2013 ) . In comparison , Google Flu Trends data was able to accurately estimate peaks of seasonal ILI activity in 2 of 6 influenza seasons ( 2009–2010 and 2010–2011 season ) , and was accurate within one week in 2 other influenza season ( 2007–2008 and 2008–2009 ) . It should be noted that in the 2010–2011 season , the CDC data peaked at the same ILI percentage at both week 4 and week 6 in 2011 , and week 6 was taken to be the true peak , as it agreed with both Wikipedia models and the GFT data . In the 2011–2012 season , the Mf and Ml models were 3 weeks early in their estimation of peak ILI activity and the GFT data was 10 weeks early . Finally , in the 2012–2013 influenza season , the GFT model was 3 weeks late and grossly over-estimated the severity by greater than 2 . 3-times .
Weekly ILI values based on Wikipedia article view counts were able to estimate US ILI activity within a reasonable range of error , with CDC data as the gold standard . While the CDC ILI data is routinely used as a gold standard , and is most often the best available source of ILI information for the country , this data source has potential biases of its own . There are over 2 , 900 outpatient healthcare providers that are registered participants of the CDC's ILI surveillance program , but in any given week , only approximately 1 , 800 provide ILI surveillance data [27] . As well , the population size/density of the area served by each outpatient healthcare provider is not uniform across locations and may lead to a skew in reporting . Additionally , increased media coverage of influenza may prompt healthcare providers to submit more samples for analysis or to report more potential ILI cases than they may have otherwise . Several models were fit to estimate ILI activity , including a model containing all 32 health-related Wikipedia articles investigated , a Lasso regression model which selected 24 health-related Wikipedia articles of significance , and each of these models were run without high media-awareness time periods representing the beginning of the H1N1 pandemic in spring of 2009 and the higher-than-normal ILI rates of the 2012–2013 influenza season . These models were compared to official CDC ILI values as well as GFT data . Comparing the Mf and Ml models , the AIC value was slightly smaller for the Ml model , as was its range of estimation residuals . With a highly non-significant likelihood ratio test between the two models , there is no evidence to suggest that the Mf model performs better than the Ml model , which may be preferred here . However , since there is no cost or energy associated with collecting additional variable information , the full model may warrant continued use to account for the potential event where more health-related Wikipedia articles become useful in ILI estimation . Mf and Ml models that did not include data for the 2009 spring pH1N1 season and the 2011–2012 peak season resulted in slightly smaller AIC and residual values compared to their full-data counterparts , but did not show large enough improvements in estimates to suggest that higher than normal Wikipedia page view traffic or ILI activity were major factors in the models' ability to estimate ILI activity . This result exemplifies the Wikipedia model's ability to perform well in the face of increased media attention and higher than normal levels of ILI activity , whereas GFT has been shown on several occasions to be highly susceptible to these types of perturbations . In comparison to GFT data , there are some areas where the Wikipedia models were superior , but others where they were not . Full Wikipedia models were able to estimate the week of peak activity within a season more often than GFT data . Out of the 6 seasons for which data was available , GFT estimated a value of ILI that was more accurate ( regardless of whether or not the peak timing was correct ) than the Mf or Ml models in 4 seasons , while the Wikipedia models were more accurate in the remaining 2 . These analyses and comparisons were carried out on GFT data that was retrospectively adjusted by Google after large discrepancies between its estimates and CDC ILI data were found after the 2012–2013 influenza season , which was more severe than normal . Even with this retrospective adjustment in GFT model parameters , the peak value estimated by GFT for the 2012–2013 is more than 2 . 3-times exaggerated ( 6 . 04% ) compared to CDC data , and was also estimated to be 4 weeks later than it actually was . For this same period , the Mf model was able to accurately estimate the timing of the peak , and its estimation was within 0 . 76% compared to the CDC data . This study is unique in that it is the first scientific investigation , to the authors' knowledge , into the harnessing of Wikipedia usage data over time to estimate the burden of disease in a population . While Google keeps GFT model parameters confidential , the Wikipedia article utilization data in these analyses are freely available and are open to be modified and improved upon by anyone . Although it has not been investigated here , there is potential for this method to be altered for the monitoring of other health-related issues such as heart disease , diabetes , sexually transmitted infections , and others . While the above mentioned conditions do not have the same time-varying component as influenza , overall burden of disease may potentially be estimated based on the number of people visiting Wikipedia articles of interest . This is an open method that can be further developed by researchers to investigate the relationship between Wikipedia article views and many factors of interest to public health . Data regarding Wikipedia page views is updated and available each hour , though data in this study has been aggregated to the day level , and then further aggregated to the week level . This was done so that one week of Wikipedia data matched one week of CDC's ILI estimate . In practice , if this Wikipedia based ILI surveillance system were to be implemented on a more permanent basis , it is possible that updates to the Wikipedia-estimated proportion of ILI activity in the United States could be available on a daily or even hourly basis , although this application has not yet been explored . It is hypothesized that hourly updates may have trouble dealing with periods of low viewing activity , such as nighttime and normal sleeping hours , and that the benefit of an hourly update versus a daily update might not be worth the effort involved in its perpetuation . Daily estimates are likely to be of greater use than hourly and hold potential for use as a tool for detecting outbreaks in real-time , by creating an alert when the daily number of Wikipedia article views spikes over a set threshold . As with any study using non-traditional sources of information to make estimations or predictions , there is always some measure of noise in the gathered information . For instance , the number of Wikipedia article views used in this study represent all instances of article views for the English language Wikipedia website . As such , while the largest proportion of these article views comes from the United States ( 41% , with the next largest location being the United Kingdom representing 11% ) , the remaining 59% of views come from other countries where English is used , including Australia , the United Kingdom , Canada , India , etc . Since Wikipedia does not make the location of each article visitor readily available , this makes the relationship between article views and ILI activity in the United States less reliable than if the article view data was from the United States alone . To investigate this bias , it may be of interest to replicate this study using data that is country and language specific . For instance , obtaining Wikipedia article view information for articles that exist only on the Italian language Wikipedia website and comparing that data to specific Italian ILI activity data . Alternatively , the timing and intensities of influenza seasons in English-Wikipedia-using countries apart from the United States could be investigated as potential explanations of model performance . Depending on the timing of influenza activity in other countries , their residents' Wikipedia usage could potentially bolster the presented Wikipedia-based model estimations ( if their influenza seasons are similar to that of the United States ) , or it could negatively impact estimations ( if their influenza seasons are not similar to those of the United States ) . This is an interesting method of comparison and may potentially be explored in future iterations of this method . If these models continue to estimate real-time ILI activity accurately , there is potential for this method to be used to predict timing and intensity in upcoming weeks . While re-purposing these models could potentially be a significant undertaking , we are interested in pursing this avenue of investigation in future works . There has been much discussion in popular media recently about the potential future directions of Wikipedia . It has been noted in several papers and reviews that the number of active Wikipedia editors has been slowly decreasing over the past 6 years , from its peak of more than 51 , 000 is 2007 to approximately 31 , 000 in the summer of 2013 . [19] , [28] It has been speculated that the efforts made by the Wikimedia Foundation and it's core group of dedicated volunteers to create a more reliable , trustworthy corpus of information has limited the ability of new editors to edit or create new articles , thereby decreasing the likelihood that a new contributor will become a trusted source of information . Compounding this decrease in active editors , it has become increasingly evident that the vast majority of articles on the English Wikipedia website are both male and Western and European-centric , with comparatively few articles dealing with highly female-oriented topics or other geographic areas . Despite these concerns , the articles relating to influenza that have been investigated in this study are within the scope of the type of Wikipedia articles that are routinely and adequately maintained by long-time editors . The authors hypothesize that any decreases in the number of editors in the Wikimedia domain are unlikely to create significant changes in viewership of the articles of interest for estimating or predicting influenza-like illness , and therefore should not contribute meaningfully to the pursuit of this type of surveillance . Due to an error in Wikipedia data collection , there were no article view data available between July 13 , 2008–July 31 , 2008 , inclusive , resulting in a time gap of just over 2 . 5 weeks . Fortunately , this time gap occurred in a traditionally low ILI prevalence time of year , and is not suspected to meaningfully impact analyses . The application of Wikipedia article view data has been demonstrated to be effective at estimating the level of ILI activity in the US , when compared to CDC data . Wikipedia article view data is available daily ( and hourly , if necessary ) , and can provide a reliable estimate of ILI activity up to 2 weeks in advance of traditional ILI reporting . This study exemplifies how non-traditional data sources may be tapped to provide valuable public health related insights and , with further improvement and validation , could potentially be implemented as an automatic sentinel surveillance system for any number of disease or conditions of interest as a supplement to more traditional surveillance systems .
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Although influenza is largely avoidable through vaccination , between 3 , 000–50 , 000 deaths occur in the United States each year that are attributed to this disease . The Centers for Disease Control and Prevention continuously monitor the amount of influenza that is present in the American population and compiles this information in weekly reports . However , because it can take a long time to collect and analyze all of this information , the data that is being reported each week is typically between 1–2 weeks old at the time of publishing . For this reason , we are interested in developing new techniques to determine the amount of influenza in the population that are accurate , can return results in real-time , and can be used to supplement traditional monitoring . We have created a method of estimating the amount of influenza-like illness in the American population , at any time of year , by analyzing the amount of Internet traffic seen on certain influenza-related Wikipedia articles . This method is able to accurately estimate the percentage of Americans with influenza-like illness , in real-time , and is robust to influenza seasons that are more severe than normal and to events that promote much media attention , such as the H1N1 pandemic in 2009 .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
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"public",
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2014
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Wikipedia Usage Estimates Prevalence of Influenza-Like Illness in the United States in Near Real-Time
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Attempts to detect genetic population substructure in humans are troubled by the fact that the vast majority of the total amount of observed genetic variation is present within populations rather than between populations . Here we introduce a new algorithm for transforming a genetic distance matrix that reduces the within-population variation considerably . Extensive computer simulations revealed that the transformed matrix captured the genetic population differentiation better than the original one which was based on the T1 statistic . In an empirical genomic data set comprising 2 , 457 individuals from 23 different European subpopulations , the proportion of individuals that were determined as a genetic neighbour to another individual from the same sampling location increased from 25% with the original matrix to 52% with the transformed matrix . Similarly , the percentage of genetic variation explained between populations by means of Analysis of Molecular Variance ( AMOVA ) increased from 1 . 62% to 7 . 98% . Furthermore , the first two dimensions of a classical multidimensional scaling ( MDS ) using the transformed matrix explained 15% of the variance , compared to 0 . 7% obtained with the original matrix . Application of MDS with Mclust , SPA with Mclust , and GemTools algorithms to the same dataset also showed that the transformed matrix gave a better association of the genetic clusters with the sampling locations , and particularly so when it was used in the AMOVA framework with a genetic algorithm . Overall , the new matrix transformation introduced here substantially reduces the within population genetic differentiation , and can be broadly applied to methods such as AMOVA to enhance their sensitivity to reveal population substructure . We herewith provide a publically available ( http://www . erasmusmc . nl/fmb/resources/GAGA ) model-free method for improved genetic population substructure detection that can be applied to human as well as any other species data in future studies relevant to evolutionary biology , behavioural ecology , medicine , and forensics .
At what degree genetically homogeneous groups of human individuals exist is a long-standing and yet unsolved debate in the scientific community [1] . Answering this question is important for better understanding recent human evolutionary history [1] , for reducing the amount of false positives in gene mapping studies [2] and other medical issues [3] , and for inferring the bio-geographic origin of unknown persons in forensic investigations [4] . In general , for any species , detecting genetically homogeneous groups can be of relevance in answering questions in evolutionary biology and behavioural ecology . Previously developed methods for estimating average genomic ancestry and detecting genetic population substructure can be broadly classified into two types: model-based ancestry estimation and algorithmic ancestry estimation [5] . The former type aims to estimate the contribution of hypothetically existing ancestral populations to the genome of each specimen tested; popular implementation methods include STRUCTURE [6] , ADMIXTURE [5] , and FRAPPE [7] . The latter type uses hypothesis-free multivariate techniques , such as Principal Component Analysis ( PCA; [8] ) , classical multidimensional scaling ( MDS ) , or principal coordinates analysis [9] , to position each specimen tested in a reduced Euclidean space [10] , so that the proximity between specimens can be interpreted as genetic affinity [8] . The coordinates proposed by algorithmic ancestry methods tend to correlate with the geographic sampling location of the tested individuals when applied to human genetic data [11] . Recently , a method called SPA [12] was proposed; it exploits the geographic dependency between allelic frequencies and space to infer the coordinates in a 2D/3D space of a given set of individuals . However , detecting genetic population substructure can be complex depending on the evolutionary history of the species in question , and certainly in the case of humans . Certain processes such as isolation by geographic distance [13] , local genetic adaptation to environmental factors [14] , and other factors including cultural ones [15] , all impact on the amount of genetic differences observable between individuals within and between populations [16] . In particular , the recent origin of the human species and the even more recent dispersal out of the African continent [17] played a major role in shaping the neutral variation of the human genome with dramatic consequences for the detection of genetic population substructure . Due to our single recent origin , the vast majority ( ∼85% ) of the total genetic differences is explained by variation between individuals within populations [1] . Moreover , the genetic differences between populations usually follow clinal geographic patterns [18] , which typically are in agreement with major past migration routes [19] , rather than showing sharp discontinuities . For instance , within the European continent , the genetic differentiation between European subpopulations ( with exceptions such as European Romani , [20] ) is small [21] compared to that found among worldwide populations , and even smaller when sampling within specific sub-regions of Europe [22] . Furthermore , long identical-by-descendent ( IBD ) genomic tracks that are shared between geographically distant European individuals have been found , suggesting a recent common ancestry of European populations [23] . Finally , individuals from one population tend to have their best genetic-matching partner ( as defined by the Best Overall Match ( BOM ) ) far away from their sampling population [24] . Nevertheless , a remarkable correlation between genetic and geographic distance as well as a clinal distribution of genetic diversity on the continental [21] , [25] and sub-regional level ( i . e . , [22] ) have been observed within Europe . Overall , the fact that the vast majority of human genetic variation exists among individuals within populations [21] limits the capacity of existing methods to resolve genetic population substructure at a fine geographic scale and asks for the development of alternative methods for detecting population substructure and genetic ancestry in humans that can also be applied to other species . Recently , a new algorithm implemented in the fineSTRUCTURE software [26] analyzes the shared haplo-blocks between previously phased pairs of individuals . However , genome phasing can be computational intensive [27] , especially when a large number of individuals and markers is used . Moreover , despite the current state-of-art of phasing algorithms [27] , errors are unavoidable , especially when considering variants at low frequency [28]; furthermore , some prior population information is usually desired [27] . Finally , genomic SNP density is only considerable in the case of humans ( and not for all the geographic regions [29] ) ) , whereas in other species , such as cattle , a relatively limited number of markers have been described thus far [30] . In the present study , we propose a new matrix distance transformation with the aim to reduce the within-population variation . We conducted extensive computer simulations under two demographic models to test if this aim is achieved . We additionally implemented a genetic algorithm which , in combination with AMOVA statistics , allows searching for the optimal genetic clustering configuration of specimens and populations . We practically test the performance of this new , model-free approach using a genome-wide dataset comprising 2 , 457 individuals from 23 geographically dispersed subpopulations of Europe . We make this new method for improved genetic population substructure detection publically available as software package for free use .
Our algorithm starts with a genetic distance matrix D computed for each possible pair among N individuals , which in this study is derived from the T1 statistic [31] . The T1 statistic has been shown to be informative for detecting hidden genetic relatedness [32] , independently of the ( unknown ) allelic frequencies in each population [31] . T1 is defined for a given pair of individuals i and j as: ( 1 ) where nxx , yy denotes the number of SNPs of a particular genotype pattern ( i . e . n00 , 11 refers to SNPs where the first individual is homozygous for one allele ( 0 ) and the second individual is homozygous for the alternative allele ( 1 ) ) . Under Hardy-Weinberg equilibrium ( HWE ) , the expectancy E ( T1 ) = 2/3 if both individuals are unrelated from the same population , E ( T1 ) < 2/3 if the individuals are from different populations and E ( T1 ) > 2/3 if they are more related than by chance . We define the distance matrix as D = 1-T1 . That is , we set di , j = 1-T1i , j in order to obtain a genetic distance between individuals i and j . Individuals can then be classified into populations , and the genetic differentiation between populations quantified using this individual distance by applying the Analysis of Molecular Variance ( AMOVA [33] ) framework . In analogy to the Analysis Of Variance ( ANOVA ) , the AMOVA framework decomposes the total sum of squares ( SS ( T ) ) from the individual distance matrix in sum of squares among populations ( SS ( AP ) ) and sum of squares within populations ( SS ( WP ) ) , so that: ( 2 ) ( 3 ) ( 4 ) where K is the number of groups . The estimated values of population differentiation can be transformed into Fst-like statistics , and reflect demographic parameters such as migration rate or time of population split , among others [34] . However , it has been suggested that within population variance can be as high as the total variance for highly polymorphic markers , resulting in very low values of SS ( AP ) even if the compared populations have no alleles in common [35] . Meirmans [35] proposed a standardized version of the AMOVA under different scenarios . However , several other genetic dissimilarity statistics can also be used for estimating genetic relatedness between diploid individuals [36] , [37] . Here we attempt to reduce the within-population variation and maximize the between-population variation without a priori knowledge of the clusters ( that is , only using individual pairwise distances ) and without any distance restriction . We do this by transforming the genetic distance D matrix into a new dissimilarity one V , where Vij = Vji = var[di . –dj . ] taking into account that dii – dij and djj – dji are excluded during the variance computation . The rationale for proposing the V matrix transformation is as follows: Following the AMOVA framework , individual relationships are modelled using a list colouring of graph [38] , so each vertex can be either assigned to an individual , a non-admixed population , an admixed population , or a group of populations ( see Figure 1 A ) ; therefore , for a pair of individuals i , j , the distance di , j can be decomposed in within- and between-population distances ( see Figure 1B ) : ( 5 ) where di , I is the distance of individual i to his group I ( ) , dj , J is the distance of individual j to his group J ( ) , dI , J is the distance between group I and J and ε is a random error in the estimation of any d . , . which we assume follows a normal distribution and is identical for all the individual pairwise distances ( ) . If i and j share the same adjacency vertex ( i . e . and ) , then: ( 6 ) ( 7 ) for any individual k ( k≠i; k≠j ) . The mean of the difference between distances is then: ( 8 ) with expected variance: ( 9 ) Therefore , the variance of the difference of distances for a given pair of individuals from the same group to all the other individuals becomes independent of the distance of each individual to his group , and it is the same for all the elements of the group . In contrast , it can be expected that the distances between individuals from different populations will depend on the topology of the graph and the number of individuals that belong to the same population . For example , consider the simplest case of a graph of two populations ( Figure 1B ) ; if i and j do not share the same adjacency vertex ( i . e . they are from a different populations ) , Vij becomes: ( 10 ) And ( 11 ) Where is the number of individuals that belong to population I and is the number of individuals that belong to population J . In this case , the variance of the difference of distances includes an additional term to the error in the estimation proportional to the distance between the two groups and their respective sample sizes . As previously , the within-population variance ( i . e . the distance of the individual to his population ) is cancelled , which can therefore improve the detection of population differentiation . If = , it can be seen that Vij = d2I , J + 2σ2 . Also , notice that if = 1 or = 1 , then Vij = 2σ2 . Therefore , in this example population differentiation could only be detected by this statistic when there are at least two individuals in each population so that the distance of each individual to his population can be estimated . The pseudocode for computing V is provided in Text S1 . The AMOVA framework has been previously applied to identifying the best genetically homogeneous sets of geographically related populations [39] by trying to maximize the amount of genetic differentiation among groups of populations ( conversely minimizing the variance within groups of populations ) . Since exploring the entire solution space is unfeasible even for a reduced number of populations , Dupanloup et al . [39] applied a simulated annealing algorithm . The method was devised to detect spatial barriers between already defined populations . However , a similar heuristic approach can also be applied for clustering individuals into populations , rather than populations into groups of populations . In particular , we propose to use a continuous genetic algorithm [40] with Crossover Pair SubClusterSwap_TWO_NEW [41] movement in order to explore the space of possible combinations and recover the optimal ( or suboptimal ) combination that maximizes the SS ( AP ) statistic ( conversely minimizes SS ( WP ) ; see Text S1 ) . In order to test the V matrix transformation in a known graph model , we performed simulations on four populations of 10 individuals each , modelling a situation of three parental populations and one admixed population ( see Figure S1 ) . In each simulation , we varied at random the distance of each individual to its population , the distances between populations , and whether the distances of the individuals to their populations were larger than the distances between populations . We performed 1000 simulations for each of the 8 possible combinations , and for each simulation computed the distance between each pair of individuals according to formula ( 5 ) , including an error term following a normal distribution with mean = 0 and standard deviation = 0 . 05; for each simulation the D and V matrix and the percentage of SS ( AP ) explained was computed . We also conducted two sets of simulations of increasing demographic complexity to check whether V is more sensitive for detecting population substructure than D and to analyse to what extent the use of V improves the geographic sampling location prediction compared to the use of D . Both demographic models were implemented with the ms software [42] and simulated 25 populations , 10 diploid individuals per population ( that is , 20 chromosomes per population ) and either 10 , 000 or 100 , 000 independent SNPs sampled from fragments of 50 kb and assuming a mutation rate of 2 . 5*10−8 per nucleotide and generation [43] . In the first demographic scenario , we model the colonization of a one-dimensional space from a starting founder population by splitting the youngest population in two new ones every t generations [44] ( see Figure 2A and Text S1 for details ) . The second scenario considers spatial structure and migration between neighbour populations following an isolation by distance model [13] ( see Figure 2B and Text S1 ) . For each simulated dataset , the sensitivity of D and V towards the real sampling location was quantified by means of SS ( AP ) /SS ( T ) . We further analysed the performance of V for improving the percentage of best genetic-matching partners in the same population by computing the percentage of BOM . We used a previously published dataset comprising 309 , 790 SNPs and 2 , 457 individuals from 23 European subpopulations genotyped with the Affymetrix 250K Xba and 250K Sty SNP microarrays , [21] ( see Table S1 ) . Previous data cleaning of that dataset included removing individuals showing a higher or smaller genetic differentiation compared to the rest of the individuals from the same subpopulation , and excluding SNPs showing a statistically significant HWE deviation in at least one subpopulation ( see [21] for a complete description of the data cleaning procedure ) . Since most of the applied methods assume linkage equilibrium among SNPs , a Linkage Disequilibrium ( LD ) pruned SNP subset of 133 , 363 was computed with plink software [45] with the default plink --indep 50 5 2 command , and was used for method-comparison analyses . Also , since multivariate techniques such as PCA have shown that unequal sample size can affect the outcome [46] , all analyses were performed twice , once considering the original sample size and once considering 19 sample sites or subpopulations with a sample size of 40 individuals ( after excluding Lisbon-Portugal , Dublin-Ireland , Budapest-Hungary and Bucharest-Romania ) polymorphic at 124 , 134 SNPs . We attempted to apply five of the previously proposed methods for inferring groups of genetically homogeneous individuals ( for example , see [47] ) to this dataset . When not included in the original algorithm , we applied the algorithm Mclust [48] to obtain the clusters . This algorithm assigns individuals to clusters by fitting multivariate normal distributions using the coordinates of the proposed dimensions and proposes the best clustering based on the Bayesian Information Criterion ( BIC ) . Mclust has been put forward as a clustering algorithm for the output of Principal Component Analysis using genetic data [49] . The first analysis consisted of a Classical Multidimensional Scaling ( MDS;[9] ) performed using either the D or the V distance matrix between pairs of individuals using the cmdscale function of R statistical package [50] , and adding a constant to avoid negative eigenvalues [51]; Mclust clustering was performed using the first 10 dimensions , and setting the number of clusters from 1 to 60 . The second analysis consisted of a spatial ancestry analysis ( SPA ) [12] conducted to infer the geographic ancestry of each individual in two spatial dimensions . Clusters of individuals were then inferred by means of Mclust using the proposed SPA coordinates , also ranging from 1 to 60 . The third analysis was performed with the clusterGem algorithm implemented in the GemTools package which uses spectral graph theory to propose clusters of individuals [52] . Recently , a new software called LOCO-LD [53] has been proposed for estimating the geographic locations of a set of individuals . Similar in essence to SPA ( i . e . for each SNP it is assumed that there is an allelic gradient ) , LOCO-LD additionally incorporates LD patterns into the model , which has been suggested to improve ancestry detection . However , the fact that it necessarily requires a training dataset where the localization of some individuals is known a priori ( personal communication with the authors ) has precluded its use for comparative purposes , as all the other used algorithms are unsupervised . We also aimed to run fineSTRUCTURE , another software that uses LD patterns [26] , on the same dataset , after phasing it with the Beagle software [54] . However , computing the shared chunk matrix of all individuals with the default parameters of ChromoPainting [26] turned out to be extremely computationally intensive , even after splitting the genome into chromosomes for parallel computing . As an example , chromosome 22 , the smallest human chromosome comprising only 3 , 698 SNPs in this dataset , has a computational complexity according to the ChromoPainter manual of 96 , 589 , 584 , 000 steps for only one E-M iteration . The authors of this software reported in the ChromoPainter manual computation times of 2-3 hours for a computational complexity of 115 , 543 , 296 steps using a computer of similar characteristics as the one we used here ( 8 cpus , 24 GB of RAM ) . Therefore , it can be expected that the computational time for this chromosome is going to be ∼83* ( 2 to 3 ) hours . Given that the authors suggest to run ChromoPainter considering different numbers of E-M iterations and parameters , running all 22 chromosomes of this dataset appears beyond reasonable practicability with the computer resources available . Because of this , we decided to exclude this software from comparison . Pie map plots were constructed for each method and each proposed clustering using the R packages map and mapplots . The Cramer's V value [55] was used for summarizing the goodness of fitness between the proposed clusters and the labelled population origin of the individuals . Cramer's V is a classical measure of association of two variables in a contingency table and is defined as: ( 12 ) where χ2 is the chi-squared value from Pearson's chi-squared test , N is the total of observations , k is the number of rows or the number of columns if less than the number of rows . Cramer's V ranges from 0 , which corresponds to random assignment of the individuals of each population to the different clusters , to 1 , meaning that each proposed individual cluster perfectly matches one population . Also , in order to quantify how well the genetic clusters proposed by each method differentiate each sampling location or subpopulation from all the others , we computed the Informativeness of Ancestry ( In ) statistic [37] between each pair of sampling locations using the obtained frequency of the proposed clusters by each method: ( 13 ) Where K is the number of proposed clusters , psc is the frequency of the cluster c in sampling location s and ptc is the frequency of the cluster c in sampling location t . In ranges between 0 ( i . e . the proposed clusters cannot distinguish individuals from the two populations ) to log ( 2 ) , indicating that the two sampling locations are perfectly differentiable based on the proposed clusters . Therefore , if a sampling location is perfectly differentiated from any other sampling location based on the proposed clusters , the minimum value that is going to be obtained for all the possible ( sub ) population comparisons of that particular sampling site is log ( 2 ) . In contrast , if the sampling location is identical based on the proposed clusters to at least one of the other sampling locations , the minimum In value is going to be 0 . The complete methodological pipeline is depicted in Figure S2
We started comparing V and D matrices in explaining the between-population variation in a simple case modelling four populations under different scenarios of distances between individuals and populations . As can be seen in Figure S1 , SS ( AP ) computed with the D matrix strongly varies depending on which distance model assumptions are applied . In contrast , the SS ( AP ) values obtained with the V matrix are close to 1 in all cases , and are in all the cases larger than those obtained for the same simulation with the D matrix . Next , we analysed the behaviour of the V and D matrix in genetic data by means of extensive simulations using two of the most commonly applied models in human populations , considering either 10 , 000 SNPs ( see Figure S3 ) or 100 , 000 SNPs ( see Figure 2 ) . In the two-dimensional stepping-stone grid model the amount of genetic differentiation between populations increased proportionally to the decrease in the number of migrants among neighbour populations when using either D or V matrix , regardless of the number of considered SNPs ( see Figure 2A . 2 ) . Nevertheless , the between-population differentiation increased much faster in the case of V than in the case of D and even faster when simulating 100 , 000 SNPs ( see Figure 2 and Figure S3; Wilcoxon signed paired rank test p-value between SS ( AP ) estimated from V matrix with either 10 , 000 SNPs or 100 , 000 SNPs = 0 . 001953 ) . In contrast , SS ( AP ) values estimated with the D matrix were similar , independently of the number of considered SNPs ( Wilcoxon signed paired rank test p-value between SS ( AP ) estimated from D matrix with either 10 , 000 SNPs or 100 , 000 SNPs = 0 . 4316 ) . A similar trend of results , both for the V matrix and the D matrix , was observed when simulating the data under the sequential split model and increasing the time of separation between populations ( see Figure 2B . 2 ) . Furthermore , the percentage of BOM from the same sampling population increases when the migration rate decreases ( in the case of the stepping-stone model ) and the time of split increases ( in the case of the sequential split model ) , independently of the number of SNPs or type of considered distance matrix ( see Figure 2A . 3 and 2B . 3 ) . However , the percentage of BOM from the same sampled population increases faster when using V than when using D after a certain parameter threshold in both models ( see Figure 2 . A . 3 and Figure 2 . B . 3 ) . Furthermore , this threshold depends on the number of considered SNPs ( see Figure S3 ) : a smaller migration rate for the sequential split model and a larger time of population split for the stepping stone model is required in order to detect differences in the percentage of BOM from V or D matrix using 10 , 000 SNPs compared to when using 100 , 000 SNPs . Overall , our simulation experiments demonstrate that V can be used to detect further genetic-geographic population substructure in the cases where the amount of genetic differentiation is particularly small compared to within each population , such as is expected and partly known already in human populations from the European continent . Given these promising results obtained in the computer simulations , we applied our newly developed approach to a previously collected dataset comprising 2 , 457 individuals from 23 European subpopulations using 133 , 363 LD pruned genome-wide SNPs [21] . We first observed that the mean distance T1 of each individual to all the other individuals collected at the same geographic site ( i . e . belonging to the same subpopulation ) was 0 . 331 ( 95% CI from 0 . 322 to 0 . 342 ) . Thirty-three percent of the individuals showed a mean T1 distance to their sampling population >1/3 , suggesting that they belonged to a different random mating population [31] . Moreover , this proportion was not constant among European subpopulations ( ranging from 0% in Budapest-Hungary to 63% in Madrid-Spain; see Table S2 , two sided Fisher exact test p value < 0 . 0005 after 2000 replicates ) , indicating that some European subpopulations are more genetically heterogeneous than others . The percentage of individuals with BOM in the same subpopulation using the T1 matrix was 25 . 93% , a value similar to the one obtained previously when using Identical By State distance between pairs of individuals [24] . This value ranged from 0% in Bucharest-Romania , Copenhagen-Denmark , Lyon-France , Prague-Czech Republic and Warsaw-Poland to 78 . 7% in Helsinki-Finland ( Table S3 ) . In contrast , the BOM computed from the V distance matrix increased to 52 . 83% , ranging from 6% in Lyon to 97 . 87% in Helsinki ( see Table S4 ) . This improvement is much higher than the one observed in the simulated datasets for BOM of 20% computed with the D matrix . Furthermore , the SS ( AP ) was estimated to be 1 . 62% when using the D matrix , while it increased to 7 . 98% when using the V matrix . Hence , also when applied to real genomic data our newly developed approach revealed increased genetic population differentiation . We further focused on studying to which extent unsupervised clusters of individuals inferred from the genetic data would match the geographic site of their sampling origin or subpopulation ( see Table 1 ) . In the case of MDS , the first two dimensions using the D matrix and considering all the individuals explained 0 . 733% of the total variance ( see Figure S4 ) , 2% when considering an equal sample size of 40 individuals per subpopulation . In contrast , the first two dimensions of the MDS using V and all the individuals explained 15 . 133% of the total variance , 20 . 64 times more , and increased to 30 . 45% when using unbiased sample size among populations . These results supports that the V transformation reduces the amount of non-shared ( i . e . particular of each individual ) variation , and highlights the differences among groups of individuals . The best supported clustering by Mclust using the first 10 MDS dimensions using the D matrix was 26 genetic clusters ( Figure 3A ) . Cramer’s V statistic between the proposed clusters and the sampling sites or subpopulations was 0 . 655 . The average amount of minimum sampling site differentiation based on these 26 clusters was 0 . 231 , with Helsinki-Finland being the mostly differentiated of all European subpopulations considered ( In = 0 . 642 , see Figure 4 ) and Lisbon-Portugal , Madrid-Spain and Barcelona-Spain appearing as non-distinguishable from each other ( In = 0 ) . In contrast , Mclust using the first 10 MDS dimensions from the V matrix proposed 37 different genetic clusters , all the populations sharing at least one of the proposed clusters ( see Figure 3B ) . Cramer’s V increased to 0 . 71 , and the average In increased to 0 . 304 , again suggesting that V provides a better population sampling resolution than the original D matrix . The strongest improvement in European subpopulation differentiation was observed in Ancona-Italy ( In using the D matrix ( In–D ) = 0 . 122 compared to In using the V matrix ( In–V ) = 0 . 434 ) and Rome-Italy ( In–D = 0 . 122 to In–V = 434 ) . Running Mclust on SPA based on the original genotype matrix suggested 13 clusters ( Figure 3C ) ; the average amount of subpopulation differentiation provided by these genetic clusters was quite poor ( average In = 0 . 127; see Figure 4 ) , and none of the sampling subpopulations improved their differentiation compared to all the other methods . Nevertheless , it must be taken into account that these results are not directly comparable , since SPA models the observed data in a very limited number of dimensions ( two in our case ) , whereas MDS+Mclust analyses were based on 10 dimensions . Indeed , the MDS+Mclust analysis using the first two dimensions provided similar results to the ones observed with SPA ( results not shown ) . GemTools analysis proposed 56 different genetic clusters ( see Figure 3D ) . However , despite this increase in the number of proposed clusters , the average minimum differentiation among subpopulations ( average In = 0 . 268 ) was smaller than the one obtained when running MDS-V+Mclust . Compared to MDS-V+Mclust , the proposed clusters by GemTools increased the differentiation of Barcelona-Spain , Ancona-Italy , Augsburg-Germany , and Innsbruck-Austria but reduced it in Belgrade-Serbia , Bucharest-Romania , North Greece , Forde-Norway , and particularly in Warsaw-Poland ( see Figure 4 ) . We used the genetic algorithm maximizing AMOVA's SS ( AP ) statistic either with the D or the V matrix ( the latest comprising the GAGA approach ) to the same genetic dataset , setting K = 56 allowed clusters ( see Figure S5 for results using K = 2 , 5 , 10 , 15 and 23 with V matrix ) , the same number of clusters as identified by GemTools ( see Figure 3E ) . The average amount of minimum genetic differentiation of the proposed clusters by the genetic algorithm + D matrix was In = 0 . 254 , the second worse value after MDS-D+Mclust . Only in the case of Belgrade there was an improvement compared to all the other methods ( see Figure 4 ) . In contrast to these results , when the genetic algorithm uses the V matrix , the average differentiation among European subpopulations increased to In = 0 . 316 , the largest value of all the applied methods . Hence , GAGA was able to increase the geographic resolution compared to other methods . Furthermore , in the case of Budapest-Hungary , North Greece , Helsinki-Finland , Prague-Czech Republic and Rome-Italy ( Figure 4 ) , GAGA provides the best values of subpopulation differentiation in this European genomic dataset . We further analysed the effect of different sample size in the outcome of the different methods . We repeated all the analyses with a subset of 19 populations ( after excluding Lisbon-Portugal , Dublin-Ireland , Budapest-Hungary and Bucharest- Romania ) with equal sample size of 40 individuals . The percentage of closest genetic neighbours in the same ( sub ) population is similar to the ones when considering all the individuals ( BOM = 58 . 03% for the V matrix , 22 . 37% for the D matrix ) . Nevertheless , the association between the proposed clusters and the ( sub ) population samples increases in all the methods . This is particularly pronounced in the case of GemTools ( see Table 1 ) . We wondered whether this difference in performance of GemTools is due to the excluded four populations and/or to the use of equal sample sizes , so we performed all the analyses considering the same subset of 19 subpopulations but with their original sample size . The values of minimum informativeness differentiation of GemTools increased to 0 . 385 , thus suggesting the influence of the four excluded populations in the final results . We have described a new matrix distance transformation that tends to minimize the within-population variance without knowing a priori the ( sub ) populations , and have shown , by means of computer simulations and application to real European genetic data , that this new approach improves the differentiation among ( sub ) populations compared to the original distance matrix . A practical result of our analyses is that this matrix transformation improves the output of MDS , both at the level of explained variance and resolution , as well as from the AMOVA estimations . In the present paper we show that GAGA performs reasonably well when using the K proposed by GemTools . One could also consider estimating the K based on parameterized Gaussian mixture models [56] such as implemented in Mclust . Nevertheless , the choice of K is rather arbitrary depending on the required resolution and subject of further study . Most importantly , our findings of previously undetected fine-scale human population substructure down to the level of sampling sites or subpopulations within Europe , has important implications for various basic and applied fields of life science . With relevance for genetic epidemiology , our results suggest that the genetic homogeneity detection desired in case-control studies should be preferably established by analyzing the relationships of pairs of individuals in the context of all other individuals tested , rather than by analyzing how genetically similar individuals are , as usually done . The GAGA approach we introduce here is now available for application to all types of genetic data . The GAGA algorithm was implemented in JAVA ( Sun Microsystems ) and is publically available for widespread use at http://www . erasmusmc . nl/fmb/resources/GAGA .
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Understanding genetic population substructure is important in evolutionary biology , behavioral ecology , medical genetics and forensic genetics , among others . Several algorithms have recently been developed for investigating genetic population substructure . However , detecting genetic population substructure can be cumbersome in humans since most of the genetic diversity present in that species exists among individuals from the same population rather than between populations . We developed a Genetic Algorithm for Genetic Ancestry ( GAGA ) to overcome current limitations in reliably detecting population substructure from genetic and genomic data in humans , which can also be applied to any other species . The method was validated by means of extensive demographic simulations . When applied to a real , human genome-wide SNP microarray dataset covering a reasonable proportion of the European continent , we identified previously undetected fine-scale genetic population substructure . Overall , our study thus not only introduces a new method for investigating genetic population substructure in humans and other species , but also highlights that fine population substructure can be detected among European humans .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results/Discussion"
] |
[
"evolutionary",
"biology",
"genetics",
"population",
"genetics",
"biology"
] |
2014
|
GAGA: A New Algorithm for Genomic Inference of Geographic Ancestry Reveals Fine Level Population Substructure in Europeans
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Microbial biofilms are a dominant feature of many human infections . However , developing effective strategies for controlling biofilms requires an understanding of the underlying biology well beyond what currently exists . Using a novel strategy , we have induced formation of a robust biofilm in Escherichia coli by utilizing an exogenous source of poly-N-acetylglucosamine ( PNAG ) polymer , a major virulence factor of many pathogens . Through microarray profiling of competitive selections , carried out in both transposon insertion and over-expression libraries , we have revealed the genetic basis of PNAG-based biofilm formation . Our observations reveal the dominance of electrostatic interactions between PNAG and surface structures such as lipopolysaccharides . We show that regulatory modulation of these surface structures has significant impact on biofilm formation behavior of the cell . Furthermore , the majority of clinical isolates which produced PNAG also showed the capacity to respond to the exogenously produced version of the polymer .
Biofilms are an integral component in the life-cycle of many microorganisms . Compared to their planktonic complement , however , bacterial biofilms have remained poorly understood , mostly due to the inherent complexities associated with biofilm studies , including spatial heterogeneity of the biofilm structure , longer generation time , and uncharacterized growth parameters [1] . Bacterial biofilms are characterized by the presence of an extracellular polymeric matrix , which encases the cells . The physicochemical properties of this matrix , including its charge , porosity , and architecture are prominent determinants of biofilm lifestyle . The matrix , for example , could act as a protective barrier by interacting with large , charged , or reactive biocidal agents and neutralizing them [1] . One major component of matrix in various bacterial species is a homopolymer of N-acetylglucosamine . In fact , poly-N-acetylglucosamine ( PNAG ) is the major virulence factor of Staphylococcus epidermidis [2] . There is increasing evidence that this polysaccharide is produced by a variety of other pathogens including Bordetella , Yersinia , Staphylococcus , Actinobacillai , and certain pathogenic Escherichia coli strains as well . It was reported that enzymatic hydrolysis of poly-N-acetylglucosamines disrupts biofilm formation by Yersinia pestis , Pseudomonas fluorescens , Aggregatibacter actinomycetemcomitans , pathogenic E . coli strains , and various Bordetella species [3]–[7] . This suggests that PNAG is a critical component of the biofilm structure made by all these bacteria . Furthermore , a recent study showed that most E . coli strains isolated from urinary tract and neonatal bloodstream infections possess the pga locus required for PNAG biosynthesis , and almost all of them produce immunologically detectable levels of PNAG [8] . Involvement of PNAG-based biofilms in the pathogenesis of various bacterial species makes it an important phenomenon to study [4] , [8]–[11] . Even though the properties of PNAG-based biofilms have been extensively studied in Staphylococcus species [2] , [12] , the existence of a PNAG-based matrix in biofilm structures from other species , including E . coli , has been reported only recently [3] , [13] , and is not as well characterized as it is in Staphylococcus species . However , there are some features of PNAG-based biofilm like spatial distribution of the cells in PNAG-based biofilms that are better studied in E . coli [14] . In E . coli K-12 , expression of PNAG biosynthesis genes is not high enough to support the formation of a robust biofilm structure under laboratory conditions [15] , complicating the analysis of this phenotype . Therefore , in order to study the genetic basis of PNAG-based biofilm formation , we decided to enhance this phenotype in E . coli K-12 by either increasing the level of endogenous PNAG or providing an exogenously produced form of PNAG . PNAG production in E . coli can be enhanced by manipulation of the genetic elements involved in pga locus regulation [16] , [17] . For example , E . coli csrA mutants overproduce the PNAG polymer [13] , [17] . However , csrA is a master regulator of the carbon storage system , and csrA mutants show highly pleiotropic phenotypes . An alternative approach for enhancing PNAG-based biofilm formation would be to use a functionally active exogenous source of PNAG to induce biofilm formation in E . coli . In the csrA mutant background , cells secrete PNAG polymer into the growth media [18] . Therefore , the spent media from the csrA mutant culture can be used as a potential source of PNAG for inducing biofilm formation . We observed that application of exogenously produced PNAG , isolated from ΔcsrA cells saturated culture of csrA mutants , led to robust biofilm formation in the E . coli K-12 background . This provided us a unique opportunity to extensively characterize the underlying genetics of the PNAG-based biofilm formation phenomenon . Given our observations , we favor a model in which electrostatic interactions between this polysaccharide and cell surface structures , such as lipopolysaccharide ( LPS ) , are critical for PNAG-induced biofilm formation by E . coli . We also show that although response to PNAG polymer is a purely structural phenomenon , it can be modulated by multiple pathways including LPS biosynthesis , the acid tolerance system , capsule biosynthesis , and regulation of cell morphology .
Escherichia coli csrA mutants secrete poly N-acetylglucosamine polysaccharide into the culture medium [18] . In order to see whether the secreted polysaccharide is functional , we grew ΔcsrA cells to stationary phase , discarded the cells , and added the cell-free spent media to wild-type MG1655 cells in the presence of fresh media . Interestingly , we observed that the cell-free spent media of saturated ΔcsrA cultures made wild-type cells form a biofilm on a rapid time-scale ( Figure 1 , compare i and ii ) . Presence of a carbon source , as expected , was required for formation of mature visible microcolonies by living wild-type cells ( Figure 1 , compare iv and vii to v and vi ) . Synthesis of PNAG in E . coli requires the gene products of the pgaABCD operon [13] , [17] . To test whether the observed biofilm-inducing activity is associated with the secreted PNAG ( sPNAG ) , we generated four double mutants each harboring csrA deletion together with the deletion of one of the four genes present in the pga locus . There was no detectable biofilm-inducing activity in the cultures of any of the four double mutants . Furthermore , the biofilm-inducing activity was lost after treatment of ΔcsrA culture spent media with Dispersin B , an enzyme that specifically cleaves PNAG [3] , [5] , [19] , confirming that the biofilm inducing factor is an N-acetylglucosamine containing polysaccharide . Differential up-regulation of the pga locus transcription between the ΔcsrA and wild-type cells was also confirmed by a reporter assay ( Figure S1 ) . In order to confirm that the observed biofilm formation phenotype is specific to sPNAG and rule out the possibility of involvement of other biofilm-inducing agents that might be present in the ΔcsrA spent media , we purified sPNAG from spent media . As part of the purification steps , sPNAG was treated with various enzymes , including DNase , RNase , α-amylase , and Proteinase ( see Materials and Methods section ) . The purified sample showed identical biofilm-inducing activity , suggesting that sPNAG is sufficient for inducing biofilm formation . The purified polysaccharide was also characterized by mass spectrometry . As shown in Figure 2 , almost all prominent molecules found on the mass spectrum corresponded to N-acetylglucosamine oligomers with different levels of acetylation or to their monomers . In S . epidermidis , deacetylation of PNAG polymer introduces positive charges in the otherwise neutral polymer [20] . Our results also indicate that a considerable fraction of sPNAG building units is deacetylated , which should leave a net positive charge on the polymer . Purified PNAG isolated from Staphylococcus aureus strain MN8m [21] showed similar biofilm-inducing activity when applied to wild-type MG1655 cells , further confirming that PNAG is sufficient for the observed biofilm-inducing activity . The presence of various identical peaks in the mass spectra of PNAG from E . coli and S . aureus ( Figure S2 ) indicates that they are closely related molecular species . The response of wild-type E . coli cells to sPNAG was so fast that we decided to study the early stages of the process . Using time-lapse microscopy ( Video S1 ) , we observed that wild-type cells started seeding microcolony structures on a glass slide in less than an hour of exposure to sPNAG . The pace of microcolony formation observed here was much faster than the previously reported behavior by csrA mutants [15] . The microcolonies expanded in size due to both growth of pre-existing cells and continued incorporation of new cells . No similar activity was observed in the absence of sPNAG ( Video S2 ) . These results show that sPNAG enhances both cell-cell and cell-surface interactions . SEM images of the biofilm structures formed by wild-type cells in the presence of sPNAG also confirmed the presence of an extracellular matrix encasing the cells ( Figure S3 ) . We used a microarray-based genetic footprinting strategy [22] to study the genetic basis of sPNAG-mediated biofilm formation . Since formation of biofilm by wild-type cells in the presence of sPNAG is robust and fast , the phenotype is highly amenable to genetic analysis . Starting from a close to saturation Tn5-based library of approximately 5×105 independent transposon insertional mutants , generated in wild-type E . coli MG1655 , we devised a selection strategy to enrich for mutants defective in responding to sPNAG . Roughly 1010 cells of the abovementioned library were exposed to sPNAG in LB medium . After 12 hours of incubation , cells which were present in the liquid phase of the culture and were not part of the biofilm were isolated , grown up to log phase , and transferred to a new container with fresh media and fresh sPNAG , in order to enrich for mutants impaired in responding to sPNAG . After four rounds of serial enrichment , no visible biofilm formation activity was present in the enriched population . A schematic representation of the enrichment procedure is shown in Figure 3 . To quantify the contribution of different loci to this impaired biofilm formation phenotype , the insertion sites of the transposon in the enriched population were mapped using a microarray-based approach [22] . The histogram in Figure 4A shows the normalized average output of the hybridization data from two experimental replicates . The z-score for each ORF is indicative of the abundance of transposon insertion events in that ORF ( or its vicinity ) in the enriched population of mutants . More detailed information regarding the calculation of z-score is provided in the supporting information section ( Dataset S1 ) . Interestingly , the majority of the genes that were highly enriched in our selection were involved in two major biological processes: LPS core biosynthesis and regulation of cell shape and morphology . Most of these candidate genes belong to two long operons ( Figure 4B ) . In other words , genetic perturbations caused by transposon insertion in many components of LPS biosynthesis or cell shape regulation made wild-type cells lose their ability to form a biofilm in the presence of sPNAG . The dominance of genes involved in the synthesis and regulation of exposed structural components suggested that physical interaction between sPNAG and these surface structures may be a major determinant of biofilm formation capacity . sPNAG pre-treatment of the cells , however , did not cause any change in the migration of their extracted LPS samples on SDS-PAGE gels ( Figure S4 ) . Incomplete disruption of the targeted genes and polar effects are characteristics of transposon insertion events . In order to get around these complications and get a fine-scale perspective of genetic perturbations that prevent cells from responding to sPNAG , we generated in-frame deletions of some of the candidate ORFs , obtained from our transposon mutagenesis screen , in the MG1655 background [23] , [24] . We then studied the behavior of these mutant strains in the presence of sPNAG ( Table S2 ) . Among the candidate genes , deletion of rfaY , rafP , or rfaQ , diminished sPNAG-based biofilm formation ( Figure 1 , compare i and iii ) . As shown in Figure 4C , the product of these three genes are directly or indirectly involved in addition of phosphate groups to the inner core of LPS [25] . Since the major common point in the LPS structure of rfaY , rfaP , and rfaQ mutants is the lower density of negative charge ( phosphate groups ) on their LPS outer core , these phosphate groups are likely to be critical for this interaction . Given the positive charge of sPNAG , we favor a model in which electrostatic interaction between the positively charged polysaccharide and the negatively charged phosphate groups on LPS is the major determinant of sPNAG-mediated biofilm formation . Electrostatic interactions were also proposed to be responsible for PNAG-based biofilm formation in S . epidermidis [20] . It is postulated that neighboring LPS molecules can be cross-linked by divalent cations due to the presence of phosphate groups in the LPS structure [25] . Therefore , any presumable electrostatic interaction between phosphate groups and sPNAG should be sensitive to increasing concentrations of divalent cations . As shown in Table S3 , response to sPNAG is lost in Ca2+ concentrations higher than 100 µM , which could be considered as an additional support for our electrostatic interaction model . However , changing calcium concentration might also change cell viability . Exposure to this concentration of calcium , however , did not have any effect on the viability of the cells , as measured by viable counting and CFU determination . Identical results were obtained when the experiment was repeated with other divalent cations ( manganese or magnesium ) . Based on the microarray , transposon insertions in genes involved in regulation of cell shape and morphology should also interfere with sPNAG-base biofilm formation . However , since all the genes in this category are essential , we could not introduce those deletions into the wild-type background and check their phenotype . Transposon insertion events in this operon presumably occurred in either regulatory regions ( e . g . promoter ) or dispensable parts of essential genes . As could be inferred from the data , there was no significant correlation between capacity of producing PNAG and responding to it . However , in order to confirm this , we studied sPNAG-induced biofilm formation phenotype of all four ΔcsrA Δpga double-mutants ( ΔcsrA pgaA::kan , ΔcsrA pgaB::kan , ΔcsrA pgaC::kan , and ΔcsrA pgaD::kan ) and also all four Δpga single-mutants and found it to be indistinguishable from that of wild-type cells . We were curious to know whether deletion of rfaY , rfaQ , or rfaP abolished the response to sPNAG due to downstream signaling events or the phenotype was a simple consequence of the structural modifications imposed on LPS . Therefore , we decided to systematically identify extra-genic suppressors which can restore biofilm formation capacity of ΔrfaY cells and test whether there are any known or putative signaling pathway components among such suppressors . To identify suppressors of rfaY deletion , a transposon insertion library was generated in the ΔrfaY background ( i . e . a strain with clean deletion of rfaY ORF ) , and enriched for double mutants that recovered their ability to form a biofilm in the presence of sPNAG ( opposite to what was demonstrated in Figure 3 ) . A glass slide was provided as the biofilm formation surface and at the end of each round , the slide was transferred to a new container with sPNAG and fresh media . After four rounds of enrichment , macroscopic microcolony structures could be detected on the glass slide . The transposon insertion sites in the enriched population were mapped by the same footprinting strategy described previously ( Dataset S2 ) . Surprisingly , transposon insertions in many of the LPS biosynthetic genes were significantly enriched in this selection ( Figure 5A ) To validate our microarray predictions , we generated in-frame deletions of the candidate genes in the ΔrfaY background and studied their behavior in the presence of sPNAG ( Table S4 ) . We found that deletion of rfaC , rfaF , rfaI , pgm , galU , rfaH , rfbD , rfbC , and adiY reverted the phenotype of ΔrfaY cells . rfaC , rfaF , rfaI , pgm , rfbC , rfbD , and galU are involved in the synthesis of LPS core structure or its precursors ( Figure 5B ) . rfbC and rfbD are involved in rhamnose biosynthesis which is a component of the second major LPS glycoform in E . coli K12 [26] , which is not shown in Figure 5B . rfaH is a transcription anti-terminator which is required for full-length transcription of long operons , including rfaQ-K operon [27] . adiY is the positive regulator of the arginine decarboxylase system and will be discussed later . Overall , these mutants make truncated versions of LPS , an expectation we verified for a subset of them ( Figure 5C ) . We hypothesized that in these truncated structures , inner phosphate groups of lipid A or possibly other negatively charged cell-surface moieties ( which tend to be buried by longer LPS chains in the wild-type cells ) are now more exposed and available for interaction with sPNAG . Deleting any of the four genes in the pga operon did not restore the biofilm formation capacity of the ΔrfaY cells , as was also inferred from the microarray data . We also used fluorescence microscopy to characterize the dynamics of biofilm formation in a heterogeneous population composed of cells either capable ( represented by ΔrfaY ΔrfaF cells ) or defective ( represented by ΔrfaY cells ) in biofilm formation . To this end , ΔrfaY cells , expressing RFP fluorescent marker , were competed against ΔrfaY ΔrfaF cells expressing GFP with the starting ratio of 1∶1 for making a biofilm on a glass slide in the presence of sPNAG . After 12 hours , the biofilm structure formed on the glass slide was visualized by fluorescence microscopy . As shown in Figure 5D , top row , microcolonies in biofilm structure were mostly formed by ΔrfaY ΔrfaF double mutants ( i . e . GFP expressing cells ) . The same result was obtained by swapping the fluorescent labels ( Figure 5D , bottom row ) . Transposon insertions typically lead to a loss of function phenotype . In order to complement our transposon insertion based approach , we used an over-expression library in the ΔrfaY background . This library contained ∼2 . 5×105 independent mutants each carrying a 1–3 kb long genomic fragment of E . coli cloned into the pBR322 plasmid . The over-expression library was enriched for mutants responding to sPNAG , similar to the approach used for studying the ΔrfaY transposon insertion library . After four rounds of enrichment , the over-expressed fragments represented in the enriched population were identified by microarray hybridization ( Dataset S3 ) . As expected , when comparing the results from ΔrfaY transposon insertion library with ΔrfaY over-expression library , LPS biosynthesis genes showed the opposite behavior ( Figure 6A ) . Many LPS biosynthetic genes like rfaG , rfaJ , rfaI , rfaP , kdtA , rfbX , rfaZ , and rfaL were found to be among the top 10% highly enriched category in the ΔrfaY background transposon insertion library while in the over-expression library , they belonged to the top 10% most depleted group . The microarray results also revealed that mutants over-expressing certain genes associated with the acid tolerance system in E . coli were abundant in the enriched population . In order to clarify how this system contributes to biofilm formation , we further characterized the phenotypic consequences of over-expressing these genes in individual cells . To this end , we isolated individual clones from the enriched population . One of the isolated mutants was found to have the genomic region corresponding to three genes , gadW , gadY , and gadX . GadX and GadW are dual regulators of the glutamate-dependent decarboxylase acid-resistance system of E . coli . [28] . GadY is a small RNA which acts as a positive regulator of gadX [29] . Our previous results demonstrated that LPS modification was a dominant mechanism in regulating response to sPNAG . Therefore , we first investigated the effect of gadXYW over-expression on LPS structure . As shown in Figure 6B , ΔrfaY pBR322-gadWYX strain contained some smaller LPS variants as compared to the parental ΔrfaY cell . This suggests that the reversion of ΔrfaY phenotype upon over-expression of gadWYX gene cluster is a consequence of this truncated LPS structure . To test whether this change in LPS structure was due to a transcriptional regulatory event , we measured the transcription of the rfaQ-K operon in both pBR322-gadWYX and pBR322 ( empty vector ) backgrounds by a β-galactosidase assay and found it to be almost 3-fold lower in the gad-over-expressing cells ( Figure 6C ) . Evidence regarding the existence of a cross-talk between acid tolerance system and LPS regulation has been observed before , and gadE , the transcriptional regulator of the acid resistance system in E . coli , was reported to be a potential activator of the rfaQ-K operon [30] . We also found that over-expression of gadY alone was sufficient to cause the phenotype , although not as strongly as the over-expression of gadXYW . A strain harboring the pBR322-argR plasmid was also isolated from the enriched ΔrfaY library . However ArgR , the negative regulator of arginine biosynthesis system , acts as a weak suppressor of ΔrfaY biofilm formation deficiency . Putting all these observations together , four of the suppressors found in the transposon insertion and over-expression libraries , adiY , gadX , gadW , and argR , were directly or indirectly associated with the amino acid decarboxylase systems , involved in acid tolerance in E . coli . The biological function of these suppressors ( Table S5 ) and the distribution of acid tolerance genes in the over-expression library ( Figure S5 ) suggest that down-regulation of the acid stress response , or more specifically the amino acid decarboxylase systems , positively contribute to biofilm formation in the ΔrfaY background , presumably due to the changes imposed on LPS . Overall , our observations support the existence of a physical interaction between sPNAG and LPS . As such , biofilm formation in the presence of sPNAG may be a purely structural phenomenon , occurring as a simple consequence of passive interactions between sPNAG and LPS . Based on this model , even dead cells with intact outer membrane structure should still be capable of responding to sPNAG . To test this , we killed wild-type cells by either UV-irradiation or exposing them to formaldehyde , and visualized their behavior upon exposure to sPNAG . Time-lapse microscopy showed that these dead cells start nucleating microcolony structures , similar to living cells ( Video S3 ) . All together , our observations argue that sPNAG-mediated biofilm formation can be considered as a two-step process , starting with the nucleation event which is a purely structural phenomenon , followed by microcolony expansion and maturation which is a growth-dependent process ( Figure 1 , compare iv and vii to v and vi ) . Most natural and clinical isolates of E . coli produce different serotype-specific surface structures including O-antigen and capsular polysaccharide , also known as K-antigen , which are absent in E . coli MG1655 [31] , [32] . We were curious to know how variations in composition of these surface antigens might affect the response to sPNAG . Therefore , we chose 11 strains , which were reported to be competent of endogenous PNAG production [8] , for further analysis . Among these strains , 7 formed biofilms in the presence of sPNAG ( Table S6 ) , and two of the latter group were also O-antigen− ( Figure S6 ) . In case of K-antigen , we focused on K1 capsule , a homopolymer of α- ( 2-8 ) -linked polysialic acid [33] , which is the predominant capsule found in a major subset of these clinical isolates . Three K1+ isolates used in this study were also capable of responding to sPNAG ( Table S6 ) . Natural and clinical isolates of E . coli possess uncharacterized surface structures other than K1 , e . g . fimbriae and other capsular polysaccharides . These structures could significantly affect the physicochemical properties of the cell surface . Consequently , cell response to sPNAG in these clinical isolates could not be solely judged based on their O-antigen structure or presence of K1 capsule . Furthermore , the limited number of strains tested in this study and their non-isogenic background make these observations preliminary and they should be followed up with future studies . Therefore , we decided to study the role of O- and K-antigen in the well-characterized K-12 background . We generated all possible combinations for presence or absence of O16 antigen and K1/K92 capsule in the K-12 background . K92 is a polysialic acid capsule very similar to K1 , and its biosynthetic gene cluster can be transferred on a plasmid . As shown in Table 1 , only non-capsulated O16+ cells were impaired in sPNAG-based biofilm formation . In the presence of both O16 and K1/K92 antigen , however , cells were capable of responding to sPNAG , which is not surprising considering that capsule is a more exposed surface structure than O-antigen [32] . Since K1 and K92 capsules confer a high density of negative charge to the E . coli cell surface , their presence could contribute to establishing any potential electrostatic interaction with sPNAG . These data suggest that loss of O-antigen ( O16 ) or presence of K1 capsule is associated with sPNAG-induced biofilm formation in the E . coli K-12 . However , in order to confirm the involvement of these structures in sPNAG-induced biofilm formation , targeted genetic experiments together with more careful characterization of their role in physiochemical properties of outer membrane are required . Biofilms can afford protection from a variety of environmental challenges including phagocytosis , extreme pH , and antibiotic exposure [1] . We were curious to characterize some of these biofilm-specific features in sPNAG-based biofilms . In order to investigate their antibiotic tolerance , we challenged the cells with two different antibiotics: ampicillin and polymyxin B . As shown in Figure 7 , cells in the context of sPNAG biofilm showed ∼10 fold higher tolerance to polymyxin B compared to planktonic cells , whereas no significant difference was observed in tolerance to ampicillin . Polymyxin B resembles antimicrobial peptides , an integral component of the innate defense system in many organisms , in terms of both structure and mechanism of action [34] . Considering sPNAG as a positively charged matrix encasing the cells , electrostatic repulsion between this polysaccharide and polymyxin B could potentially protect the bacteria by reducing the local concentration of the drug in the vicinity of the cells . However , we still consider the involvement of other as yet unknown sPNAG-induced physiological response in this phenomenon . Considering other biofilm-specific phenotypes , we found no significant change in the rate of F-plasmid conjugation in sPNAG-based biofilms ( Figure S7 ) . We also did not observe any evidence supporting the existence of a phase-variable mechanism regulating sPNAG production in E . coli ( Figure S8 and Figure S9 ) . We were also curious whether sPNAG can induce biofilm formation in species other than E . coli , because this could potentially facilitate interspecies interactions , e . g . conjugation , between E . coli and other microorganisms . Therefore , we exposed Salmonella typhimurium LT2 cells to sPNAG similar to what was done for E . coli , but we did not observe any detectable biofilm formation . We reasoned that if sPNAG-mediated biofilm formation has a structural basis , then a mutant Salmonella with modified surface characteristics may be capable of forming a biofilm in the presence of sPNAG . To test this hypothesis , we applied sPNAG to a transposon insertion library of S . typhimurium LT2 with approximately 105 mutants and enriched for mutants that form biofilms . After four rounds of enrichment , macroscopic microcolony structures were formed by cells . We mapped the transposon insertion sites in 4 of the mutants capable of forming biofilms in a sPNAG-dependent pattern . In all cases , the transposon mapped to different positions in the rfaK gene , involved in LPS biosynthesis . Salmonella rfaK mutants are lacking most of the O-antigen structure [35] , providing further support , beyond E . coli , for our physical interaction model between sPNAG and LPS .
Poly-N-acetylglucosamine , the major virulence factor of Staphylococcus epidermidis , has recently been found in many other pathogenic bacteria [7] , including E . coli , but PNAG-based biofilms in these pathogens are poorly characterized relative to Staphylococcus species [2] . Here , we carried out a systematic genetic analysis of poly-N-acetylglucosamine-induced biofilm formation in E . coli . However , instead of working in a biofilm-permissive genetic background , in which the time scale of biofilm formation is slow , we applied the functionally active secreted version of PNAG ( sPNAG ) to wild-type E . coli MG1655 cells and observed rapid and reproducible biofilm formation . The fast kinetics and robustness of sPNAG-induced biofilm formation phenomenon allowed us to comprehensively characterize its underlying genetic basis . Our observations support the notion that electrostatic interaction between positively charged sPNAG and different cell surface antigens with negative charge is responsible for the formation of the biofilm structure . This is consistent with the generally accepted intuition that physicochemical properties of the matrix , including its charge , geometry , ion selectivity , and pore size contribute significantly to biofilm formation [1] , [18] . The composition and physicochemical properties of cell surface structures can be modulated by multiple biological pathways and environmental factors . Therefore , although response to sPNAG seems to be the consequence of simple electrostatic interactions , it can be regulated by a complex interplay between LPS structural dynamics , the presence of serotype-specific capsular polysaccharide , the acid tolerance system , and cell morphology as shown in this work . There are also other relevant pathways , not fully active in our laboratory strain , that might contribute to this phenotype , such as addition of positively charged 4-amino-4-deoxy-L-arabinose to lipid A involved in polymyxin B and other cationic antimicrobial peptide tolerance in E . coli and S . typhimurium . Interestingly , a polymyxin B resistant mutant isolated from our transposon insertion library was also impaired in responding to sPNAG ( data not shown ) . Since resistance to polymyxin is usually concomitant with higher density of positive charge on the outer membrane [36] , polymyxin resistant mutants are expected to be defective in interacting with sPNAG . Given that antimicrobial peptides generated by the host immune system are a major challenge for the survival of pathogens , encasement in a positively charged matrix or biofilm serves as a protective mechanism against antimicrobial peptides for sensitive cells , while resistant cells can survive without it . Since in this study , selections were performed with pools of mutants rather than with clonal populations , phenotype of different mutants should be interpreted as a spectrum of different capacities for forming a biofilm rather than a simple binary phenomenon . The results of our selection , carried out on the over-expression library in the ΔrfaY background , strongly support this notion . ΔrfaY cells are defective in responding to sPNAG , so it is reasonable to assume that only a handful of genes in the over-expression library should be capable of suppressing its phenotype and the majority of the library mutants should be equally defective in biofilm formation . However , the fact that a considerable fraction of genes involved in LPS biosynthesis were significantly depleted in that selection ( Figure 6A ) indicates that even ΔrfaY cells might have residual biofilm formation activity and that over-expression of some LPS biosynthetic genes might reduce this partial activity . The weak biofilm formation activity of ΔrfaY cells could be due to their entrapment in the microcolony structures formed by other mutants in the population . This is in part shown in Figure 5D in which ΔrfaY cells are mostly colocalized with microcolonies rather than being uniformly distributed . Imposed modifications in LPS structures caused by over-expression of some LPS biosynthetic genes [37] might interfere with this partial activity . In this study , we have demonstrated the involvement of different surface structures and regulatory systems in sPNAG-mediated biofilm formation . However , there might exist a myriad of other uncharacterized regulatory systems , surface structures , and environmental factors that may influence this phenotype through changes in the physicochemical properties of the cell surface . Covalent modifications of lipid A with phosphoethanolamine in a Ca2+-dependent manner , or with 4-amino-4-deoxy-L-arabinose in response to Mg2+ and pH , represent just a few examples [38] . Therefore , even if a natural isolate of E . coli is incapable of responding to sPNAG in the laboratory environment , it may show a different phenotype in its natural environment . There is also the possibility of higher level cooperation between different cell types in the population , with one subpopulation producing sPNAG while another is responding to it . The producer subpopulation may even be defective in the initiation of the biofilm formation process , but could be incorporated into preformed structures . Examples of population heterogeneity in biofilm communities has been reported before [1] . Furthermore , it is likely that different cells produce different versions of the PNAG polymer , with different degrees of acetylation [39] or different polymer length distributions , which could make response to sPNAG more strain-specific . A better understanding of sPNAG-based biofilms acquired from studies like this could be useful for development of new therapeutic strategies against pathogens that use this polysaccharide as a virulence factor . The systematic framework presented in this study , along with the acquired insights , should also benefit the study of microbial biofilms formed by other species .
All strains used in this study are listed in Table S7 , bacteriophages and plasmids are mentioned in Table S8 . All the experiments were performed in LB ( 1% tryptone , 0 . 5% yeast extract , 1% NaCl ) , supplemented as required with the following antibiotics: ampicillin , 50 µg/ml; tetracycline , 25 µg/ml; spectinomycin , 100 µg/ml; chloramphenicol , 30 µg/ml and kanamycin , 100 µg/ml , unless otherwise mentioned . β-galactosidase measurements were performed in triplicate as described before [40] . Transposon mutagenesis and microarray based genetic footprinting were carried out as described before [22] . Chromosomal deletions were created using the previously described method [23] and transferred by generalized transduction with P1 phage as required . lacZ reporter strain was generated using the plasmid pCE37 [41] . The over-expression plasmid library construct was a kind gift of Joseph Sklar . Each plasmid contained a 1–3 kb long fragment of E . coli MC4100 genome ( average fragment size of 2 kb ) cloned into the β-lactamase coding sequence of the pBR322 vector . The plasmid pool was electroporated into E . coli MG1655 ΔrfaY cells , leading to ∼2 . 5×105 independent over-expressing mutants . For PCR amplification of DNA constructs for cloning , Pfu Ultra polymerase ( http://www . stratagene . com ) was used , whereas ExTaq DNA polymerase ( http://www . takara-bio . com ) was used for all other PCR reactions . Restriction endonucleases and T4 ligase were obtained from New England Biolabs ( http://www . neb . com ) . DNA purification kits were obtained from QIAGEN ( http://www1 . qiagen . com ) . Primer sequences are available upon request . Structural analysis of LPS samples was performed as described before [42] . LPS was purified from cell lysates after phenol-ether extraction , separated on 14% tricine-SDS-PAGE gel and visualized after silver staining . All fluorescence and light microscopy experiments were performed using Zeiss AxioVision 4 . 5 . Time-lapse microscopy was performed in the FC81 flow cell apparatus from Biosurface Technologies Corp ( in the absence of flow ) . SEM analysis was performed using a Philips XL30 Field Emission Scanning Electron Microscope . For the sake of consistency , a large batch of ΔcsrA spent media , grown in LB , was prepared and used as the source of sPNAG for all the experiments throughout this study . The amount of sPNAG present in this stock solution , was estimated by measuring its hexosamine content using 3-methyl-2-benzothiazolone hydrazone hydrochloride ( MBTH ) method [43] and found to be ∼0 . 175 mg equivalent of hexosamine per ml of saturated ΔcsrA culture supernatant . The quantity of sPNAG in each milliliter of this stock solution was defined to be 1 arbitrary unit ( U ) . Control experiments were also carried out using sPNAG from different preparations of ΔcsrA spent media and the results were reproducible . Spent media from 1 liter of saturated ΔcsrA culture in LB was passed through a 0 . 22 µ filter ( http://www . nalgene . com/ ) . The cell-free supernatant was concentrated 200 fold by Centriplus YM-100 columns ( http://www . millipore . com ) . The concentrated sample was treated with DNase I ( 2 mg ) , RNase A ( 10 mg ) , and α-amylase ( 20 mg ) and incubated for 2 hours at room temperature followed by 2 hours at 37°C . Next , sample was treated with Proteinase K ( 20 mg ) for 1 hour at 37°C and 1 hour at 55°C . Enzymes and other proteins were removed by pre-warmed ( 55°C ) phenol: ether extraction , followed by ethanol precipitation . The precipitate was re-suspended in water and fractionated on a S-300 Sephacryl column . The fractions with biofilm-inducing activity were pooled together and concentrated by Centriplus YM-100 columns . The sample was treated with Dispersin B ( 20 µg ) for 4 hours at 37°C . After removing the enzyme by phenol: chloroform extraction , the sample was dialyzed with Spectra/Por cellulose ester membranes with MWCO = 500 ( http://www . SPECTRUMLABS . com/ ) to remove the salt present in Dispersin buffer . Finally , the sample was analyzed by an ESI-LTQ Orbitrap Hybrid mass spectrometer from Thermo Fisher Scientific . For all biofilm formation assays , cells and sPNAG were added to fresh LB in such a way as to obtain ∼5×108 cells and 0 . 1 U of sPNAG per milliliter of the mixture . Biofilm structures were studied or transferred ( in case of serial enrichments ) 12 hours after exposure to sPNAG at 25°C . Plasmids from ∼109 cells of the enriched population ( or in case of references , from the maximally diverse unselected library ) were extracted using QIAGEN plasmid miniprep kit . The extracted plasmid pool was amplified with two separate PCR reactions , using primer pair pBR_Lib_T7_L and pBR_Lib_R or pBR_Lib_T7_R and pBR_Lib_L . The sequences of these primers are as follows: The incorporated T7 promoter in pBR_Lib_T7_L and pBR_Lib_T7_R is underlined . Cycling conditions for PCR were 94°C for 2 min; 30 cycles of 94°C for 30 s , 55°C for 30 s , and 72°C for 5 min; and 72°C for 10 min , using ExTaq DNA polymerase . The PCR products from these two reactions were pooled together . The T7 promoter incorporated into the pBR_Lib_T7_L and pBR_Lib_T7_R primer sequences was used to generate RNA from the pooled PCR product in an in vitro transcription reaction using Ambion MEGAscript T7 Kit ( http://www . ambion . com ) . Finally the RNA from the previous reaction was reverse transcribed to cDNA , using Cy3-labeled nucleotides with Invitorgen SupreScript II Reverse Transcriptase ( http://www . invitrogen . com ) . The fluorescently labeled cDNA was used for microarray hybridization versus Cy5-labeled fragmented ( nebulized ) MG1655 genomic DNA . The hybridization data of the two maximally diverse unselected libraries were used as the reference . Production of PNAG in S . epidermidis could be discriminated on congo red indicator plates [44] . In order to extend this to E . coli , congo red indicator plates ( 3% tryptic soy broth , 1% glucose , 0 . 08% congo red , and 1 . 5% agar ) were supplemented with 200 mM NaCl which was reported to enhance pga locus transcription in E . coli [16] . Different dilutions of stationary phase cultures of ΔcsrA mutants were plated on these plates and incubated for two days at 37°C . PNAG-producing ΔcsrA cells formed dark brown colonies whereas ΔcsrA mutants defective in producing PNAG were red . To map the location of the mutation which abolished PNAG production , red colonies were picked individually and transduced with a P1 phage lysate obtained from the maximally diverse transposon insertion library . Kanamycin resistant transductants which recovered their PNAG production were identified by replica plating on congo red plates . Dark colonies were picked , and the location of transposon insertion , which should have been linked to the mutation site , was mapped using the same footprinting strategy . The exact location of the mutation was determined after PCR amplification of the candidate genomic locations and subsequent sequencing of the PCR product .
|
Both in the wild and in the clinical setting many bacterial species live within surface-attached communities called biofilms . It is still unclear the extent to which the biofilm lifestyle and its associated phenotypes , such as hyper-tolerance to antimicrobial agents , can be attributed to structural characteristics of the biofilm community or to intrinsic biofilm-specific physiological programs . In order to address this longstanding question , we focused on poly-N-acetylglucosamine ( PNAG ) –based biofilms , a clinically relevant phenotype of many bacterial pathogens , including E . coli . Instead of working in a biofilm-permissive genetic background , in which the timescale of biofilm formation is slow , we applied the functionally active secreted version of the PNAG exo-polysaccharide ( sPNAG ) to wild-type E . coli cells , generating robust biofilms on the timescale of hours . In this way , we managed to uncouple upstream regulatory processes and matrix preparatory phase of biofilm formation , focusing specifically on the latter part . By using a powerful genome-wide technology , we identified the genes and pathways involved in sPNAG-based biofilm formation . Our results revealed that structural interactions between sPNAG and surface structures such as lipopolysaccharides are the crucial determinants of biofilm formation and that multiple pathways including acid-tolerance , capsule biosynthesis , and regulation of cell morphology modulate this phenotype .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/bacterial",
"infections",
"microbiology/microbial",
"growth",
"and",
"development",
"genetics",
"and",
"genomics",
"microbiology"
] |
2009
|
Genetic Dissection of an Exogenously Induced Biofilm in Laboratory and Clinical Isolates of E. coli
|
Dengue appears to be endemic in Africa with a number of reported outbreaks . In February 2013 , several individuals with dengue-like illnesses and negative malaria blood smears were identified in Mombasa , Kenya . Dengue was laboratory confirmed and an investigation was conducted to estimate the magnitude of local transmission including a serologic survey to determine incident dengue virus ( DENV ) infections . Consenting household members provided serum and were questioned regarding exposures and medical history . RT-PCR was used to identify current DENV infections and IgM anti-DENV ELISA to identify recent infections . Of 1 , 500 participants from 701 households , 210 ( 13% ) had evidence of current or recent DENV infection . Among those infected , 93 ( 44% ) reported fever in the past month . Most ( 68 , 73% ) febrile infected participants were seen by a clinician and all but one of 32 participants who reportedly received a diagnosis were clinically diagnosed as having malaria . Having open windows at night ( OR = 2 . 3; CI: 1 . 1–4 . 8 ) , not using daily mosquito repellent ( OR = 1 . 6; CI: 1 . 0–2 . 8 ) , and recent travel outside of Kenya ( OR = 2 . 5; CI: 1 . 1–5 . 4 ) were associated with increased risk of DENV infection . This survey provided a robust measure of incident DENV infections in a setting where cases were often unrecognized and misdiagnosed .
Dengue is a mosquito-borne acute febrile illness caused by one of four dengue viruses ( DENV-1-4 ) . It is endemic throughout the tropics and sub-tropics [1 , 2] where more than 40% of the world’s population is at risk and approximately 400 million DENV infections are estimated to have occurred in 2010 alone [3] . While the majority of infections are asymptomatic or result in a mild febrile illness , about one-quarter of infected people have signs and symptoms consistent with dengue or severe dengue [3 , 4] . Infection with any DENV type may result in dengue , an illness characterized by fever , headache , retro-orbital eye pain , myalgia , arthralgia , minor hemorrhagic manifestations , and rash [4] . Although most dengue patients will recover within one week , 5 to 10% of patients in endemic areas will progress to severe dengue , which includes dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) , and is characterized by thrombocytopenia , plasma leakage due to increased vascular permeability , severe organ involvement , and/or clinically significant bleeding [5–8] . Of severe dengue patients , 0 . 1–10% will not survive , which is dictated in part by the timing and quality of medical care . Medical care with appropriate intravascular volume repletion and supportive intensive care has been shown to lower mortality associated with severe dengue [4 , 9 , 10] . Dengue is likely endemic throughout much of Africa where the mosquito vector is widely present [3] . However , from 1960–2010 , only 22 of 52 African countries reported sporadic cases or dengue outbreaks because of a general lack of awareness of the disease , surveillance or availability of diagnostic testing [11] . Although recent estimates suggest a substantial disease burden in Africa with an estimated 65 million annual DENV infections , they also demonstrated the uncertainty of the geographic distribution of DENV transmission and disease [3] . Reports of dengue occurrence from Africa constitute only a small percentage ( <4% ) of all globally reported disease and only a single country in Africa has reported cases to the World Health Organization ( WHO ) [3] . In countries such as Kenya , where no dengue cases were reported from 1986 to 2013 , population-based studies are needed to better understand the epidemiology and true incidence of disease and infection [11–18] . In February–March 2013 , 71 patients with an acute febrile illness and negative malaria blood smears were identified in Mombasa , Kenya , and reported to the National Ministry of Health ( MOH ) . Nearly half ( 46% ) were confirmed to have an acute DENV infection by real-time , reverse transcriptase-polymerase chain reaction ( RT-PCR ) . The Kenya MOH in collaboration with the Centers for Disease Control and Prevention ( CDC ) Center for Global Health , CDC Dengue Branch , and Kenya Medical Research Institute ( KEMRI ) responded by conducting an outbreak investigation that included establishing enhanced hospital surveillance in Mombasa followed by a serological survey of the population in Tudor district , an area of initial case detection , in order to provide an estimate of local dengue incidence and identify risk factors for DENV infection .
Mombasa , the second largest city in Kenya , has an approximate population of 939 , 370 residents and is a major port and international tourist destination in East Africa [5] . Hospital surveillance was established in Mombasa , which is comprised of six constituencies and thirty districts . Tudor district of Mombasa was selected as the site for a seroincidence survey because initial dengue cases were detected from area residents . The Tudor district is located at 4°2′22″S 39°39′48″E and has a population of approximately 10 , 000 residents [5] . Hospital surveillance activities , which included active case finding and chart reviews , were performed to follow temporal dengue trends in Mombasa and determine the effectiveness of control activities . Enhanced surveillance for dengue-like acute febrile illnesses was initiated at seven hospitals: three public ( Tudor Hospital , Coast Province General Hospital , Port Reitz District Hospital ) and four private ( Aga Khan Hospital , Mombasa Hospital , Pandya Memorial Hospital , Bomu Hospital ) . Physicians , nurses , and laboratory staff at these hospitals were trained in clinical recognition and management of dengue , and asked to submit serum specimens and patient information from suspected dengue cases . Suspected case-patients were defined as patients with an acute febrile illness ( i . e . , fever ≥38°C for ≤7 days ) , absence of cough , a negative malaria blood smear , and at least one additional sign or symptom consistent with dengue including retro-orbital pain , headache , rash , myalgia , arthralgia , leucopenia , or a hemorrhagic manifestation . Dengue diagnostic testing was performed as described below ( see Laboratory Diagnosis ) . A sample size of 1 , 500 individuals was calculated based on an assumed prevalence of recent DENV infection of approximately 4% , an absolute error of +/- 3% , and a design effect of two to account for intra-household clustering . Assuming that an average of 1 to 2 persons per participating household would consent to provide a blood specimen , 986 households were randomly selected for enrollment in 5 geographic areas within Tudor district . The areas were selected based on their population density , housing type , and socioeconomic diversity . The primary sampling unit for the household survey was a housing plot . In four of the five areas , housing plots were enumerated using a combination of a local and Google Earth map ( Google , Inc . ) , and a random number generator ( R v2 . 15 . 1 ) was used to select housing plots for inclusion . In one area , each plot contained a single house and no subsampling was performed . In three areas , multiple households were located on a single plot and random sampling of households was carried out by enumeration of households with subset selection using a random digits table carried by the field teams . The fifth area contained high density housing in which housing plots could not be identified; field teams were instructed to use the random digits tables to select distances and direction to households . All selected households were visited from May 3–11 , 2013 by a team consisting of one community health worker , village elder , interviewer , and a phlebotomist . Unoccupied homes on the first visit were revisited a single time , and households that did not agree to participate in the investigation were not replaced . After the head of household agreed to participate , all household members were asked to participate . Consenting household members were asked to complete a questionnaire that inquired about demographics , recent illness , travel history , and potential risk factors for DENV infection . In addition , one adult per consenting household also completed a questionnaire to obtain household demographics , recent history of sick household members , house construction , use of windows and/or screens , and presence of potential mosquito breeding containers in their yard . Religion was asked due to differences in clothing body coverage between the different religious groups . Responses to the household and individual questionnaires were linked to each participating individual for data analysis . All household members were asked to provide a 10 mL venous blood specimen which was allowed to clot in cold-pack refrigerated field containers; serum was separated in a Mombasa field laboratory within 4 hours of collection , stored and transported at 4°C to KEMRI where it was then stored at -80°C until tested . Post hoc weighting of individuals within households was performed if all individuals within a household did not participate in the survey . Confidence intervals ( CI ) accounted for sampling design and finite population correction factors . Logistic regression was used to assess risk factors for DENV infection , and resulting inferences accounted for the sampling design . Tests were performed with an overall Type I error of α = 0 . 05 . All analyses were performed in R v2 . 15 . 1 . All specimens for both hospital surveillance and the seroincidence survey were tested by enzyme linked immunosorbent assay ( ELISA ) to detect IgM anti-DENV antibodies ( InBios International , Inc . , Seattle , WA ) [19–21] , and by a previously described multiplex DENV-type specific , real-time RT-PCR assay to detect DENV nucleic acid [22–24] . A current DENV infection was defined by detection of DENV nucleic acid by RT-PCR . A positive IgM anti-DENV ELISA result was defined by an index value >2 . 84 and was considered evidence of a recent DENV infection . An IgM ELISA result between 1 . 65 and 2 . 84 was indeterminate . All indeterminates were retested , and if it was indeterminate upon repeat testing , it was classified as negative . The investigation protocol was reviewed by human subjects review experts from the Institutional Review Boards ( IRB ) at CDC and KEMRI . CDC review determined the activity was consistent with a non-research public health response . A full IRB review of the protocol was required by KEMRI and approval was obtained . Informed written consent for survey participation and blood collection was obtained from all adult participants 18 years of age and older . Verbal assent and written consent from parents/guardians was obtained from minors ( children 6–17 years old ) and written consent from parents was obtained for children ≤ 5 years .
A total of 267 suspected dengue cases were detected at hospitals from January–May , 2013 , of which 155 ( 58% ) were confirmed to have a current DENV infection and included a single fatal case . The RT-PCR positive cases ranged in age from 3 to 75 years and all cases were residents of coastal areas of Mombasa . Identified DENV types were DENV-1 , DENV-2 , and DENV-3 ( Fig 1 ) . Of 986 randomly-selected Tudor households , 701 ( 71% ) households including 1 , 500 individuals participated in the seroincidence survey ( median = 2 participants per household [range: 1–11] ) . Median age of study participants was 28 years ( range: 0 . 1–94 ) , and most were Christian ( 67% ) and female ( 60% ) , and thus similar to Mombasa’s population [5] . In total , 210 ( 13%; 95% confidence interval [CI] = 10–16% ) participants had evidence of DENV infection with 101 current infections , including 12 participants who were both RT-PCR and IgM positive , and 109 recent DENV infections . Of the 101 RT-PCR positive participants , DENV-1 and -2 were detected in 51 ( 50% ) and 48 ( 48% ) of all cases , respectively , and two ( 2% ) DENV-1 and DENV-2 co-infections were detected . Participants with evidence of current or recent DENV infection were distributed throughout the Tudor district , and there was no statistically significant clustering by area ( Fig 2 ) . There was no significant difference in infection rate between study areas within the Tudor district . The median age of the 210 DENV infected participants was 27 years ( range: 2–86 ) , the majority were female ( 60% ) , and Christian ( 66% ) . Nearly half , 44% ( 95% CI = 39%–57% ) of the 210 infected participants recalled having a fever in the 30 days before being interviewed , and 68 ( 73% ) of these febrile participants reported being seen by a clinician for their illness . Approximately half ( 47% ) of these 68 participants reported receiving a diagnosis , and all but one reported being diagnosed by their doctor with malaria while one of the patients was correctly diagnosed with dengue . Three ( 4% ) febrile infected participants were hospitalized , and two ( 1% ) reportedly had bleeding manifestations . Participants with current or recent DENV infection were nearly three times more likely to report fever in the 30 days before being interviewed than non-infected participants ( OR = 2 . 8; CI = 1 . 9–4 . 2 ) . Participants that reported bruising were more likely to have a current or recent DENV infection ( OR = 6 . 3; CI = 1 . 4–33 . 0 ) ( Table 1 ) . No other signs or symptoms were significantly associated with having a current or recent DENV infection . Risk factors significantly associated with having evidence of current or recent DENV infection included report of having windows open at night ( OR = 2 . 3; CI = 1 . 1–4 . 8 ) , travel outside of Kenya in the past month ( OR = 2 . 5; CI = 1 . 1–5 . 4 ) , and failure to use mosquito repellent daily ( OR = 1 . 6; CI = 1 . 0–2 . 8 ) ( Table 2 ) . Of those 12 participants who reported travel outside of Kenya in the month before being interviewed , 8/12 ( 73% ) reported their travel destination and 7/8 ( 88% ) had traveled to Tanzania , an area experiencing a current dengue outbreak . Of 701 participating households , the most common self-reported water containers that could serve as mosquito breeding sites in the yard were buckets ( 47% ) , septic tanks ( 40% ) , and water cisterns ( 31% ) .
Our survey estimated that 13% of residents of Tudor Mombasa , Kenya were infected with DENV during the 2013 dengue outbreak , or 13 , 782 DENV infections per 100 , 000 residents . Of those infected , 44% recalled having a recent acute febrile illness . While few participants reportedly required hospitalization , the majority of febrile DENV infected participants were sick enough to utilize outpatient healthcare services . Importantly , our survey found evidence of under recognition of dengue among the febrile , DENV infected participants who sought outpatient care . Similar to previous studies that found malaria being overdiagnosed in patients with acute febrile illness [25–29] , we found that among participants who could recall their diagnosis , all but one reported being diagnosed with malaria . While some of our DENV-infected participants could have been co-infected with malaria , previous studies have found the incidence of malaria/DENV co-infections to be about 3% [30–32] . In our survey , the majority of febrile DENV infected participants may have been misdiagnosed as having malaria . This is important because not only is clinician awareness essential to successful disease surveillance , but early clinical recognition of dengue allows the clinician to offer accurate anticipatory guidance and timely referral for lifesaving supportive inpatient care . These secondary prevention measures have been shown to reduce medical complications and mortality among severe dengue patients from 10 to <1% [4 , 9 , 10] . Little information is available regarding the incidence of dengue in Kenya as only one comparable seroincidence survey has been conducted during an ongoing outbreak , and disease surveillance has been limited [11–18] . Our attack rate was similar to that found ( 14% IgM anti-DENV positive ) during a dengue outbreak affecting refugee camps in neighboring Somalia almost 10 years earlier [33] , and comparable to the rate of recent dengue infection during an outbreak investigation in Haiti which used the same methods [34] . The only other serosurvey was conducted in western Kenya using banked , paired serum samples from 354 afebrile children aged 12–47 months [12] , which identified three anti-DENV IgG seroconversions in ~1 year period for an estimated incidence of 850 DENV infections per 100 , 000 persons . Regardless of the paucity of dengue incidence data , there is some evidence of dengue endemicity in at least the coastal areas of Kenya . Recent seroprevalence studies conducted during non-outbreak periods have found that ~34–53% of residents of coastal areas have evidence of prior DENV infection despite the absence of any reported outbreaks [12] . In comparison , only 1–8% of residents in non-coastal areas of Kenya have evidence of prior DENV infection; however these studies were conducted in the 1980-90s [17 , 18] . In areas where dengue surveillance does not exist , conducting seroincidence surveys to determine the prevalence of incident DENV infections during an apparent outbreak can be used to estimate attack rates , the extent of the outbreak and the burden of dengue [35 , 36] . Such an approach is now feasible because a single serum specimen can be tested by RT-PCR to detect a current DENV infection and by IgM anti-DENV ELISA to detect a recent DENV infection . This is in contrast to testing acute and convalescent specimens for IgM anti-DENV seroconversion , as was done in the past . In this outbreak , the serologic survey for incident DENV infections is especially useful for obtaining a “snapshot” of its magnitude . Recent experience using this method has shown that when combined with interview information , such surveys can be used to differentiate individuals with symptomatic or asymptomatic DENV infection , elucidate health care-seeking behaviors to estimate the degree of misdiagnosis , and identify risk factors for DENV infection . Although such serosurveys must be carefully planned to ensure representativeness of the population in question , when appropriately conducted they appear to be powerful tools to better understand and plan for the response to dengue outbreaks in areas with limited surveillance capacity . Several risk factors for DENV infection were identified during this investigation . First , having open windows at night was a plausible risk factor because female Aedes aegypti mosquitoes have been shown to enter houses at night to find resting sites , and then they may take a blood meal from household members the following morning [37–39] . Second , travel outside of Kenya , specifically to Tanzania where a simultaneous dengue outbreak was occurring , may have enabled importation of DENV into Mombasa and secondary cases . This observation indicates that even in dengue endemic areas , travel to other endemic areas may constitute a risk factor for infection and highlights the need for that prevention message [40] . Lastly , daily use of mosquito repellent was found to be protective against DENV infection . Education campaigns should encourage residents to use mosquito repellent and other mosquito avoidance strategies including wearing long sleeves and pants and using permethrin-impregnated clothing . Our investigation had several limitations . First , we made several assumptions as to the representativeness of the Tudor district when the site was selected for the seroincidence survey . Tudor district was chosen primarily because of its socioeconomic and demographic diversity , however it was also chosen because it was one of the most logistically accessible sites . Second , we were only able to estimate the incidence of DENV infection for a 90-day period prior to specimen collection based on our testing methods , which likely underestimated the true incidence of infection over the duration of the outbreak . Lastly , because there was no dengue surveillance before the outbreak and only limited surveillance after the seroincidence survey was conducted , it is unclear whether our estimates represented the beginning , peak or end of the outbreak and we were unable to directly compare these data to previous outbreaks . Our investigation provides additional support for high level DENV transmission in coastal Kenya and important evidence for the under recognition and misclassification of dengue cases . One of the strengths of our survey to detect incident DENV infections was that it was conducted immediately following the declaration of the outbreak , which may have allowed for better recall and higher participation rates . While this outbreak illustrates the need for establishing on-going dengue surveillance to detect outbreaks , there is also the need to use ongoing dengue surveillance to describe the epidemiology of dengue in East Africa .
|
Dengue appears to be endemic in Africa with a number of reported outbreaks . In February 2013 , several individuals with dengue-like illnesses and negative malaria blood smears were identified in Mombasa , Kenya . Dengue was laboratory confirmed and an investigation was conducted to estimate the magnitude of local transmission including a serologic survey to determine incident dengue virus ( DENV ) infections . Consenting household members provided serum and were questioned regarding exposures and medical history . RT-PCR was used to identify current DENV infections and IgM anti-DENV ELISA to identify recent infections . Of 1 , 500 participants , 13% had evidence of current or recent DENV infection . Among those infected , 44% reported fever in the past month . Most ( 73% ) febrile infected participants were seen by a clinician , and all but one of the 32 participants who reportedly received a diagnosis were clinically diagnosed as having malaria . Having open windows at night , not using daily mosquito repellent , and recent travel outside of Kenya were associated with increased risk of DENV infection . This survey provided a robust measure of incident DENV infections in a setting where cases were often unrecognized and misdiagnosed .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
A Household Serosurvey to Estimate the Magnitude of a Dengue Outbreak in Mombasa, Kenya, 2013
|
Insects that feed by ingesting plant and animal fluids cause devastating damage to humans , livestock , and agriculture worldwide , primarily by transmitting pathogens of plants and animals . The feeding processes required for successful pathogen transmission by sucking insects can be recorded by monitoring voltage changes across an insect-food source feeding circuit . The output from such monitoring has traditionally been examined manually , a slow and onerous process . We taught a computer program to automatically classify previously described insect feeding patterns involved in transmission of the pathogen causing citrus greening disease . We also show how such analysis contributes to discovery of previously unrecognized feeding states and can be used to characterize plant resistance mechanisms . This advance greatly reduces the time and effort required to analyze insect feeding , and should facilitate developing , screening , and testing of novel intervention strategies to disrupt pathogen transmission affecting agriculture , livestock and human health .
The invention of an electronic method for monitoring the feeding behavior of sucking insects [1–4] provided a potentially powerful tool to describe the cryptic behavior of the mouthparts of fluid-feeding phytophagous insects inside a host plant ( Fig 1 ) . Coupled with histological studies to correlate specific waveforms with the mouthparts’ position within the host [5 , 6] , electronic monitoring allows researchers to follow the sequence of events that lead to ingestion and , in the case of insect vectors , to acquisition and transmission of pathogens . The method , variously referred to as electronic feeding monitor or electrical penetration graph ( EPG ) , has been applied to various studies of host plant resistance and pathogen transmission [6–12] . It has also been used to characterize feeding by blood-feeding mosquitoes and ticks [11 , 13 , 14] . A major constraint to the utility of the method is the amount of time required to interpret the waveforms produced . Currently , a trained human observer is required to characterize each waveform and assign the corresponding feeding state on a second-by-second basis . During a typical experiment , EPG recordings generate gigabytes of data . Classification of these data into insect feeding states corresponding to intercellular passage , cell sampling , salivation , phloem ingestion , xylem ingestion and other activities associated with feeding or pathogen transmission is typically accomplished by comparison to published standards [6] . Computer classification methods based on motif recognition have been devised , but suffer from low accuracy [15] . Most analysis currently requires expert training and manual annotation that preclude high-throughput analysis . This onerous and time-consuming process is a major limitation to the broader and more in-depth application of this otherwise useful technique . We focused on removing the data analysis bottleneck through application of machine learning algorithms designed to teach a computer program to recognize and learn from insect feeding states with little or no human input [16] . To do so , we relied on EPG recordings from an insect-plant-pathogen model system where automated processing and analysis of insect feeding data could have an immediate and measurable impact on development of effective intervention strategies through screening of plant varieties resistant to pathogen transmission . In this system , the Asian citrus psyllid , Diaphorina citri ( Hemiptera: Liviidae ) transmits the phloem-limited and persistently propagated bacterium Candidatus Liberabacter asiaticus ( CLas ) , implicated as the causative agent of citrus greening disease [17–19] . Citrus trees infected with this pathogen rapidly develop debilitating symptoms affecting tree health and fruit quality; the pathogen kills the tree within three to five years [20] . Since the first report of this pathogen in Florida in 2005 , this vector-pathogen complex has devastated the United States citrus industry . The Florida citrus industry alone has seen five years of unprecedented decline resulting in billions of dollars of lost revenue and jobs [21] . In 2015 , the U . S . Department of Agriculture predicted a precipitous drop in citrus production in 2016 to 69 million boxes in Florida , well below a peak of 242 million boxes as recently as 2004 [22] . All citrus varieties are susceptible to CLas . Citrus production in Florida including fresh fruit and juice is facing a complete collapse if significant progress is not achieved soon [23] . Management of this pathogen-insect vector complex has been extremely challenging . Intensive pesticide management has done little to halt the spread [24] and currently it is believed that 100% percent of Florida citrus groves are infected with the disease [25] . Critical to reversing the spread of this pathogen and recovering productivity of Florida citrus groves is development of pathogen transmission intervention strategies such as development of resistant citrus genotypes that prevent or reduce insect feeding [26] . Here we use random forests , hidden markov models , and heirarchical cluster analysis to reduce the time required to analyze EPG data . In addition , these analyses point to the presence of additional undescribed feeding states suggesting that the behavior of psyllid stylets within the host plant is more complex than has been recognized .
To evaluate such pathogen transmission intervention strategies , we first sought to remove the data analysis bottleneck present in the current paradigm for monitoring feeding of insects using EPG recordings . To do so , we taught the computer to recognize insect feeding states using pattern recognition algorithms . Specifically , we developed high-throughput automated classification of insect feeding states using supervised classification of Fourier-transformed raw EPG data with random forests models . Random forests models are an ensemble machine learning method that relies on bootstrap aggregation of decision trees [27] . These models have been successfully applied for diverse classification tasks including land cover classification and 3D facial recognition [28 , 29] . The computer learned to recognize patterns of insect feeding remarkably well . Overall classification accuracy of random forests models trained on the six human recognized feeding states ( Table 1 ) can reach 97 . 4±0 . 1% ( 95% CI ) when compared with human expert annotation ( Fig 2; confusion matrix and accuracy statistics in S1 , S2 and S3 Tables ) . Accuracy improved , and can reach close to 100% , when these models are simply asked to identify phloem feeding . Phloem feeding was our primary interest in this case because ingestion and salivation in phloem sieve elements are when pathogen acquisition and inoculation of CLas are presumed to occur ( Fig 3 ) . Importantly , these supervised classification algorithms achieved high accuracy when trained on a random 5% subsample of the full dataset . This obviated the need for human manual annotation of 95% of the data and engenders timesavings that begin to enable high-throughput analysis . Ideally , automated classification of EPG recordings would obviate all human input and allow for real-time monitoring of insect feeding states within the plant or vertebrate subject . This may be possible . Greater than 95% accuracy was achieved using a leave-one-out classification scheme wherein a supervised random forest classifier was trained on a random 5% subsample of 26 of 27 available recordings and then used to classify the remaining recording ( S1 Fig ) . In some cases , accuracy decreased due to variation in waveform patterns generated by insect feeding on different varieties ( S2 Fig ) . Further development of more sophisticated machine learning algorithms should enhance our ability to accurately classify insect feeding and pathogen transmission in real time to more precisely follow stylet behavior within the host . In addition to the abilities of machine learning algorithms to enable high-throughput screening of pathogen transmission intervention strategies , such models can be used to extend our understanding of the dynamics of insect feeding . We can learn from the computer how to recognize additional patterns of insect feeding . Currently , six distinct feeding states are recognized from EPG recordings of the Asian citrus psyllid based on human observation of waveform patterns correlated with histological studies [6] . We wondered if unsupervised pattern recognition models could identify additional , as yet unrecognized , feeding states . To do so , we applied hidden Markov models to Fourier-transformed raw EPG data without supplying the algorithm any information about human-annotated insect feeding states . Hidden Markov models use Markov processes to model and uncover hidden states affecting given observations [30] and are used in natural language processing and in predicting protein topology [31–33] . We provided the model with Fourier-transformed time series data from EPG recordings and asked it to classify the data into as many as 12 feeding states ( Fig 4 ) . By doing this , the computer could recognize and highlight additional feeding states not discerned through histological studies . Eight-state hidden Markov models successfully resolved phloem feeding states ( when pathogen transmission occurs in this system ) and identified two additional feeding states within the human-recognized C feeding state thought to correlate with insect stylet passage through plant tissue [6] ( Fig 4 ) . These two additional feeding states suggest that the insect is performing two rapidly alternating tasks during passage of the stylets through nonvascular tissues . Additionally , Bayesian information criterion scores from multistate hidden Markov models [34] suggest that there may be many more than the six currently recognized feeding states further emphasizing the dynamic nature of phloem , xylem , and potentially blood feeding in piercing/sucking arthropods ( Fig 4 ) . More information regarding insect feeding patterns can be obtained by applying pattern recognition algorithms to the six human-recognized waveforms identified by histology [6] . Applying hierarchical cluster analysis to frequency distributions extracted from Fourier-transformed EPG data for each feeding state revealed similarities within ingestion ( G , E1 , and E2 ) feeding states ( Fig 5: left dendrogram ) [35] . The frequencies ( Fig 5: density plots ) produced by psyllid ingestion from xylem ( feeding state G ) , were not significantly different ( P > 0 . 05 , from heirarchical cluster analysis ) from those produced by phloem salivation and ingestion ( E1 and E2 , respectively ) . In contrast , probing and non-probing feeding states ( NP , C , and D , respectively ) during which ingestion does not occur , produced significantly different frequency patterns compared with those of states associated with pathogen transmission ( G , E1 , and E2 ) . These results suggested that ingestion from xylem and phloem by the Asian citrus psyllid is accomplished by mechanically similar means . Further analysis of feeding states provided insight into the nature of pathogen transmission and allowed identification of characteristics that render certain plant varieties more resistant to pathogen infection . Development of resistant citrus genotypes is of primary interest to citrus growers as other methods of controlling citrus greening have proved unsuccessful [24] . Trifoliate genotypes ( Table 2 ) , such as Poncirus trifoliata and its hybrids , are under consideration for commercial development . These have been noted for their tolerance to citrus greening [18] . The level of tolerance is yet to be determined , however . When directly inoculated with CLas by graft inoculation with infected buds , trifoliate varieties displayed symptoms of disease progression similar to susceptible Citrus trees [36] . In contrast , under field conditions where trifoliate varieties were only subjected to infection by insect transmission , trifoliate varieties displayed reduced or delayed symptoms . [37] . To compare and contrast insect feeding on different genotypes of trifoliate and non-trifoliate citrus varieties , we applied a hierarchical cluster analysis to 27 recordings of Asian citrus psyllid feeding on nine citrus genotypes [35] . Despite receiving no information on human-annotated feeding states , the computer recognized differences in insect feeding across genotypes . Cluster analysis tended to group recordings of the same variety ( Fig 5: top dendrogram ) . Poncirus ( trifoliate ) citrus genotypes in particular were more similar to each other and grouped together; multidimensional Euclidean distances within trifoliate genotypes were on average 8 . 1% ( 95% CI: 2 . 2 , 13 . 3% ) less than between-variety differences . These groupings of genotypes correspond to patterns of insect feeding ( Fig 5: Heatmap ) . Genotypes that experienced little to no phloem feeding ( states E1 and E2 ) were grouped together ( Fig 5: red box ) . Those genotypes with limited opportunity for pathogen transmission tended to be trifoliates or trifoliate hybrids that experienced significantly ( α = 0 . 05 ) less phloem feeding by the psyllid compared with other genotypes ( Fig 6 ) . The observed low incidence of phloem feeding on P . trifoliata and trifoliate hybrids suggests a mechanism to explain the observed tolerance of citrus genotypes in the field , despite demonstrated susceptibility to the pathogen by graft inoculation [36 , 37] . Poncirus trifoliata may possess physical traits that confer resistance to transmission by interfering with the vector’s ability to attain the phloem . Our results suggest that psyllid feeding may be hindered by physical barriers to stylet passage conferred by fibrous rings of sclerenchyma cells associated with vascular tissue in P . trifoliata [38] .
These analyses hold direct implications for prevention of transmission of CLas by its hemipteran insect vector , the Asian citrus psyllid . The low incidence of phloem feeding on varieties of P . trifoliata genotypes and Poncirus x Citrus hybrids confirms these genotypes as sources of resistance for cultivar development , and suggests a potential mechanism for their resistance to infection that can be selected for in the future through traditional breeding or genetic modification [26] . Further development of these strategies and resistance mechanisms will benefit from high-throughput screening and analysis using machine learning algorithms . While this type of analysis provides insights directly applicable to preventing the spread of greening disease in citrus through high-throughput screening and identification of resistance mechanisms , analysis of insect feeding as described here holds implications for all insect vector-pathogen systems . These results are broadly applicable to development of resistant varieties [39 , 40] and management of other plant diseases , including Zebra chip that affects the staple crop potato and is caused by a bacterium closely related to citrus greening disease [41] . Insights into the dynamics of insect feeding gained from machine learning analysis of electrical penetration graphs can be used to design novel intervention strategies to disrupt transmission of insect-transmitted pathogens of agricultural crops , livestock , and humans . Testing and screening of strategies such as genetic manipulation , RNAi , or chemical deterrents to feeding and transmission will benefit from high-throughput , human independent classification via machine learning . These electrical penetration graph analyses that extend human insight and reduce time investment will engender advances in both basic and applied investigation of insect transmitted pathogens and advance discovery of tools to prevent the spread of disease in agricultural crops , livestock , and humans .
EPG recordings were performed using a Giga-8 DC-EPG system ( Wageningen , the Netherlands ) to record the feeding activities of adult Asian citrus psyllids on nine trifoliate and citrus varieties . Psyllids were tethered to recording equipment using fine gold wire and silver conducting glue then settled on the adaxial midrib of a leaf ( Fig 1 ) . To complete the circuit , a second electrode electrode ( ground electrode ) was inserted into the saturated soil ( 70–80% moisture content ) of the pot containing the citrus plant . EPG recordings were conducted within a Faraday cage in a climate-controlled laboratory ( 25 ± 1°C , 60 ± 5% RH ) for 8 to 21 h under lighted conditions . Waveforms were classified by visual inspection by a trained expert according to previous reports [6 , 42] into six feeding states: salivary sheath secretion and stylet passage ( C ) , first contact with phloem ( D ) , salivation at phloem ( E1 ) , phloem ingestion ( E2 ) , xylem ingestion ( G ) or no probing ( NP ) . Twenty-seven EPG recordings totaling 470 hours on nine different citrus varieties were used to explore machine learning for waveform recognition . Raw voltage data from psyllid feeding were recorded using WinDaq Data acquisition and Playback software ( DataQ Instruments ) . Data were classified by visual inspection and annotated using the WinDaq data browser then exported to comma separated value files . Raw data from comma-separated values were then loaded in the R version 3 . 2 . 2 computing environment [43] and converted from the time domain to the frequency domain using fast fourier transform [44] . The six frequencies with the highest magnitudes , often harmonics , were extracted for use in machine learning algorithms . Fast Fourier transformed data were randomly split into training and test sets for each recording . A random five percent subset of each recording was used to train a supervised random forests model with 3 repeated 10 fold cross validation and was then tested on the remaining ninety five percent of the recording . This procedure was used to classify all six human recognized feeding states , and to differentiate between phloem ( E1 and E2 ) and nonphloem ( C , D , NP , and G ) feeding states . Out of sample accuracy , based on comparison to human expert classification of the test set , and ninety-five percent confidence intervals averaged for all feeding states are reported . 50:50 , and 95:5 training to test set schemes were also considered for the analysis and did not produce differences in overall accuracy . A 5% training to 95% test set was considered most advantageous in terms of reducing human labor while maintaining high accuracies . Using randomly sampled training sets less than 5% of the overall dataset increased the likelihood of missing certain feeding states and lowered classification accuracy accordingly . A leave one out classification scheme was pursued to determine the possibility of classification without additional human input . To that end , a random five percent subsample of each feeding state from each of 26 human annotated recordings was used to train a random forests model with 3 repeated 10 fold cross validation . The model was then asked to classify the 27th recording; results of such classification were compared with human expert annotation to determine out of sample accuracy . This procedure was then repeated and used to classify each of the 27 recordings , one of which was left out each time . To explore the possibility of additional insect feeding states beyond those six currently recognized by humans , hidden Markov models were applied to the dominant frequencies extracted from Fourier transformed data and asked to separate the electrical penetration graph time series into up to 12 feeding states . Parameter estimation for the hidden Markov models was accomplished through use of the expectation maximization algorithm and the posterior state sequence was recovered by the Viterbi algorithm [45–47] . Bayesian information criterion was used to penalize additional feeding states [34] . To explore similarities between varieties and insect feeding states , hierarchical cluster analysis was applied to density distributions of dominant frequencies extracted from Fourier transformed electrical penetration graph recordings . Variety similarity was determined through bootstrapping 1000 times the difference in Euclidean distance among and between frequency density distributions of trifoliate varieties . Comparison of unsupervised classification using hierarchical clustering to human annotated states was accomplished through construction of a heatmap presenting the percent median feeding bout time scaled within each feeding state . Comparison of phloem feeding between trifoliate and non-trifoliate varieties was accomplished through bootstrapping 1000 times the difference in median phloem ( feeding states E1 and E2 ) feeding time . After exportation from the WinDaq data collection and browser software , all data were loaded into R version 3 . 2 . 2 for further analysis [43] . RStudio was used as a development environment [48] . Packages provided additional functionality and facilitated analysis: data . table [49] , dplyr [50] , tidyr [51] , and pryr [52] for data management , caret [53] and randomForest [54] for implementation of random forest models , foreach [55] , doParallel [56] , and doMC [57] for parallel implementation of analysis , pvclust [58] and ggdendro [59] for hierarchical cluster analysis , depmixS1 [60] for implementation of Hidden Markov Models , and ggplot2 [61] for developing graphics .
|
Insect vectors acquire and transmit pathogens causing infectious diseases through probing on host tissues and ingesting host fluids . By connecting insects and their food source via an electrical circuit , computers , using machine learning algorithms , can learn to recognize insect feeding patterns involved in pathogen transmission . In addition , these machine learning algorithms can show us novel patterns of insect feeding and uncover mechanisms that lead to disruption of pathogen transmission . While we use these techniques to help save the citrus industry from a major decline due to an insect-transmitted bacterial pathogen , such intelligent monitoring of insect vector feeding will engender advances in disrupting transmission of pathogens causing disease in agriculture , livestock , and human health .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
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2016
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Machine Learning for Characterization of Insect Vector Feeding
|
Lateral organ position along roots and shoots largely determines plant architecture , and depends on auxin distribution patterns . Determination of the underlying patterning mechanisms has hitherto been complicated because they operate during growth and division . Here , we show by experiments and computational modeling that curvature of the Arabidopsis root influences cell sizes , which , together with tissue properties that determine auxin transport , induces higher auxin levels in the pericycle cells on the outside of the curve . The abundance and position of the auxin transporters restricts this response to the zone competent for lateral root formation . The auxin import facilitator , AUX1 , is up-regulated by auxin , resulting in additional local auxin import , thus creating a new auxin maximum that triggers organ formation . Longitudinal spacing of lateral roots is modulated by PIN proteins that promote auxin efflux , and pin2 , 3 , 7 triple mutants show impaired lateral inhibition . Thus , lateral root patterning combines a trigger , such as cell size difference due to bending , with a self-organizing system that mediates alterations in auxin transport .
Developmental biologists often conceptualize patterning mechanisms in uniform fields of cells , but in reality , positional information may be generated and translated in dynamic circumstances in which cells divide , grow , and change shape . These circumstances allow for unexpected feedbacks , making analysis a challenge . Rhizotaxis , the arrangement of lateral organs along plant roots , is a good example of a patterning process that occurs in a dynamic context . The mechanisms that regulate rhizotaxis have long remained a mystery , although the origin of lateral roots was described as early as 1888 [1] . In Arabidopsis , lateral roots arise from two files of pericycle cells that lie adjacent to the protoxylem [2] , and the pattern of emerged lateral roots can be described in terms of the longitudinal spacing along a file and the left/right component ( Figure 1A ) . The longitudinal pattern is variable and cannot be explained by mechanisms that require a fixed amount of time , distance , or number of pericycle cells between initiations [3] . There is , however , a strong tendency for lateral roots to arise on the outside , i . e . , convex side , of the curve [4] . This tendency correlates with above-average auxin response at the proximal end of the meristematic zone ( MZ ) well before the first asymmetric divisions [5] . Lateral root formation can be induced by global increases in auxin content [6] , and more specifically , by local activation of auxin synthesis in pericycle cells [7] . Mutations that render plants less sensitive to auxin reduce lateral root numbers [8 , 9] . Additionally , chemical or genetic inhibition of auxin transport can decrease lateral root density [10–12] . These observations indicate that lateral root formation is influenced by auxin , but they do not reveal the underlying mechanism . Here , we combine experimental and multilevel modeling approaches to unravel the molecular and biophysical mechanism that regulates rhizotactic patterning .
Consistent with prior reports of a correlation between lateral root formation and the presence of curves , we observed that a waving growth pattern increases the average density of emerged lateral roots ( 1 . 6 and 1 . 8 vs . 2 . 5 and 2 . 6 emerged lateral roots/centimeter for “straight” grown vs . waving roots in each of two experiments , where by “straight , ” we refer to vertically grown roots that curve less than those grown on slanted agar ) . To determine how the degree of curvature impacts lateral root formation , we inverted Arabidopsis seedlings for variable lengths of time before returning them to an upright position ( Figure 1B–1D ) . The degree of curvature and the probability that a lateral root is located on that curve both increased with time ( Figure 1E ) . Strikingly , roots with curves of more than 45° all developed associated lateral roots , indicating a strong correlation between the presence of a sharp curve and lateral root induction ( Figure 1E and Protocol S1: Figure S1A and S1B for details ) . To determine whether lateral root positioning is influenced by the mechanisms that produce undulating root growth or by curvature itself , we bent roots in the shape of a J , 0 . 5 cm from the root tip , in regions where cells are fully differentiated . Lateral root formation was examined in the curved region and around the comparable position that was marked on straight-grown control roots . Lateral roots arose along the entire length of the curve , effectively extending the zone of lateral root formation closer toward the root tip ( Figure 1F , left ) . The average distance from the center of the curve to the closest lateral root is less than from the control mark to a lateral , demonstrating that curves promote lateral root formation . Furthermore , formation of new lateral roots was strongly biased toward the outside of the curve ( Figure 1F , right ) . We conclude that curvature of a root by itself focuses lateral root density along the longitudinal axis and establishes the left/right position of lateral roots . Roots that curve all along the longitudinal axis do not display lateral roots in the MZ and the elongation zone ( EZ ) , indicating that only the differentiation zone ( DZ ) responds to curvature with lateral root formation . To investigate whether curving roots in the DZ induces changes in auxin distribution , roots expressing the auxin response marker DR5::GFP [13] were manually curved and subjected to dynamic confocal imaging . Straight roots reveal strong auxin response maxima in the MZ [14] and a weaker auxin response in the vasculature of the DZ . When a curve was introduced into the DZ , fluorescence initially increased more or less symmetrically across the vasculature , but about 2 h later , the auxin response pattern became biased towards the outside of the curve ( Figure 1G and Protocol S1: Figure S1C for details ) . To facilitate dynamic analysis of lateral root formation , we developed a system for inducing lateral roots within a narrow region that could be imaged with a confocal microscope . Six-day old plants were turned 180° , left for 4 h , and then placed horizontally under the microscope . Consistent with expectation , plants carrying DR5::venusYFP , a nuclear-targeted fluorescent marker noted for its high intensity and short maturation time [15] , show increased levels of auxin response in epidermal cells of the MZ along the inside of the curving root where elongation is inhibited [16 , 17] ( Figure 1H ) . However , the situation in a curved region of the DZ is quite different . There , the highest level of auxin response is found in mature vasculature , particularly on the outside of the curve , where lateral roots form ( Figure 1G ) . This is in accordance with the results of our manual curvature experiments . In the pericycle , increased auxin response was first observed about 190 min before the first asymmetric division of the founder cells , then rapidly escalated , far surpassing the level of auxin response in all of the surrounding cells ( Figure 1I–1L; Video S1 ) . The endodermal cell located adjacent to the founder pericycle cells underwent a smaller and transient increase in auxin response . In previous studies , Stage I primordia that formed after gravistimulation appeared 3 h after strong curvature was established [18] , which agrees well with our data and implies that curvature leads to a rapid increase in auxin levels . To understand how root curvature affects lateral root initiation , we developed a model that describes the dynamics of auxin transport through the root . In the model , we consider that auxin can only diffuse freely within cells and in the cell wall , whereas passage of auxin over cell membranes is determined by permeability properties . The efflux and influx permeability values are enhanced by the presence of PIN and AUX1 expression , respectively . The model captures the following basic biophysical characteristics of the system: ( 1 ) the overall cell geometries and tissue types; ( 2 ) typical lineage- and zone-dependent PIN distributions and expression levels; ( 3 ) cell-shape changes due to a mechanical alteration of curvature of the whole organ; and ( 4 ) auxin transport itself ( see Protocol S1: Tables S1–S3 and Text S1 for a detailed discussion ) . Given the discrete nature of cells , and the manner in which free diffusion of auxin is interrupted by membranes , auxin dynamics may be strongly influenced by cell shape . The in silico root layout is therefore constructed using typical cell lengths and widths within the MZ , EZ , and DZ . In live roots , cells in the MZ are smaller , but transiently increase length when in the EZ , until reaching a maximally elongated state in the DZ . This is modeled by selecting different characteristic cell lengths for each of the three zones . Differences in width between the external cell files , and the thinner nature of the vascular cells , are also included ( Figure 2F and Protocol S1: Text S1 for details ) . Such models are necessary because simple , intuitive predictions of auxin flow based only on the location of auxin transporters neglect important factors that determine flux patterns within the context of the whole tissue , including the impact of cell size and shape and the fact that the amount of flux through the transport facilitators is determined by the substrate concentrations [19–22] . We performed a systematic analysis of the PIN expression patterns throughout the whole axis of the root ( Figure 2A–2E ) . Our model incorporates the experimentally observed PIN expression topologies , as well as the specific differences between each zone in a schematic manner , by specifying the orientation and distinguishing between strong and weak PIN expression ( Figure 2F ) . Overall PIN expression levels are weaker in the DZ than in the EZ or MZ . Because we first focus on understanding the influence of a root curvature by itself on the auxin dynamics , regulation of PIN or AUX1 expression or localization is initially left out . Using a similar system , we have previously shown that polar auxin transport is sufficient to generate and maintain the auxin maximum at the quiescent center ( QC ) [22] . PIN localization—in particular the distribution of laterally inward-oriented PINs—is of paramount importance in determining the properties of the root tip [22] . When the model is extended to include the DZ , we observe distinct differences in auxin levels and flux patterns in the three regions of the root . In the MZ , auxin flowing down the vasculature towards the root tip moves back up the external cell layers , reentering the vascular flow via lateral transport across the width of the root . This reflux loop generates a strong accumulation of auxin around the QC . High auxin concentrations in this distal region drop exponentially in the proximal direction , towards the EZ ( Figure 3A ) . The strong lateral PIN expression that allows auxin to flow from the external cell layers toward the vasculature results in very low auxin levels in the epidermis , cortex , and endodermis of the EZ . Consequently , in the vasculature , auxin concentration reaches a low , constant level , which is predominantly determined by the shoot-derived basal flux ( red line in graph of Figure 3A ) . At the transition from the EZ to the DZ , lateral PIN1 and PIN2 expression decrease ( Figure 2B and 2C ) . As a result , the basal flux through the vascular tissue , in which concentrations tend to be much higher than in the flanking external tissue files , becomes less confined ( inset of Figure 3A and Protocol S1: Figure S2 ) . Consequently , concentrations in the external cell files strongly increase in the DZ ( green line in Figure 3A ) , and due to the upward flux through the external cell files and reflux back into the vasculature , an increase occurs in the vascular auxin levels ( red line in Figure 3A ) . Although this result may seem counterintuitive ( i . e . , leakage of auxin from the vasculature into the external cell files causes an increase in the vasculature auxin concentrations ) , it can be readily understood by realizing that in equilibrium , net basal fluxes over different transverse cross-sections through the DZ , EZ , or proximal MZ should be equal . ( The auxin decay rate is low , and therefore substantial amounts of auxin are lost only in the root tip , where concentrations are the highest , such that net basal fluxes in the DZ remain constant . ) Consequently , any increase in the apical flow leads to an increase in the basal flow through the vasculature . A reflux is established , causing auxin on its way to the root tip to pass through the tissue multiple times , thereby increasing the concentration levels without changing the net downward flow ( inset of Figure 3A and Protocol S1: Figure S2 ) . At the transition from EZ to DZ , auxin levels in the exterior cells rise due to the weakened PIN expression . These higher levels are maintained throughout the rest of the DZ ( Figure 3A ) . Along the whole straight root , concentrations are transversely symmetric . In summary , PIN proteins , whose expression is controlled by auxin concentration and by the root-tip–associated PLETHORA ( PLT ) transcription factors [23 , 24] , produce different flux patterns in different root zones . We investigated whether curve formation can trigger auxin accumulation by bending roots in silico ( see Protocol S1: Text S1 ) . When live roots curve , the cells on the inside of the curve are shorter than those on the outside , with the largest differences seen when roots grow in response to a gravitropic stimulus ( Protocol S1: Table S4 ) . Similarly , bending our model root alters the size and shape of cells in the curved region . Even when cellular PIN localization and the total amount of PIN activity per cell are held constant , the auxin distribution in the region of the bend rapidly changes . A new equilibrium situation is reached within 15 min ( Figure 3B , 3C , and 3E ) . Auxin concentrations rise in both the vascular and external cell files , and a bias is established , with higher values in the outer half of the root and a maximum in the outer pericycle cells at the bend ( Figure 3B , red line ) . This outcome is robust over wide ranges of diffusion and permeability values ( Protocol S1: Figures S3 and S4 ) , as well as for different cell-wall widths ( Protocol S1: Figure S5 ) or mature cell sizes ( unpublished data ) . Moreover , when PIN density per membrane length is held constant after a cell-shape change ( rapid delivery of PINs to the membrane might provide such homeostatic control ) , bending the root still results in higher auxin concentrations in the pericycle on the outer half of the bend ( Protocol S1: Figure S6 ) . Simulations in which cell volume is held constant as the root is bent generate even stronger biases on the outer bend ( Protocol S1: Figure S7 ) . Furthermore , auxin maxima in the outer pericycle still form when the model is extended to include a Casparian strip , which might be auxin impermeable ( Protocol S1: Figure S8 ) , as lateral diffusion through the apical and basal cell walls has only a limited contribution to the overall lateral flux . Thus , our simulations indicate that curvature-induced auxin accumulation is a robust process that is not dependent on the particular choice of implementation , assumptions , or parameter values . Auxin levels rise due to the effect of cell-shape changes on auxin transport dynamics , with the extent of the increase depending on the degree of curvature ( Protocol S1: Figure S9 ) . The localized increase in auxin concentration is independent of the mechanism that induces the curve , such as the gravitropic response . Given that root bending results in cell-shape changes , it is tempting to think that auxin accumulation is the result of cell-length differences only . However , that is not a sufficient explanation . For a cell with polar PIN expression ( i . e . , an epidermal , cortical , or vascular cell ) , the mean auxin concentration is expected to increase linearly with cell length . This is strongly dependent on the fact that cells are discrete units in which the flux becomes “boosted” at each cell–cell transition due to the unidirectional auxin transport . Thus , within the cell , auxin forms a gradient along which , due to passive diffusion , auxin fluxes polarly . ( For an ideal one-dimensional vascular cell: given the cytosolic diffusion constant of auxin D , the effective polar transport P across the cell , and an auxin concentration a at the basal end , a linear gradient will establish within the cell with a slope Pa/D . Consequently , given the same auxin transport properties , the slope does not depend on the cell length , whereas the mean auxin level within the cell—given by a ( 1+ ) , where l is the length of the cell—increases with length . Similarly , under the assumption of constant basal influx i , the mean auxin level within the cell is given by i ( ) . Thus , length increase ( in both cases ) is expected to result in a linear rise in mean auxin concentration . Although it cannot be excluded that such small auxin increases could suffice to trigger lateral root initiation , this mechanism by itself is insufficient to explain all of our observations . First , geometric considerations alone would imply that the cells along the inner side of a bend , which have become smaller due to the bending , should present lower concentrations . Clearly , this is not the case: Figure 3B and 3E shows an increase in auxin in all cell files at the bend , in agreement with our experimental observations ( Figure 1G , black lines ) . Moreover , holding the degree of curvature constant over a bend , but varying the length of the region undergoing bending , progressively increases the bias in auxin concentrations ( Protocol S1: Figure S10 ) , indicating that tissue properties are also important in generating the bias . Simulations of J-hooks that cause only 6% differences in cortical cell lengths still present remarkable increases in auxin levels and bias , but only when the length of the bent tissue is sufficiently long ( Protocol S1: Figure S11 ) . The formation of a strong bias results from an increase in the amount of auxin “leaking” out of the vasculature into the external cell files . Given the large fluxes that pass through the vascular tissue , internal auxin gradients are steep within the vascular cells . Steeper gradients imply larger auxin increases due to cell-length increase , and as a result , the concentration difference between vascular and external cell files is enhanced due to bending , leading to the lateral flow . As is the case for the DZ in straight roots ( insets of Figure 3A and Protocol S1: Figure S2 ) , this higher concentration of auxin in the external files locally enhances the DZ-reflux loop , causing auxin levels to further rise in the vasculature . This is accompanied by a further increase in lateral flux , effectively recapturing even larger amounts of auxin that would otherwise pass through the region only once ( Protocol S1: Figure S2 ) . The result is an increase in auxin concentration at the bend . To untangle the relative contributions of cell shape and auxin reflux , we analyzed two situations in which transport in the bent region is locally interrupted . First , we introduced an impermeable vertical wall in the apoplastic space in between the endodermis and pericycle to the model in the region of the bend ( Protocol S1: Figure S12A and S12C ) . This prevents lateral fluxes between vascular and external cell files at the bend , and as a result , the increase in auxin is reduced in the vasculature and outer external cell files , as well as lacking in the inner external cell files . Second , we completely eliminated auxin reflux by blocking fluxes through the external cell files ( Protocol S1: Figure S12B and S12C ) . This leads to an even stronger reduction in auxin at the bend , revealing the importance of the reflux loop in this mechanism . To determine how robust the tendency for lateral root formation to occur on the outside of a curve is , we examined the position of emerged lateral roots in mutants with altered growth patterns . The wag1/wag2 double mutants have a wavy root phenotype , and rcn1–6 plants exhibit a pronounced skew , with root tips slanted to the right when plates are viewed from the front . Both mutants maintain a strong outward bias in the location of lateral root formation . All of the roots bearing emerged laterals on the curve 3 d after inversion had those laterals on the outside of the curve ( 44 out:0 in for wag1/wag2 and 46 out:0 in for Col-0 in one experiment; 33 out:0 in for both rcn1–6 and Col-0 in a second experiment ) . Because flux patterns differ substantially throughout the root tissue ( insets in Figure 3A ) , the different zones present specific responses towards bending ( Figure 3E–3G ) . These differences follow naturally from the differences in PIN abundance: The MZ , due to its large lateral fluxes and steep auxin gradient , not only fails to generate the typical bias observed in the DZ , but actually forms an inverted bias ( higher levels on the inside , i . e . , concave side ) , without the appearance of a single maximum ( Figure 3G ) . The EZ is capable of generating a small outer/inner bias ( Figure 3F ) , due to the effect of increased cell lengths ( similar to Protocol S1: Figure S12B ) , but the reflux that is essential for creating a substantial increase is suppressed by strong lateral PIN expression . These lateral PINs act like a “cordon” along the vasculature , keeping auxin levels within the external cell files very low . Thus , PIN organization in the different zones ( MZ , EZ , and DZ ) explains why lateral root formation is limited to the DZ . Classical physiological experiments have shown that tip removal induces lateral root formation , even when the shoot is decapitated , as long as either a sufficiently long root segment is preserved or external auxin is supplied [25] . We simulated the same treatment in our model , and observed auxin accumulation dynamics that corroborate the link between vascular auxin accumulation and lateral root initiation ( Protocol S1: Figure S13 ) . Seedlings with loss-of-function mutations in AUX1 have decreased numbers of lateral roots [10] ( Figure 5A ) , resulting from reduced rates of lateral root initiation [12] . To investigate how changes in AUX1 might be associated with lateral root formation , AUX1:YFP plants were gravistimulated for 4 h and then subjected to dynamic imaging . AUX1:YFP levels accumulated asymmetrically in the region of the bend , with levels on the outside of the curve being clearly higher than those on the inside ( Figure 4A–4D; Video S2 ) . Fluorescence intensity within the pericycle increased steadily before and after the first asymmetric cell divisions . Notably , the distribution of AUX1:YFP was relatively uniform within a single pericycle cell membrane ( Figure 4C ) . The first cell divisions took place 2–3 . 5 h after imaging began , thus the increase in AUX1 along the vasculature occurs no later than the increase in auxin response in the founder cells , and likely before it ( Figure 4A–4D ) . We noted striking similarities between AUX1 and auxin response reporter accumulation . Transcription of AUX1 is substantially up-regulated in roots within 90 min after auxin application [26] . Auxin application to AUX1:YFP plants results in increased levels of fluorescence in the pericycle and ectopic expression in cells outside the vasculature . These changes become visible within 2 h after auxin application , and membrane localization of AUX1 is pronounced within 3 h ( Protocol S1: Figure S14 , compare S14B , S14C , and S14F ) , indicating that auxin induces AUX1 . Because AUX1 facilitates auxin influx , this response not only provides a mechanism for increasing the auxin concentration within the responding pericycle cells , but also initiates a positive feedback loop in which those increased concentrations go on to further induce AUX1 . A major role for vascular AUX1 in lateral root formation seems at odds with previously published data , which revealed that AUX1 expressed under the control of the J0951 GAL4 enhancer trap line rescues the lateral root phenotype [5 , 18] . Near the root tip , this line drives expression in the expanding epidermis and root cap , as was reported . However , we found that in mature portions of the root , J0951 mainly drives expression in the vasculature ( Protocol S1: Figure S15 ) , consistent with the most parsimonious hypothesis from the expression data that this is the primary site of action for lateral root induction . Another concern about postulating a major role of auxin influx in lateral root initiation is that the aux1 mutation has only a mild effect on lateral root initiation . To assess potential redundancy , we administered 1-napthoxyacetic acid ( 1-NOA ) , an auxin analog that lacks auxin activity and inhibits AUX1-mediated transport . 1-NOA inhibits AUX1 and LAX3-mediated transport [27 , 28] , but polar transport of 3H-IAA in inflorescence stems remains unaffected , indicating that it does not affect PIN efflux proteins [29] . 1-NOA inhibits lateral root formation when assayed 4 d after transfer; the density of emerged lateral roots on the new growth declined steadily with increasing concentrations of 1-NOA ( Figure 4J ) . Wild-type plants showed a statistically significant reduction in lateral root formation after treatment with 3 μM 1-NOA , whereas aux1 roots do not . Thus , 1-NOA acts on AUX1 . Higher concentrations of 1-NOA do generate a significant reduction in lateral root density in aux1 plants , confirming that 1-NOA also acts on other members of the AUX/LAX family of auxin import facilitators . This suggests that such members are active in lateral root initiation , although we cannot exclude the possibility that emergence is affected , as recent evidence indicates that LAX3 promotes lateral root emergence [28] . Whereas 96% ( 23/24 ) of wild-type roots that were manually curved formed lateral roots on regular media , only 50% ( 12/24 ) of roots did so when placed on 30 μM 1-NOA . Roots that are transferred to 1-NOA and left straight only develop emerged lateral roots 32% of the time , indicating that curve formation is still a positive stimulus for lateral root formation in the presence of the 1-NOA . For roots that did form , 11/12 were on the outside of the curve , indicating that bias toward the outside was still maintained . We next investigated a positive feedback role for AUX1/LAX proteins in auxin accumulation , using the modeling approach . Simulations in which AUX1 is expressed at high levels in specific pericycle cells on the bend result in a clear increase in auxin in these cells ( Figure 4E ) . Motivated by this result , we investigated whether high expression of AUX1 in outer pericycle cells could emerge as a consequence of the flux patterns , without invoking special rules for the pericycle . We built a simple feedback into the model , in which AUX1 expression in the DZ cells is regulated by a sigmoidal response to auxin concentration ( see Protocol S1: Text S1 for details ) . A self-organizing amplification of auxin accumulation and AUX1 expression occurs through their mutual feedback . Consistent with our experimental observations ( Figure 4A–4D ) , these simulations reveal increased AUX1 concentrations only upon curvature ( Figure 4F–4I and 4K–4N , and compare Video S3 with Video S4 , for control ) . The resulting auxin maximum becomes localized at one to three outer “pericycle” cells because , as a result of these cells' strategic position at the interface of the basal-directed vascular flux with the flanking external apical fluxes , the gain of auxin due to a certain increment in AUX1 expression levels is greatest in these regions . As cells “compete” for auxin uptake through AUX1 and its auxin-dependent regulation , a winner-take-all situation manifests , with pericycle cells having a clear advantage . The AUX1 response may spread in a cell-wise fashion to distal neighbors ( Figure 4H and 4I ) within the bent region . When AUX1 expression levels become very high in a pericycle cell , this cell begins to efficiently take up auxin from the cell wall , thus depleting neighbor auxin concentrations . Subsequently , auxin concentrations within the cell accumulate and its basal efflux ( due to the basal PIN expression ) becomes sufficient to replenish the neighboring distal pericycle cell . The neighbor , through its AUX1–auxin regulation loop , can initiate a similar cycle , also reaching high auxin levels . This effect tends to be restricted to the bend , where the auxin maxima become fixed . In some simulations , we also observe an auxin maximum that travels down the root towards the meristematic region in the form of a pulse , initiated from the newly formed curvature ( Video S5 ) . This suggests that the formation of a curve could potentially trigger periodic increases in auxin concentration in regions as far away as the basal meristem region . Importantly , simulation of the AUX1 feedback cannot increase “pericycle” auxin levels in straight roots ( Video S4 ) . Collectively , our data reveal that modest increases in auxin concentration on the outside of a curve are amplified by the AUX1/LAX auxin influx facilitators , serving as a central mechanism for lateral root initiation . An initial bias , supplied here by the bending of the root , is necessary for the positive feedback . The mechanism , however , readily accepts other sources of bias , predicting in all cases auxin accumulation in the pericycle cells . The density of emerged lateral roots depends on the presence of specific auxin transporters [30] . We noted that slight changes in growth circumstances have large effects on lateral root densities , so in the following analysis , we compared the density of lateral roots in wild-type and mutant plants that were grown on the same plates . At 7 d postgermination ( dpg ) , statistically significant increases in lateral root density were seen in pin2 plants ( Figure 5A ) , whereas pin3 mutants showed a significant reduction . pin7 and pin1 loss-of-function mutations did not appreciably alter lateral root density . Although several mutations affect the frequency of lateral root formation , they do not interfere with the tendency to form a lateral root on the outside of a curve ( Table 1 ) . The effect of AUX1 on lateral root density decreases with age [3] , an observation that is substantiated by our data ( Protocol S1: Figure S16 ) , despite the fact that data in Figure 5A and Protocol S1: Figure S16 were obtained in different laboratories . Such age-related differences might be caused by compensatory mechanisms and/or changes in root geometry that occur as a result of continued growth ( see Protocol S1: Figures S10 and S11 for discussion ) . aux1 pin2 double mutants mimic the effect seen in aux1 ( Figure 5A ) , indicating that the loss of AUX1 has a stronger influence on lateral root formation than does PIN2 , supporting our conclusion that AUX1-mediated auxin influx is central to lateral root positioning . Double mutants reveal unexpected reversals in density changes ( Figure 5A ) , suggesting nonadditive interactions between auxin transporters in lateral root formation . To investigate the apparently complex influence that PIN proteins have on lateral root density , we took advantage of the possibility of the model to independently alter lateral , apical , and basal expression of PIN proteins ( Protocol S1: Figure S17A and S17B ) . The potential for forming lateral roots upon curvature is increased slightly when the lateral PIN expression component is diminished ( i . e . , “lateral” mutant ) , and increasingly so for a “basal” mutant , both due to a more effective auxin reflux . In contrast , an “apical” mutant has a somewhat decreased potential for auxin accumulation at the bend , because reduced upward flow in the external cell layers reduces the reflux . Interestingly , when combining these in different permutations , forming double and triple “mutants , ” nonadditive effects occur ( Protocol S1: Figure S17C and S17D ) . This is a consequence of the complex manner in which PIN expression modulates reflux loops within the root . We find the strongest propensity for lateral root formation in the “basal-lateral” mutant; a prediction that cannot be readily tested , as PIN2 is expressed both apically and laterally , and it is as yet impossible to uncouple these two components . Despite this difficulty in experimental validation , our simulations corroborate the observation that changes in PIN transporters play a significant and nonlinear role in the lateral spacing of root primordia . Longitudinal spacing of lateral roots is diminished in pin2 , 3 , 7 triple mutants . These mutants have marked increases in lateral root density not correlated with root length ( Figure 5A ) . In 13/100 of these roots , we observed lateral roots that formed exactly adjacent to one another or that fused at the base , separating into two distinct roots only near their tips , a phenotype that was never seen ( 0/65 ) in wild-type roots . The existence of adjacent and fused laterals in pin2 , 3 , 7 triple mutants indicates that mechanisms leading to lateral inhibition of organ formation are interrupted in those plants ( Figure 5B ) . Consistent with this finding , modeling simultaneously decreased vascular flow and reduced flow through the epidermis ( lateral-apical mutant ) largely mimics loss of PIN2 , 3 , 7 and leads to a higher accumulation of auxin at the EZ–DZ boundary as well as higher accumulations due to bending in the region of the curvature , thus predisposing a larger region for lateral root induction ( Protocol S1: Figure S17D–S17F ) . In the mature region of a straight-grown root , PIN3 and PIN7:GFP are similarly expressed in the vasculature . In cases in which single lateral roots form , the levels of PIN3 or PIN7 fluorescence decrease , and the signal becomes diffuse throughout the cytoplasm . This decrease in polar localization occurs in a region that extends across the width of vasculature , followed by the formation of a single lateral root primordium just on the shoot side of that region ( Figure 6A–6C , Video S6 ) . PIN3 and PIN7 are transcriptionally regulated by auxin [31] , and by the tip-focused PLT gradient [24] , and severing the main root from the shoot , which serves as its primary source of vascular auxin , decreases the level of PIN7:GFP within about 5 h . Interestingly , when the distribution of PIN3 or PIN7 did not decline , multiple primordia formed along the curve . Typically , all pericycle cells located on the outside of such a curve underwent an initial round of cell division ( Figure 6D–6F ) . Thus , PIN3:GFP and PIN7:GFP during lateral root initiation document a strong correlation between PIN reduction and the efficiency of lateral inhibition . Our model predicts that simply bending a root in the DZ causes auxin concentrations to drop just distal to the curve , especially in the vascular tissue ( Figure 3D , yellow line ) . This strongly suggests that bending might be the source of the decreased auxin levels distal to the curve , leading to partial depolarization of PIN3 and PIN7 , although the molecular mechanism regulating PIN modulation remains uncharacterized . In simulations with decreased basal expression and partial depolarization of vascular PINs , basal vascular fluxes are interrupted ( Figure 6G ) and directed towards the external cell files ( Figure 6H and 6I ) . As a result , more auxin is brought into the apical stream , in the direction of the shoot and the bend . Auxin reenters the vasculature at the bend , where basal PIN expression is unaffected , leading to a greater accumulation of auxin in the curved region . Importantly , this process can regulate the spacing between auxin maxima . This is consistent with our experimental observations that lateral inhibition is suppressed in the absence of this PIN3/7 fading , suggesting that the feedback between PIN and auxin can explain the suppression of nearby emerged lateral root initials . Through AUX1 feedbacks alone , these distances would not be maintained . On the contrary , multiple cells along a curve could be expected to differentiate , due to comparably high auxin values . The model further predicts that the location of the auxin maximum relative to the center of a curve depends on the relative contributions of AUX1 and PIN3/7 . If the AUX1 response occurs more rapidly than the decrease in PIN3/7 , the auxin maximum is focused on the proximal region of the bend , but is able to spread longitudinally along the outer pericycle cells at the curve ( Video S3 ) . Alternatively , if the decrease in polarly localized PIN3/7 precedes the AUX1 increase , it focuses the maximum to the distal region of the bend . This is in full agreement with the observation that decreases in polar PIN3/7 are highly correlated with formation of one lateral root just proximal to the fading .
Here , we show that curvature is causal for lateral root initiation , an observation that extends the previously observed correlations between curves and sites of lateral root formation [4 , 6 , 18] . In vivo observations indicate that auxin accumulates on the outside of the vasculature in bent regions of the root , the same area in which lateral root initiation is favored . The auxin influx carrier AUX1 is increasingly up-regulated in this region , and becomes focused in either one or several cells in tight correlation with the extent of down-regulation of two PIN efflux facilitators expressed in the vasculature . Mutation of AUX1 and pharmacological inhibition of the AUX1/LAX pathway confirm a major role for the auxin influx pathway in determining the density of root primordia . Our novel multilevel feedforward model for lateral root initiation parsimoniously explains both the patterning mechanism that restricts a new auxin maximum to a local site and the zone of competence for lateral root formation . We show that the presence of a curve leads to localized cell-shape changes , which produce a “trigger” for the auxin transport system in the DZ , which acts as an excitable system . Activation of the trigger results in increased levels of auxin response on the outside of the vasculature , exactly at the “pericycle” boundary layer where lateral roots form . Once small changes in auxin concentration occur , they can change the expression and polar localization of auxin transporters [32–34] . We observe an AUX1 positive feedback loop and down-regulation of PIN3 and PIN7 , both of which can be seen as self-organizing , cell autonomous processes , triggered by an initial bias in auxin concentration , that further amplify the trigger . Employing both experimental work and predictive modeling has proven powerful , leading to results that would not have been obtained by either method alone . Auxin fluxes are not simply a “readout” of the PIN localization , but depend on local intracellular and cell-wall auxin concentrations , as well as on global tissue structure . In addition , positive feedback and cross-regulation between auxin accumulation and AUX1/PIN transporter activity play an important role in lateral root patterning . Our model explains how a specific trigger , i . e . , curvature , can provoke developmental responses in a self-organizing fashion . Our model involves local cues that are causally linked to lateral root formation , and in this respect , differs from previous suggestions for a role for the MZ/EZ boundary region based on correlations between auxin response oscillations in this region [5] . Our study and the work of Lucas et al . [18] indicate that fixed-period oscillations are not required for lateral root formation . Our simulations show traveling pulses of auxin to the root tip associated with primordia formation , suggesting that oscillations in auxin within the vasculature at the MZ-EZ boundary , as observed by De Smet et al . [5] , might be a consequence of formation of previous lateral root primordia rather than a cause for the formation of new ones . The mechanism that we propose has two modules: the initial cell-size trigger and the excitable DZ-specific AUX1/PIN feedback loop . It is important to note that several additional triggers may act on the feedback loop to stimulate lateral root formation . For example , mechanosensory triggers or nutrient-induced changes in auxin transport and response may impinge on the activity of the DZ loop . Future research should address whether such additional inputs are needed to explain the root branching pattern in its complex natural environment . The model for explaining rhizotactic patterning that we present here is similar to current models for phyllotactic patterning [21 , 35–38] in the sense that organ formation is controlled by the abundance and location of auxin transporters that collectively generate an auxin maximum . The important role for AUX/LAX proteins in rhizotaxis is mirrored by the recent discovery that these proteins are redundantly required for phyllotaxis [38] . Whereas the initial events and the feedback loops leading to auxin accumulation maxima in the shoot are incompletely understood , there is one more intriguing parallel: it has been observed that leaf formation that follows from local auxin accumulation can be initiated by local increases in cell expansion [39 , 40] . Thus , a biophysical mechanism that alters relative cell shapes in a tissue , changing local flux patterns and followed by responsive changes in auxin transporters , may be common to both root and shoot organ formation . This suggests that a unified concept for organ initiation in root and shoot systems may be within reach .
Arabidopsis thaliana plants , ecotype Col-0 , were used for all experiments , including marker lines and auxin transport mutants . The DR5vYFP marker line was generated by fusing DR5rev [41] to nuclear targeted venusYFP coding sequence [15] in a pGREENII vector . Transgenic plants were generated by transforming Col-0 wild type as described [42] . PIN1:GFP , PIN2:GFP , PIN3:GFP , PIN7:GFP , and AUX1:YFP marker lines and auxin transport mutants were as described [43] . Unless otherwise indicated , plants were grown on vertically positioned GM plates containing 1% sucrose and 1 . 5% agar . For reversion experiments , marks were placed on the agar beside the root tips of 6-d-old plants . Plates were turned 180° and left for various periods , after which they were returned and left for 3 d . Distance from the center of the newly formed curve to the nearest emerged lateral root and degree of root curvature were measured using ImageJ ( v . 1 . 36 and 1 . 38 ) ; for more details , see Protocol S1: Figure S1 . If no lateral roots emerged within the image frame , the distance to the nearest lateral root was set to the longest recorded length . For manual root curving experiments ( J-hooks ) , 6-d-old plants were grasped with tweezers just below the cotyledons and turned in a J shape by gently dragging the root across the agar surface with the center of the curve 0 . 5 cm from the root tip . Control roots were also grasped and moved along the agar , but were left in a straight orientation . Marks were made on the agar beside each root , 0 . 5 cm from its tip . For lateral root density measurements , the number of emerged lateral roots was counted with the aid of a dissecting microscope , and root lengths were measured as described above for plants 7 dpg and 12 dpg . Germination was monitored every 12 h . We noted that lateral root density is very sensitive to changes in environmental conditions , and we controlled for this as closely as possible within each experiment . For example , wild-type and mutant plants were grown on different sides of the same plate . Data for Figure 5A and Protocol S1: Figure S16 were obtained from plants grown in different laboratories . 1-NOA treatments were performed by transferring 6-d-old plants to fresh agar plates with the indicated concentrations; addition of 30 μM 1-NOA was as described [44] , resulting in 0 . 006% DMSO in the media; control plates contained comparable amounts of DMSO . Lateral root densities were determined for the newly grown portion of the root: the entire MZ and EZ , together with the portion of the DZ formed during the interval indicated , and hence , the densities reported in Figure 5A are not comparable to those of Protocol S1: Figure S16 in which the whole length of the root was considered . Cortical cell lengths were obtained from plants that were either inverted 180° and allowed to grow their own curve , or hand curved into the shape of a J . Roots were imaged under a Zeiss confocal microscope fitted with a 25× objective . To assist in holding the curves in place , coverslips were coated with a thin layer of agar in MES prior to use . During imaging , a slab of agar was used in place of a slide or coverglass . Projection errors and root twisting make it difficult to obtain accurate measurements of cell length . To minimize that source of error , and provide a better estimate of actual cell length , cortical cells were measured only if their end walls were in focus when the xylem plane was clearly visible . Lengths were obtained from images using Zeiss software . The radius of curvature was determined by fitting a circle to three points: the center of the measured area and the two most distant end walls of the specific outer cortical cells that were measured . If only one cortical cell was measured , the region of the cortex under consideration was extended by 200 μm to minimize error in curve fitting . Confocal microscopy was performed using an inverted Zeiss confocal microscope , and image analysis was done using Zeiss LSM Pascal ( 3 . 2SP2 ) software . For live imaging , 6-d-old plants were transferred to coverslip-bottomed imaging chambers containing water or various IAA dilutions , and covered with a slab of agar . Prior to transfer , some roots were either manually curved or gravitropically stimulated by inverting the plates for 4 h . Images were taken every 10 min for a period of 14–16 h , with an autofocus system employed . Auxin dynamics . Simulations are performed by numerically solving transport equations through an implicit reaction-diffusion algorithm . It takes into account diffusion within cells and cell wall at a rate of 300 μm2/s , as well as permeability across a cell membrane: influx permeability is by default set to 20 μm/s , but is 4-fold increased when AUX1 overexpression is taken into account; efflux permeability is set to 1 μm/s in the absence of visible PINs , and to 5 μm/s or 20 μm/s in the case of weak , respectively strong PIN expression . Boundary conditions at the proximal end of the root segment are kept such as to mimic the continuation of the described segment , being linked to the rest of the plant . Further details on the implementation of auxin transport can be found in Grieneisen et al . [22] , and in Protocol S1: Tables S1–S3 and Text S1 . Root bending . Root bending has been implemented through a geometrical transformation in which curvature , axis of rotation , and length of curved segment can be specified and controlled . Bending is always applied in such a way that the length along the central axis of the root does not change . Consequently , due to the bending , cells on the inner side of the curve decrease in length , while those on the outer side expand . All cells keep their original width . To ensure that cell-length changes do not affect the total amount of PIN molecules along a cell membrane , lateral PIN-dependent efflux is diminished proportionally to the gain of length due to bending . ( Note that using an alternative assumption , in which PIN density remains constant independent of the addition of the cells perimeter , will continue to yield similar results , as we show in Protocol S1: Figure S6 ) . Graphs showing transversal and longitudinal auxin profiles of bent roots are obtained by first defining for an unbent root the horizontal lines that pass through the centers of the cell rows and the vertical lines through the centers of the cell files . During bending , these lines then undergo the same transformation as the root itself , thereby creating a link between the positions after bending and the corresponding locations along the original lines in the unbent case . In all plots that present auxin concentration profiles along cell files or cell rows , this mapping of the positions from the original ( unbent ) to the bent state is being used . This explains why the cell lengths within the inner and outer regions always appear to be the same when plotted on graphs . Although this method slightly modifies the length along bent cell files on the graph plot , it is preferred because it makes direct comparison between cells before and after bending possible . AUX1 regulation . The feedforward regulation of AUX1 by auxin is implemented by using a moving average of the intracellular auxin concentration . The variable Ci ( t ) gives the mean auxin concentration within cell i , integrated over a time window ΔTAUX1 ( typically 10 min ) . It determines the level of AUX1 expression , Ai ( t ) , of that cell i at a given moment t , through the sigmoidal relation where β is the auxin concentration at which AUX1 expression reaches half its maximum response , and n determines the steepness of the response . The AUX1 expression enhances auxin influx along all sides of the cell . To demonstrate that changes in auxin alone are sufficient to regulate AUX1 expression , we use the same functional response and the same parameters for all cells of all cell types within the DZ , both within and outside of the bent region . In short , the expression of the AUX1 auxin importer dynamically changes as a consequence of the absolute levels of auxin within the same cell . ( Note that only local information is being used , i . e . , for example relative differences in auxin levels between neighboring cells do not play a role . ) In turn , ( changes in ) auxin concentrations result from ( modifications of ) local fluxes , which are influenced by the influx rates along the membrane of the cell and its close neighbors . At each position along the membrane of a cell i , the influx is given by where Pin is the default influx permeability , Ai the permeability due to AUX1 expression , and Cout the local concentration within the cell wall , just outside the membrane . This feedforward loop , however , is not inherently unstable ( see Video S4 ) . This is because AUX1 up-regulation reduces the reflux , and thereby the total auxin throughput .
|
Plant architecture is determined by where shoots or roots form along the main axis , but the mechanism responsible for lateral root initiation has long puzzled biologists . Here , we show that stretching root cells initiates changes in hormone transport , leading to lateral root initiation in plants , thereby solving a 120-year-old mystery: the mechanism of lateral root initiation . Our data reveal that physical tissue deformation is sufficient to induce chemical changes that unleash biological responses leading to new organ formation . When roots bend , concentrations of the plant hormone auxin increase along the outside of the bend . A complex auxin flux pattern is generated that further enhances auxin levels through localized reflux loops . Auxin importers—AUX1—and efflux carriers—PIN proteins—are known to be regulated by auxin . AUX1 up-regulation enhances the auxin maxima that specify the lateral root founder cells at the bend , while PIN down-regulation modulates the lateral spacing of the roots along the main root axis . This study shows that the biological regulation behind pattern formation can be a result of entangled hierarchies , explaining both the inner/outer spacing , lateral inhibition , and dynamics of lateral root initiation .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"developmental",
"biology",
"plant",
"biology",
"computational",
"biology"
] |
2008
|
Root System Architecture from Coupling Cell Shape to Auxin Transport
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The levels of telomeric proteins , such as telomerase , can have profound effects on telomere function , cell division and human disease . Here we demonstrate how levels of Stn1 , a component of the conserved telomere capping CST ( Cdc13 , Stn1 , Ten1 ) complex , are tightly regulated by an upstream overlapping open reading frame ( oORF ) . In budding yeast inactivation of the STN1 oORF leads to a 10-fold increase in Stn1 levels , reduced telomere length , suppression of cdc13-1 and enhancement of yku70Δ growth defects . The STN1 oORF impedes translation of the main ORF and reduces STN1 mRNA via the nonsense mediated mRNA decay ( NMD ) pathway . Interestingly , the homologs of the translation re-initiation factors , MCT-1Tma20/DENRTma22 also reduce Stn1 levels via the oORF . Human STN1 also contains oORFs , which reduce expression , demonstrating that oORFs are a conserved mechanism for reducing Stn1 levels . Bioinformatic analyses of the yeast and human transcriptomes show that oORFs are more underrepresented than upstream ORFs ( uORFs ) and associated with lower protein abundance . We propose that oORFs are an important mechanism to control expression of a subset of the proteome .
The bulk of the genome is duplicated precisely once each cell cycle but telomeres are replicated differently . DNA polymerases cannot replicate the ends of linear molecules and therefore different strategies are needed to replicate telomeric DNA . The majority of eukaryotes use a reverse transcriptase based enzyme , telomerase , to elongate DNA at telomeric ends . Telomerase activity is tightly regulated . Most human somatic cells express low levels of telomerase , therefore the telomeres shorten with each cell cycle , eventually leading to cell cycle arrest and senescence [1] . In contrast , many cancers over-express telomerase and hyper-elongate telomeres , a process that facilitates uncontrolled cell division . Indeed , point mutations in the telomerase ( TERT ) promoter , which increase TERT expression , are the most commonly identified non-coding mutations found in human cancer [2 , 3] . Yeast cells also express telomerase and maintain stable telomere length . Typically , yeast telomeric DNA shortens over several cell cycles , due to the end replication problem , or sometimes more acutely , due to DNA replication failure . Short telomeres are preferential substrates for telomerase ensuring that in yeast telomere length is comparatively stable [4 , 5] . In addition to telomerase , numerous other proteins contribute to telomere length homeostasis . The conserved CST complex has affinity for the G-rich single stranded DNA ( ssDNA ) at the very 3’ terminus of telomeres [6] . CST is encoded by CTC1 , STN1 and TEN1 in human cells and mutations in CTC1 and STN1 are associated with Coats plus disease , one of the heritable telomere syndromes [7 , 8] . In budding yeast , the equivalent proteins are Cdc13 , Stn1 and Ten1 , and each is essential for telomere function and cell viability . CST has many functions , including protecting telomeres from the harmful effects of the DNA damage response , regulating telomerase activity and recruiting DNA Pol α to complete lagging strand replication [9 , 10] . Individual CST proteins play complex roles in telomerase recruitment , telomerase inhibition and telomere capping [9 , 11] . In particular , there is evidence that levels of Stn1 are important . Increased expression of STN1 leads to short telomeres , which is thought to be due to inhibition of telomerase [12 , 13] . Additionally , overexpression of Stn1 can compensate for partial loss of telomere capping in strains defective in Cdc13 [14] . Finally , there is evidence that increased levels of Stn1 inactivate the S phase checkpoint that responds to genome-wide stalled replication forks [15] . Thus , Stn1 levels affect telomere function and the DNA damage response . Protein levels can be affected by numerous mechanisms affecting transcription , translation or degradation and each type of mechanism has associated costs and benefits [16] . Interestingly , STN1 transcript levels are strongly reduced by the nonsense mediated mRNA decay pathway ( NMD ) and STN1 is among the top 2% of NMD targets [12 , 17] . This indicates that RNA degradation is an important mechanism for reducing Stn1 levels . However , how the NMD pathway targets STN1 transcripts is unclear . Early studies indicated that the STN1 upstream regulatory sequence ( URS , 300 bases upstream of the STN1 CDS ) conferred NMD-dependent control [12] . More recently it was suggested that NMD targets STN1 via a programmed -1 ribosomal frameshifting signal in the C-terminal half of the CDS [18] . Here we establish that the yeast STN1 transcript is targeted by NMD principally because it contains an upstream overlapping ORF ( oORF ) , a special class of upstream ORF ( uORF ) . Furthermore , this oORF is also the route by which the conserved translation re-initiation heterodimer , Tma20MCT-1/Tma22DENR reduces expression of STN1 . Importantly , inactivation of the STN1 oORF has numerous strong effects on yeast telomere function . Interestingly , the human STN1 transcript is also dramatically reduced by the presence of an oORF , suggesting that there is conservation of oORF-dependent mechanisms for reducing Stn1 levels . uORF translation is widespread and associated with reduced expression in both yeast and mammalian cells [19 , 20] . Our analyses of yeast and human genomes shows that oORFs have been more heavily selected against than uORFs , and are associated with lower protein abundance . STN1 is therefore likely to be just one striking example of how an oORF affects levels of a key regulatory protein .
It is well established that Stn1 levels are affected by NMD and affect telomere function [12] . We noticed that the STN1 upstream regulatory sequence ( URS ) has two uORFs . These encode a 16 amino acid uORF and a 6 amino acid oORF , the latter terminating 2 bases after the STN1 coding sequence ( CDS ) start codon ( Fig 1A ) . To examine the effects of the uORF and oORF on expression , the initiation codons were mutated , creating STN1-u2 and STN1-u1 , respectively . STN1-u2 , 78 nucleotides upstream of the STN1 CDS , is a point mutation in the uORF initiation codon while STN1-u1 ( 13 nucleotides upstream ) is a point mutation in the oORF initiation codon ( Fig 1A ) . A dual luciferase reporter plasmid was used to measure STN1 URS driven expression . STN1-u2 slightly increased expression ( 1 . 2 fold ) while STN1-u1 dramatically increased expression ( 10 fold ) ( Fig 1B ) . These data suggest that the STN1 oORF is a strong inhibitor of Stn1 expression , while the STN1 uORF has a milder effect . We next determined the effects of the uORF and oORF in the genome . STN1-u1 and STN1-u2 point mutations were introduced into the STN1 chromosomal locus , upstream of a C-terminal epitope-tagged STN1 construct . Western blot analysis of Myc-tagged Stn1 protein , which retains function and has been widely used [21 , 22] , confirmed that STN1-u1 has a much stronger effect on gene expression ( 12-fold ) than STN1-u2 ( no detectable increase ) ( Fig 1C and 1D ) . These results , concordant with the luciferase assays , allow us to conclude that the STN1 oORF is a potent inhibitor of gene expression . The STN1 oORF could reduce expression by impeding translation , reducing mRNA levels , or both . To test these possible mechanisms STN1 transcript levels were measured . A 4-fold increase in STN1 transcript levels was caused by STN1-u1 ( Fig 1E ) , significantly less than its 12-fold effect on protein levels ( Fig 1D ) . This difference suggests that the STN1 oORF affects gene expression by reducing transcript levels and translation of these transcripts . Interestingly , mild overexpression of STN1 , due to a single copy ( centromeric ) plasmid containing STN1 genomic DNA , suppressed temperature sensitive ( ts ) , telomere-defective , cdc13-1 cells and was the first reported connection between STN1 and telomeres [14] . Therefore , it seemed likely that the STN1 oORF affected fitness of cdc13-1 cells . To test this STN1-u1 was combined with cdc13-1 . Interestingly , growth of cdc13-1 cells at 26°C and 29°C was dramatically improved by STN1-u1 , demonstrating that the STN1 oORF acts to reduce the fitness of telomere defective cdc13-1 cells ( Fig 2A ) . The strong effect of STN1-u1 on fitness of cdc13-1 cells also allowed us to test whether STN1-u1 was dominant or recessive . Interestingly , although perhaps as to be expected given that STN1-u1 increases expression , STN1-u1 is dominant over the wild type STN1 allele in diploid cdc13-1/cdc13-1 cells ( S1 Fig ) . To test the effects of the STN1 oORF in the context of other telomere defects STN1-u1 was combined with yku70Δ . yku70Δ cells have short telomeres and are temperature sensitive due to an accumulation of single stranded DNA near telomeres at higher temperatures [23] . Mild overexpression of STN1 , using a low copy centromeric vector , has previously been shown to reduce fitness of yku70Δ cells at 36°C [24] . Interestingly , STN1-u1 strongly reduced growth of yku70Δ cells at all temperatures tested ( Fig 2B ) . The strong negative effect of STN1-u1 on yku70Δ cell growth across the range of temperatures is unusual . For comparison , nmdΔ mutations , which also increase Stn1 levels , only inhibit yku70Δ growth at high temperatures [24] . Increased Stn1 levels , due to plasmid-induced overexpression or inactivation of the nonsense mediated mRNA decay pathway have been reported to reduce telomere length [12 , 25] . Consistent with these results we observed that telomeres of STN1-u1 cells were as short as those of nmd2Δ mutants ( Fig 2C ) . Interestingly , a further reduction in telomere length was observed when STN1-u1 was combined with nmd2Δ or yku70Δ mutations ( Fig 2C ) . This data suggests that Nmd2 and Yku70 act at least somewhat independently of Stn1 levels to affect telomere length . In conclusion , high levels of Stn1 caused by STN1-u1 strongly reduce telomere length . The STN1 oORF affects Stn1 levels and has at least three telomere related phenotypes , including strong suppression of cdc13-1 . Published genome-wide cdc13-1 suppressor analyses identified nam7Δ , nmd2Δ and upf3Δ , affecting the three central components of nonsense mediated decay , and tma20Δ and tma22Δ as similarly strong suppressors of cdc13-1 ( S2 Fig ) [24] . nmdΔ mutations suppress cdc13-1 principally by increasing Stn1 levels [22 , 24] . TMA20 and TMA22 encode homologues of Drosophila/Human heterodimeric translation re-initiation factors MCT-1Tma20 and DENRTma22 ( S2 Fig ) , but how they affect cdc13-1 cell fitness is unclear [27 , 28] . MCT-1Tma20 and DENRTma22 have been shown to promote expression of genes with uORFs [29 , 30] . Tma20 and Tma22 were therefore plausible candidates to interact with the STN1 uORF or oORF to affect Stn1 expression . To confirm that Tma20/Tma22 affected cdc13-1 fitness similarly to the NMD pathway , the interactions between tma20Δ , tma22Δ or nmd2Δ and cdc13-1 were tested by spot tests . By this assay , growth of cdc13-1 cells was indeed improved by tma20Δ and tma22Δ but the effects were much less than the effect of nmd2Δ ( Fig 3A ) . There was no difference in the fitness of cdc13-1 tma20Δ , cdc13-1 tma22Δ or cdc13-1 tma20Δ tma22Δ cells , in agreement with data suggesting that Tma20 and Tma22 function as a heterodimer , similarly to MCT-1Tma20 and DENRTma22 [27 , 28] ( S2 Fig ) . tma20Δ or tma22Δ did not further improve fitness of cdc13-1 nmd2Δ cells ( Fig 3A ) , suggesting that Tma20/Tma22 affect fitness of cdc13-1 cells by a similar mechanism to the NMD pathway . Overall these data are consistent with the hypothesis that like Nmd2 , Tma20 and Tma22 affect fitness of cdc13-1 cells by decreasing levels of Stn1 . One hypothesis to explain why tma20Δ had weaker effects than nmd2Δ on cdc13-1 cell fitness was that higher levels of Stn1 better suppressed cdc13-1 , and that tma20Δ increased Stn1 levels less than nmd2Δ . To measure the comparative effects of tma20Δ and nmd2Δ on Stn1 expression , western blots were performed . As previously reported Stn1 levels were several fold higher in nmd2Δ cells [22] , and as predicted , slightly higher in tma20Δ cells ( Fig 3B and S3 Fig ) . nmd2Δ tma20Δ cells had marginally higher Stn1 levels than nmd2Δ single mutants , suggesting that Nmd2 and Tma20 affect Stn1 levels independently ( Fig 3B ) . It is known that NMD affects Stn1 levels by affecting RNA abundance [17] , and since Tma20 and Tma22 homologs affect translation initiation [29 , 30] , it seemed plausible that Tma20/Tma22 instead affected STN1 translation . Consistent with this hypothesis , tma20Δ did not significantly increase STN1 transcript levels ( Fig 3C ) , whereas , nmd2Δ increased transcript ( and protein levels ) about 6-fold ( Fig 3B and 3C , S3 Fig ) . These data show that Tma20 affects Stn1 protein levels without strongly affecting transcript levels . To test if Nmd2 and Tma20 affect Stn1 expression via the uORF or oORF , luciferase assays were used . Expression driven by STN1 , STN1-u2 and STN1-u1 URSs in wild type , tma20Δ and nmd2Δ backgrounds was measured . Consistent with the western blot analysis of strains with integrated alleles there was a small increase in expression of STN1-luc in tma20Δ cells , and a larger increase in nmd2Δ cells ( Fig 3B and 3D ) . The STN1-u2-luc construct , lacking the most upstream uORF , showed a similar pattern of expression to the native STN1 construct in wild type , tma20Δ and nmd2Δ cells ( Fig 3D ) . These results suggest that Nmd2 and Tma20 do not reduce STN1 expression via the STN1 uORF . In contrast , however , STN1-u1-luc , lacking the oORF , showed a very different pattern , with very similar levels of gene expression being observed in wild type , tma20Δ and nmd2Δ cells ( Fig 3D and S3 Fig ) . These data indicate that both Tma20 and Nmd2 reduce Stn1 expression principally via the oORF . Consistent with these plasmid-based luciferase assays , we did not observe any further increase in the levels of Stn1-Myc when nmd2Δ was combined with STN1-u1 ( S4 Fig ) . Furthermore , no notable changes in STN1 transcript levels were observed between nmd2Δ and nmd2Δ STN1-u1 cells ( S4 Fig ) . Overall , these data support the view that Tma20 and Nmd2 affect Stn1 levels principally via the oORF and that their effects on Stn1 levels can explain their effects on fitness of cdc13-1 telomere-defective cells . The STN1 oORF terminates just 2 nucleotides after the main STN1 ATG and we wondered if this proximity contributed to the effectiveness of the oORF in reducing gene expression . To test this notion , a point mutation in the oORF termination codon was introduced , thus creating STN1-111 , which increases the length of the oORF from 6 to 32 amino acids ( Fig 4A ) . Interestingly , STN1-111 increased levels of Stn1 ( Fig 4B and 4C ) , suggesting that either the short length of the natural STN1 oORF , or the proximity of the oORF stop codon to the STN1 CDS initiation codon , contributes to the effectiveness of the STN1 oORF in reducing gene expression . Furthermore , the levels of Stn1 are similar in STN1-111 , tma20Δ and STN1-111 tma20Δ cells , suggesting that Tma20 decreases STN1 expression by a mechanism that is dependent on the context of the native oORF termination codon ( Fig 4C and S4 Fig ) . The effects of Nmd2 on expression of STN1-111 were also examined . While nmd2Δ increased STN1-111 mRNA and protein levels the effect was proportionately less than on wild type STN1 mRNA and protein levels ( Fig 4B and 4C and S4 Fig ) . These data suggest that the STN1-111 transcript is a weaker target for the NMD pathway than the STN1 transcript and explain why the STN1-111 transcript is more abundant than STN1 ( Fig 4D ) . To explore how Tma20 , Nmd2 and the Stn1 oORF interact to affect fitness of telomere-defective stains we combined nmd2Δ and tma20Δ with STN1-u1 and STN1-111 in a cdc13-1 background . Consistent with the idea that Tma20 reduces fitness of cdc13-1 cells via the STN1 oORF , tma20Δ does not further increase the fitness of cdc13-1 STN1-u1 or cdc13-1 STN1-111 cells ( S5 Fig ) . However , the correlation between Stn1 expression levels in nmd2Δ and STN1-u1 cells and the effect of these mutations on cdc13-1 fitness was less clear . nmd2Δ increased Stn1 less than STN1-u1 but better improved the fitness of cdc13-1 cells ( S4 and S5 Figs ) . It is known that NMD affects levels of hundreds of transcripts , including many encoding telomerase components and regulators , such as Ten1 [17] . We rationalized that cdc13-1 nmd2Δ mutants may be fitter than cdc13-1 STN1-u1 mutants because nmd2Δ causes overexpression of both Stn1 and Ten1 while STN1-u1 only affects Stn1 levels [22] . Consistent with this hypothesis , plasmid driven overexpression of Ten1 further improved fitness of cdc13-1 STN1-u1 cells ( with already very high levels of Stn1 overexpression ) while having barely any effect on cdc13-1 cells ( S5 Fig ) . We conclude that suppression of cdc13-1 telomere defects can be improved by coordinated overexpression of its two partner proteins , Stn1 and Ten1 . It is clear that the yeast STN1 oORF plays an important role in maintaining low levels of Stn1 , which in turn contributes to critical telomere functions , such as capping and length control . Given that Stn1 has conserved functions in human cells , we wondered if an oORF was also found in the human STN1 transcript leader ( TL ) . Indeed , analysis of the human STN1 revealed that there are two , encoding overlapping 15 and 19 amino acid oORFs , sharing a stop codon ( Fig 5A ) . The third nucleotide of the ORF stop codons is the first nucleotide of the STN1 CDS ( Fig 5A ) . To test whether the oORFs of STN1 reduce expression in human cells STN1 URS activity was measured using dual luciferase assays . Point mutations introduced into each of the oORF initiation codons , STN1-no-oORF , increased expression 3 . 4 fold in human cells . Therefore , human STN1 , like yeast STN1 , is regulated by overlapping open reading frames that strongly reduce STN1 expression . The yeast STN1 oORF more strongly reduces gene expression than the STN1 uORF and therefore it was possible that this is a general phenomenon . To systematically explore the effects of uORFs and oORFs in other contexts , yeast and human genomes were analyzed to calculate the fraction of transcripts that contain uORF and oORFs . To infer the effects of natural selection these numbers were compared to the number of uORFs and oORFs calculated to occur in individually scrambled TL sequences ( Fig 6A ) . In yeast the proportion of transcripts with uORFs or oORFs observed was far less than expected based on analysis of scrambled sequences ( 12 . 6% observed vs 35% expected for uORFs , 7 . 4% vs 34% for oORFs ) . In humans the proportion of transcripts with uORFs or oORFs was also less than expected ( 48 . 5% vs 54% for uORFs , 24 . 5% vs 49% for oORFs ) . uORFs and oORFs are more common in human than yeast TLs , presumably because human TL sequences , median length 173 nucleotides , are longer than yeast , median length 49 nucleotides . However , the relationship between TL length and the likelihood of uORFs or oORFs being present was unclear . To explore this relationship , the theoretical number of uORFs and oORFs found with increasing length of yeast TL sequences was calculated . As expected uORFs and oORFs increase in frequency as TL length increases , but the observed frequency was always less than expected from analysis of randomized sequence , presumably reflecting natural selection ( Fig 6B ) . Importantly , at TL lengths longer than 50 bases , or so , there are always proportionately fewer oORFs than uORFs ( S6 Fig ) , consistent with the idea that oORFs have been more strongly selected against than uORFs . Finally , in yeast there is a significant reduction in protein abundance associated with genes that encode oORFs , in comparison with those that encode uORFs ( Fig 6C ) . Together these data are consistent with the view that , on average , oORFs more strongly reduce gene expression than uORFs .
We have shown that levels of Stn1 are dramatically reduced by upstream oORFs . In yeast , STN1 contains both a uORF and an oORF but the latter is a far more potent inhibitor of gene expression . The STN1 oORF reduces gene expression by at least two routes . First , the STN1 oORF targets the transcript for degradation by NMD , and second , it reduces translation efficiency in a Tma20/Tma22 dependent manner . Reducing the efficacy of the STN1 oORF by mutating its initiation codon , its stop codon , or by inactivating NMD2 or TMA20 results in a range of different telomere related phenotypes showing how important the Stn1 oORF is to telomere function . In yeast , it is clear that low Stn1 levels are critical for normal telomere function [12] . Furthermore , it has been suggested that when overproduced Stn1 performs additional functions , is recruited to non-telomeric sites and overrides the S-phase checkpoint [15] . We now show the yeast STN1 oORF is key to maintaining low levels and appropriate functions . Analogously , in human cells , where STN1 has been shown to have a role in replication of non-telomeric DNA , perhaps oORF regulation affects the balance between telomeric and non-telomeric roles of Stn1 [32 , 33] . The NMD pathway was originally defined as targeting mRNAs that contained premature termination codons ( PTCs ) , but the list of NMD targets is expanding . It now includes mRNAs that contain uORFs , long 3’UTRs , frameshifts , unspliced introns , aberrant transcript isoforms and otherwise normal transcripts with low translation efficiencies due to out-of-frame translation or lower than average codon optimality [17 , 34 , 35] . In this case we have established that STN1 mRNA targeting by NMD depends on both the start and stop codons of the oORF , suggesting that the short length of the oORF and/or the close proximity of the oORF termination to the STN1 CDS initiation are important for NMD to target the STN1 transcript . In Drosophila and mammalian cell-based experiments MCT-1Tma20/DENRTma22 contributed to increased expression of transcripts that contain uORFs , whereas we found that Tma20/Tma22 reduced expression of STN1 via the oORF [29 , 30] . Biochemical experiments show that MCT-1Tma20/DENRTma22 promote the dissociation of the 40S ribosome subunit following translation termination [36] . One hypothesis that may explain the effects of Tma20/Tma22 on STN1 expression in yeast is that Tma20/Tma22 reduce translation re-initiation by promoting 40S ribosome subunit dissociation ( S7 Fig ) . This hypothesis is supported by the finding that Tma20/Tma22-dependant regulation of Stn1 levels requires the STN1 oORF stop codon to be in close proximity to the STN1 CDS initiation codon . Furthermore , MCT-1Tma20/DENRTma22 have been shown to affect translation re-initiation on some bicistronic viral transcripts [37] . It has become increasingly clear that uORFs are efficiently translated and associated with lower levels gene expression [19 , 20] . Mutations in uORF initiation and termination codons have been linked to human malignancies [38] and a mutation introducing a uORF into the TL of CDKN2A causes familial predisposition to melanoma [39] . In specific cases uORFs have been shown to act as regulatory mechanisms to control gene expression under conditions of stress . The best studied examples are ATF4 and GCN4 which encode homologous transcription factors in human and yeast cells [40 , 41] . The levels of each are regulated by uORFs and oORFs which facilitate translational upregulation in response to nutrient stress [40 , 41] . In a more general sense , uORFs have been suggested to permit the preferential translation of specific transcripts following DNA damage [42] . The STN1 oORF provides , in principle , a powerful mechanism to rapidly increase Stn1 levels . It was recently shown that growth at high temperature increases yeast Stn1 levels [43] , and it would be interesting to determine whether the STN1 oORF contributes to this type of upregulation . The STN1 oORF clearly provides a powerful mechanism to reduce Stn1 levels appropriately , to positively affect telomere function . More generally it seems that oORFs are more potent than uORFs at inhibiting gene expression . oORFs may therefore be part of a powerful mechanism of reducing protein levels across biology .
Standard procedures for yeast culture , mating and tetrad dissection were followed . Strain genotypes are in S1 Table . W303 strains were used and YEPD was supplemented with adenine ( 75 mg/l ) . Gene deletions were performed using one-step PCR to replace TMA22 with a marker and confirmed by PCR . STN1 point mutations were introduced into the genome using integrative plasmids based on pRS406 . These strains contained duplicated copies of the STN1-PDC2 URS separated by a URA3 gene . The URS adjacent to STN1 contained uORF point mutations and the URS adjacent to PDC2 did not ( S8 Fig ) . As a control , the same construct without mutations was also integrated . Primers and plasmids are listed in S2 and S3 Tables . A pool of colonies ( >10 ) were grown in 2 mL liquid YEPD or SC media overnight at 23°C . Serial dilutions , as indicated in legends , were made in water and spotted onto round or rectangular agar plates using a replica plating device . Plates were incubated for indicated number of days at the indicated temperatures before being photographed . Protein extractions and western blots were performed essentially as described previously [44] . 10 mL mid log phase cells were washed twice with 2 mL of 20% TCA and re-suspended in 100 μL of 20% trichloroacetic acid ( TCA ) . Cell pellets were frozen at -80°C . Once thawed 100 μL glass beads were added to the pellets and cells mechanically lysed using a Precellys ( 2x 15s 6 , 500rpm ) . 200 μL of 5% TCA was added and samples briefly vortexed . Samples were centrifuged at 13 , 000 rpm for 10 minutes at 4°C and pellets re-suspended in 100 μL Laemmli loading buffer ( Bio-Rad ) with 5% β-mercaptoethanol . 20 μL 1M Tris was added to samples to neutralise the pH . Samples were boiled for 3 minutes , centrifuged at 13 , 000 rpm for 10 minutes and the supernatant transferred to a clean tube . 10 μL of protein extracts were loaded onto a gradient ( 4–15% ) precast gel ( Bio-Rad Mini-Protean TGX ) and run for 90 minutes at 100V in Tris/Glycine/SDS running buffer ( Bio-Rad ) . Proteins were blotted , for 30 minutes , onto nitrocellulose membranes using the Trans-Blot Turbo Transfer System ( Bio-Rad ) according to the manufacturer’s protocol . Anti-C-Myc ( 9E10 ( 1 in 2000 in 1% milk ) from Abcam ) was used to detect Stn1-MYC and anti-tubulin ( 1 in 2000 ) antibodies from Keith Gull , Oxford , UK , was used to detect Tubulin as a loading control . Proteins were detected using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate according to the manufacturer’s instructions and imaged on a G-box imager ( Syngene ) . RNA purification and RT-PCR was carried out as described previously [45] . RNA was purified using the RNEasy Mini Kit ( QIAGEN , 74104 ) and by DNase I digestion ( Invitrogen , 18068–015 ) . Quantitative RT-PCR was carried out using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit ( Invitrogen , 11736–059 ) . 2 μL RNA sample ( 80 ng/μL ) was added to 8 μL of the reaction mix ( 10 μM F primer ( 0 . 2 μL ) , 10 μM R primer ( 0 . 2 μL ) , Superscript III Platinum Taq Mix ( 0 . 2 μL ) , 2x SYBR Green Reaction Mix ( 5 μL ) , ROX reference dye ( 0 . 2 μL ) , DEPC-treated water ( 2 . 2 μL ) ) in a 96 well plate . An ABI Systems StepOnePlus thermal cycler was used ( 1 cycle: 50°C for 3 minutes , 1 cycle: 95°C for 5 minutes , 40s cycle: 95°C for 15 seconds then 60°C for 30 seconds , 1 cycle: 40°C for 1 minute ) . RNA samples were normalized relative to the BUD6 loading control . Southern blot analysis was used to assess telomere length as previously described [46] . Genomic DNA was extracted , digested with XhoI and run overnight on a 1% agarose gel at 1 V/cm . Southern transfer was performed using a BioRad Vacuum Blotter according to manufacturer’s instructions . Probe labelling , hybridization and washing were performed according to the DIG High Prime DNA Labelling and Detection Starter Kit II ( Roche ) instructions . A probe that annealed to the Y’ elements and TG telomere repeats was made using DNA from a plasmid ( pDL987 ) that contained 120 bp of TG repeats and 752 bp of the upstream Y’ element from telomere VIII-R . Loading control pictures were taken from each gel , before transfer , using SYBR Safe to stain the total DNA . HCT116 F/F CTC1 cells ( obtained from Carolyn Price and containing loxP sites at the CTC1 locus ) were maintained in McCoys 5a media ( supplemented with foetal bovine serum ( 10% ) , PenStrepGlut ( Corning ) and puromycin ( 100 ng/μL ) ) under 5% CO2 in a humidified incubator . 1 μg plasmid DNA was transfected into 1 x 106 cells using a Nucleofector ( Lonza ) according to manufacturer’s protocol and incubated for 24 hours in a 6 well plate . Dual-Luciferase Reporter Assay System ( Promega ) was used to measure luciferase expressed from mammalian cells according to manufacturer’s protocol using the active lysis by scraping method . Plasmids were designed to quantify gene expression in vivo using two different reporter genes , encoding Renilla and Firefly luciferases . Firefly luciferase was fused to a constitutive promoter , PGK1 , to act as a ‘loading control’ . Renilla luciferase was fused with STN1 URSs , which encompassed the promoter and TL . To help ensure that expression of the test URS was unaffected by the loading control the genes encoding Renilla and Firefly luciferase were fused to ADH1 and CYC1 terminators respectively , and orientated in opposite orientations . 2 mL yeast cultures were grown to saturation overnight in—LEU media in a rotating wheel in an incubator at 30°C . From this a fresh 2 mL culture was inoculated with 100 μL saturated cells and rotated at 30°C until an OD600 of 0 . 6–0 . 8 was achieved ( 6 hours ) . Cultures were harvested and re-suspended in 90 μL of passive Lysis buffer from Dual-Luciferase Reporter Assay System ( Promega ) . 10 μL of cells suspension were then added to a well of a 96-well white plate . A PolarStar ( Omega ) plate reader was programmed to dispense 50 μL of LAR II ( Promega ) , shake for 5 seconds , record the luminescence signal ( 4 readings with 0 . 5 second interval times ) , dispense 50 μL Stop & Glo Reagent ( Promega ) , shake for 5 seconds , and again record the luminescence signal ( 4 readings with 0 . 5 second interval times ) . The mean of the 4 readings that were recorded was used as the final measurement . For yeast , the sequences of all genes , plus 1000 bases upstream , were retrieved from YeastMine [47] . TL lengths were obtained from [48] , using the longest isoform reported , or if unavailable from [49] . The locations of uORF and oORFs were determined computationally and are shown in S1 File . The Python scripts to identify the uORFs and oORFs within the transcript leader sequences can be found on Github ( https://github . com/vickytorrance/uAUGs ) . To infer the effects of natural selection the TL sequences were individually randomized once and the number of uORFs and oORFs in randomized sequences calculated . For human transcripts , cDNA and TL sequences were downloaded from Biomart ( Ensembl ) , only considering ‘Protein coding gene’ transcripts and the presence of uORFs or oORFs calculated as for yeast ( S2 File ) .
|
Telomeres are special structures at the ends of linear chromosomes that help protect the genetic information that chromosomes carry . The levels of telomere proteins are important and can affect diseases such as cancer and ageing . The CST complex is comprised of three proteins and binds human and yeast telomeres . Levels of Stn1 , a very low abundance protein , are of particular importance to telomere function in yeast cells . There are many ways to affect protein levels but little was understood about how Stn1 levels are controlled . We show that levels of Stn1 in yeast and human cells are reduced by the presence of an upstream overlapping open reading frame ( oORF ) . Cells lacking the oORF have short telomeres and increased fitness when combined with a defect in the Stn1-partner protein , Cdc13 . Interestingly , in another telomere defective context , yku70Δ cells missing the STN1-oORF are less fit . We show that the oORF reduces Stn1 levels by stimulating nonsense mediated mRNA decay and by reducing translation . More generally , genome-wide computational analysis shows that oORFs were strongly selected against during evolution and when present are associated with low protein abundance . We propose that oORFs are a powerful mechanism to regulate protein expression and function .
|
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"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
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"chromosome",
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2018
|
Overlapping open reading frames strongly reduce human and yeast STN1 gene expression and affect telomere function
|
The use of filter paper as a simple , inexpensive tool for storage and transportation of blood , ‘Dried Blood Spots’ or Guthrie cards , for diagnostic assays is well-established . In contrast , there are a paucity of diagnostic evaluations of dried cerebrospinal fluid ( CSF ) spots . These have potential applications in low-resource settings , such as Laos , where laboratory facilities for central nervous system ( CNS ) diagnostics are only available in Vientiane . In Laos , a major cause of CNS infection is Japanese encephalitis virus ( JEV ) . We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay ( JEV MAC-ELISA ) . Sample volumes , spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution ( PBS ) to end-limits of detection by JEV MAC-ELISA . A conventional protocol , involving eluting one paper punch in 200μl PBS , did not detect the end-dilution , nor did multiple punches utilising diverse spotting techniques . However , pre-cut filter paper enabled saturation with five times the volume of CSF-substitute , sufficiently improving sensitivity to detect the end-dilution . The diagnostic accuracy of this optimised protocol was compared with routine , neat CSF in a pilot , retrospective study of JEV MAC-ELISA on consecutive CSF samples , collected 2009–15 , from three Lao hospitals . In comparison to neat CSF , 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81 . 6% ( 65 . 7–92 . 3 95%CI ) positive agreement , 96 . 8% ( 91 . 0–99 . 3 95%CI ) negative agreement , with a kappa coefficient of 0 . 81 ( 0 . 70–0 . 92 95%CI ) . The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access . It has the potential to improve national JEV surveillance and inform vaccination policies . The saturation of filter paper has potential use in the wider context of pathogen detection , including dried spots for detecting other analytes in CSF , and other body fluids .
The last few decades have seen a substantial growth of novel and complex diagnostic tests [1] . This has not been accompanied by the same development in global laboratory infrastructure , a fundamental component of any effective healthcare system [2]–[4] . In settings with poor access to laboratories , the use of dried blood spots on filter paper ( DBS ) is now a well-established diagnostic tool for storing and transporting blood [5]–[9] . DBS obviates the need for a cold chain . The technique is also simple , economical and requires smaller sample volumes . Strikingly , there is a paucity of data on use of dried spots of other body fluids [5] . There are only three publications evaluating the use of dried cerebrospinal fluid ( CSF ) spots ( DCS ) in diagnosing infectious diseases [10]–[12] . This may in part be due to the significant level of technical expertise required to perform a lumbar puncture ( LP ) . CSF is more difficult to obtain , available in smaller volumes , often with lower concentrations of analyte , and may have lower sensitivity of corresponding diagnostic assays than for blood . Furthermore , approvals and practicalities of studies involving LPs are more challenging . It has been acknowledged that research involving CNS infections has been a neglected field [13] . However , it is also possible that more DCS methods have been tried , proved futile and were not published . Amongst CNS infections , Japanese encephalitis virus ( JEV ) is recognised as the most common causative pathogen in Asia . In the 24 endemic countries it is suggested that JEV causes 67 , 900 cases , and 20 , 000 deaths per year [14] , [15] . In the Lao PDR ( Laos ) , there is evidence to suggest that JEV is a major cause of CNS infection [16] , [17] . However , the wider epidemiology of JEV outside Vientiane is poorly documented due to the lack of laboratory facilities in the provinces . The diagnosis of JEV relies on laboratory facilities to test for anti-JEV IgM in the blood and/or CSF , with significantly increased proportion of false positive results if relying on blood alone [17] . Improved JEV laboratory networks and infrastructure have developed since 2008 , with a global central laboratory in Japan , regional centres for the Western Pacific in China and Korea and respective national centres [18] , [19] . The central laboratory performs the reference standard test , plaque reduction neutralisation assay ( PRNT ) [18]–[22] . However the mainstay of routine diagnosis involves commercial kits performing anti-JEV IgM antibody capture enzyme-linked immunosorbent assay ( JEV MAC-ELISA ) . The use of DCS in the diagnosis of JEV by JEV MAC-ELISA would have potential application in low-resource settings , for diagnostics , wider epidemiological studies on the aetiology of CNS infections and impact of vaccination . We aimed to optimise a DCS protocol for the detection of anti-JEV IgM using JEV MAC-ELISA and evaluate its performance in a pilot study of consecutive patient samples as compared to routine neat CSF .
Patient CSF and serum samples were collected at three hospitals in Vientiane , the Friendship , Children and Setthathirat Hospitals , from 2009 to 2015 ( Fig 1 ) . CSF was collected by LP from patients with suspected CNS infection , and without contraindications to LP , according to the judgment of the responsible physician . Written consent was obtained from the patient , parent and/or guardian . Samples were sent to Mahosot Hospital and stored at -80°C . The study was part of a study on the causes of CNS infections in Laos . Ethical clearance was granted by the Ethical Review Committee of the Faculty of Medical Sciences , National University of Lao , and the Oxford University Tropical Ethics Research Committee , Oxford , UK . Diagnostic testing was performed on anonymised and frozen CSF samples . LPs were performed as part of a routine diagnostic service if verbal ( 2003–2006 ) or written ( 2006–2011 ) consent was given by patients or their parents/guardian . Consent for all patients was documented on a form that had been approved by the ethics committee . The lead investigator has been trained in Good Clinical Practice , and has completed Human Tissues Training . The WHO recommended commercial JEV MAC-ELISA assay is the Inbios JE Detect ( Washington , USA ) [19] . This measures the Optical Density ( OD ) of each sample with JEV Recombinant Antigen , JERA , compared to Normal Control Antigen ( NCA ) to adjust for background nonspecific reactivity . 100μl of diluted sample ( 1/10 dilution for CSF as recommended by WHO ) are used for a single test , 50μl in the JERA well and 50μl in the NCA well . The result is an Immune Status Ratio , ISR , ( JERA OD/ NCA OD ) with qualitative interpretation , according to manufacturer’s instructions , as positive if >6 . 0 , equivocal if 4 . 0–6 . 0 , or negative if <4 . 0 . Equivocal results were classed as negative as per standard protocol in research optimising diagnostic tests [23] , [24] . Two types of filter paper were chosen , and utilised in all experiments . The 903 Protein Saver Card ( 903 ) ( Whatman , GE Healthcare Life Sciences , UK ) has been used as DBS for neonatal screening since 1963 [8] . It is made of highly purified cellulose cotton paper and is one of two FDA approved cards for DBS [5] . They have been used in the diagnosis of multiple diseases , involving elution and corresponding ELISA or PCR , including the detection of specific antibodies in CSF [25] . The 3MM Chr Blotting paper ( 3MM ) ( Whatman , GE Healthcare Life Sciences , UK ) is available as large sheets , is less expensive and more suitable for broader application in a resource limited setting . Despite being produced as a blotting paper rather than a collection paper , 3MM has been used reliably in serology testing of DBS [26] , [27] . Retrospective , consecutive analysis was performed on available CSF from all patients tested for JEV between February 2009 and July 2015 . CSF was spotted on 903 paper as per the optimised ‘pre-cut circle’ protocol detailed above , and stored at 25–30°C for 30 days as an approximation of the maximum time for the paper to reach the laboratory if this was performed in the field . DCS and neat CSF were compared on the same plate . Investigators were blinded to the previous neat CSF JEV ELISA results . The sample size ( n = 86 ) was calculated with values for sensitivity of Inbios JEV MAC-ELISA = 53% , expected positive agreement DCS with neat CSF = 75% , and expected negative agreement = 98% , programming formulae ( as per the CDC report referenced by T . Lee ) based on estimation of a kappa coefficient , into Microsoft Excel [23] , [28] . Results are presented in 2x2 tables , analysed by reporting ‘positive percentage agreement’ = ( positive by both DCS and neat CSF ) / ( positive by neat CSF ) , ‘negative percentage agreement’ = ( negative by both DCS and neat CSF ) / ( negative by neat CSF ) , overall percentage agreement ( positive or negative agreement by both DCS and neat CSF ) and kappa coefficient with respective 95% confidence intervals calculated in STATA 13 . 1 ( College Station , Tx ) [29] . The analysis acknowledges the fact that the comparison was not made with the reference standard , PRNT , that is only performed in a few reference laboratories . Further , the samples tested were paired , being taken from the same patient sample .
The conventional DBS protocol applied to DCS did not produce any positive results at the end-limit dilutions ( Table 1A displays results for 903 , for 3MM see S1 Table ) . The modified protocol with increased number of punches of the eluent , also did not produce any positive results at the end-limit dilutions ( Table 1B and S1 Table ) . Experiments were performed using CSF-substitute at x2 and x4 end-limit dilutions , to identify the limit of detection of the modified protocol . At the lowest dilution , x4 end-limit dilution , 25% of replicates were positive using a single spot . The same dilution was used with a respotting technique and produced 75% positive replicates . A third technique using the same dilution spotted as two spots , with half the punches taken from the centre of each spot , was positive in 58% and 25% % of replicates , for 903 and 3MM , respectively . The only difference between the filter paper types was more frequent detection ( 58 vs 25% ) using two spots on 903 versus 3MM . The final protocol evaluated a pre-cut circle of filter paper saturated with the maximal volume of CSF . A 1 . 49cm diameter pre-cut circle could be loaded with 250μl CSF-substitute , and this permitted detection of anti-JEV IgM in 100% of replicates ( Table 1C and S1 Table ) . In comparison , spotting 100μl on to a filter paper filled an approximate 2 . 1cm diameter circle for CSF and 1 . 3cm diameter sheet for blood [30] . Limitations of serum volume and JEV MAC-ELISA precluded testing all protocols at x2 and x4 end-limit dilutions . The pre-cut circle protocol with 903 filter paper was ultimately performed with 903 rather than 3MM filter paper in the optimised protocol as it showed marginally better results and was easier to handle . CSF was received and tested for JEV using Inbios JEV MAC-ELISA from 248 patients admitted from February 2009 to July 2015 . 132 patients had sufficient frozen CSF volumes , 10μl for neat CSF ELISA testing and 250μl to make the dried CSF spot following pre-cut circle protocol . After 30 days at room temperature ( 25–30°C ) the DCS and corresponding neat CSF were tested by Inbios JEV MAC-ELISA . The positive agreement for dried CSF spots compared to neat CSF was 81 . 6% ( 65 . 7–92 . 3 95%CI ) , negative agreement 96 . 8% ( 91 . 0–99 . 3 95%CI ) , overall agreement 92 . 4% and kappa coefficient 0 . 81 ( 0 . 70–0 . 92 95%CI ) , Table 2 . Thirty-eight neat CSF samples ( 29% ) and 34 filter paper samples ( 26% ) tested positive for JEV .
There are still considerable global deficits in laboratory capacity for diagnosing CNS infections [2] , [3] . This is seen in Laos , where laboratory diagnosis of JEV is confined to the capital , Vientiane , and elsewhere in rural Asia . DCS has the potential to provide a simple tool for improving clinical diagnostics and our understanding of the epidemiology of CNS infections . This study developed a robust protocol using DCS for the diagnosis of JEV by JEV MAC-ELISA , with excellent agreement demonstrated in a pilot study of patient samples . The use of DBS for storing , transporting and detecting anti-JEV IgM and anti-dengue IgM have already been well-documented in previous studies , and this was not repeated here . [21] , [31]–[34] The experiments performed to optimise the DCS protocol highlighted the key difference between blood and CSF , with poor performance of the conventional DBS and modified protocols likely due to low IgM concentration in the final eluate , IgM concentrations are lower in CSF than in blood [35] . This was corroborated by the improved sensitivity seen by increasing the serum concentration in the modified DCS protocol to x4 end-limit dilution of the neat CSF-substitute . This problem is compounded by the lower viscosity of CSF , leading to a wider distribution on filter paper [30] . There was no evidence to suggest a difference in the performance of the two types of filter paper , 903 and 3MM . Respotting , aiming to increase the analyte concentration , did not improve sensitivity . There is literature to suggest that analyte concentrates in the centre of a DBS [36]–[38] . However taking half ( 4 or 5 ) the punches from the centre of two spots did not demonstrate higher sensitivity . In contrast , the ‘pre-cut circle’ protocol worked very well . Saturation of filter paper could be achieved by cutting beforehand and then carefully loading with the maximum CSF volume the paper could hold . The volume of CSF that could be added to the pre-cut circle was five times that of a single CSF sample spotted on a sheet of filter paper . Prior cutting has been utilised in DBS [39] , [40] . However , while the term ‘saturation’ is frequently used in dried spot protocols , this is not true saturation , by which we mean loading of fluid to the maximal amount that the filter paper will possibly absorb before it leaks off the filter paper . The use of this saturation technique has important implications in DBS in diagnostics for diseases that have low concentrations of analyte , dried spots involving other body fluids , as well as other fields of research . Advantages of the pre-cut circle protocol include greater sampling efficiency , as it prevents loss of the analyte not punched from the filter paper . It is simpler , quicker , more reliable and reduces concerns regarding cross-contamination . The retrospective study of 132 consecutive patients recruited from three hospitals demonstrated a high level of agreement between the optimised ‘pre-cut’ circle protocol and neat CSF , with positive agreement of 81 . 6% ( 65 . 7–92 . 3 95%CI ) , negative agreement of 96 . 8% ( 91 . 0–99 . 3 95%CI ) and kappa coefficient 0 . 81 ( 0 . 70–0 . 92 95%CI ) . It is possible that kappa agreement overestimates the effect size , as the comparison involved paired samples , tested using the same diagnostic assay . Further , kappa agreement has been criticised as being dependent on disease prevalence , with a low prevalence seen in this study risking a false negative result [41] . However , it is the industry standard [28] , [29] , [42] . P values for the kappa statistic are not presented as these are considered misleading [42]–[44] . 10 of 132 ( 8% ) CSF samples had discordant ELISA results between neat CSF and pre-cut saturated paper samples . Refreezing samples may have affected results . Unfortunately there was insufficient sample volumes to investigate this by the reference standard , PRNT . Cross-reactivity with JEV MAC-ELISA occurs with other Flavivirus antibodies , and in this region , testing for dengue is crucial . We had insufficient sample volumes to test for dengue antibodies as well as JEV antibodies in the DCS . A larger study is needed to evaluate the differential diagnosis of JEV from other flaviruses , performing the protocol in ‘full’ with 500μl CSF , on a 2 . 1cm diameter circle , eluting with 400μl PBS/0 . 05% Tween-20 . Rules of thumb for the evaluation of any diagnostic test are the ‘ASSURED’ criteria , i . e . whether it is Affordable , Sensitive , Specific , User-friendly , Rapid and robust , Equipment-free and Deliverable to end-users [45] . The currently used WHO recommended standard test for JEV is the commercial Inbios JEV MAC-ELISA , CSF usually diluted 1:10 with a sensitivity under field conditions suggested to be 53% [23] . However , it is not accessible for the large populations at risk of JEV living in rural Asia . The DCS ‘pre-cut and saturated’ protocol potentially provides a simple , economical and accessible tool to store and transport CSF . The requirements for the technique are minimal , as PBS/ 0 . 05% Tween-20 and filter paper are easily available and inexpensive . Further work is crucial to evaluate the technique in larger cohorts , preparing dried CSF spots in multiple centres under field conditions , transporting the spots at the high ambient temperatures experienced in Asia , storing for longer periods ( months to years ) , and testing paired with neat CSF . Ideally this could be performed as a diagnostic accuracy study , comparing both serum and CSF anti-JEV IgM ELISA results with PRNT , and with testing for anti-dengue IgM and Dengue NS1 antigen by JEV MAC-ELISA . It is notable that DCS may improve access to diagnostics in areas with poor laboratory facilities but does not reduce the time for diagnostic processing or ‘turnaround time’ , and this is still a pertinent gap in this field . Permitting the collection , storage , and shipment of samples without the need for cold chain , this protocol could undoubtedly aid JEV surveillance by collecting data from rural hospitals with staff able to perform LPs that were not accessible before . This novel design has significant implications for use in the wider context of pathogen detection , including dried spots methods for other analytes in CSF , and other body fluids . There are no facilities for systematic , population based surveillance of CNS infections in Laos outside the capital , as is the case in many parts of the world [46] . Even in countries where surveillance is performed , there are deficits in laboratory capacity , for diagnostics of JEV and other similar pathogens [3] , [17] , [23] . This affects both clinical management , outcome and epidemiological understanding . This is illustrated by the current epidemic of Zika virus , a related Flavivirus , with an unclear and poorly characterised association with neurological complications . There is an urgent need for simple and economical solutions to improve our understanding of the aetiology and wider epidemiology of CNS infections . With the introduction of nationwide JEV vaccination , it is likely that diagnosis by CSF will be even more important , and dried CSF spots could help understand the impact of vaccination .
|
Japanese encephalitis virus infection is widespread in Asia , and primarily affects children in poor , rural areas . The virus spreads to the brain and spinal cord resulting in significant death and disability . Diagnosis requires testing for the immune response ‘antibody’ specific to Japanese encephalitis virus in the cerebrospinal fluid that surrounds the brain and spinal cord . However , in most areas where the infection occurs there are no laboratory facilities . In the absence of laboratory diagnosis , we have poor knowledge of the extent of the problem in these areas . Dried blood spots on filter paper have been used widely for many years as simple , cost-effective tools for transporting samples for testing . However , there have been few attempts at using dried cerebrospinal fluid spots . We developed a technique for using dried spots to store and test cerebrospinal fluid for antibodies to Japanese encephalitis virus . We compared the results of dried spots with routine neat cerebrospinal fluid in 132 patient samples , and demonstrated excellent agreement between the two tests . This novel method for saturating dried cerebrospinal fluid spots has the potential to enhance our knowledge of Japanese encephalitis virus epidemiology , and inform health policies where they are most needed . It could also be transferred for use in diagnosing other infectious diseases , including using other body fluid samples .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
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2016
|
Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots
|
Homologous recombination ( HR ) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes . This process initiates from single-stranded ( ss ) DNA and is driven by a universal recombinase , which promotes strand exchange between homologous sequences . The bacterial recombinase , RecA , is loaded onto ssDNA by recombinase loaders , RecBCD and RecFOR for genome maintenance . DprA was recently proposed as a third loader dedicated to genetic transformation . Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae . We firstly established that RecFOR proteins are not required for plasmid transformation , strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process . We then observed no reduction in chromosomal transformation using a PCR fragment as donor , contrasting with the 10 , 000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation . However , a ∼1 . 45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants . To account for this limited deficit , we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency ( >30% of cells ) formation of chromosome dimers as an intermediate in the generation of tandem duplications , and that RecFOR were crucial for dimer resolution . We validated this hypothesis , showing that the site-specific recombinase XerS was also crucial for dimer resolution . An even higher frequency of dimer formation ( >80% of cells ) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA , which contains numerous inversions compared to pneumococcal chromosome , each potentially promoting dimerization . In the absence of RecFOR and XerS , dimers persist , as confirmed by DAPI staining , and can limit the efficiency of transformation , since resulting in loss of transformant chromosome . These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci . These observations presumably apply to most naturally transformable species .
Homologous recombination ( HR ) is crucial for both maintenance of genome integrity and generation of diversity across all kingdoms of life . HR initiates universally from single-stranded ( ss ) DNA and involves strand exchange between homologous DNA sequences , catalyzed by homologous recombinases . In bacteria , HR is involved in repair of damaged DNA to ensure genome integrity ( reviewed in [1] ) , and is also a crucial step in genetic transformation , a widespread mechanism of horizontal gene transfer which allows acquisition of new genetic traits ( reviewed in [2] ) . Transformation involves internalization of ssDNA fragments generated from exogenous double-stranded ( ds ) DNA substrate , which can be incorporated into the chromosome via HR . This process generally occurs during a short time window called competence , during which all the proteins required for internalization and integration of ssDNA are produced ( reviewed in [2] , [3] ) . Search for homology between DNA sequences during both genome maintenance and transformation is catalyzed by the homologous recombinase RecA . This enzyme requires dedicated loaders to promote its loading onto target ssDNA ( reviewed in [4] ) . The end goal of bacterial recombinase loaders is to produce a filament of ssDNA coated in RecA , allowing HR to occur . Two main loaders are involved in genome maintenance , RecBCD targeting RecA to double strand DNA breaks ( DSB ) and RecFOR targeting it to ssDNA gaps . The latter was suggested as important for the restart of stalled replication forks , where such gaps are produced [5] . A third loader , DprA , has recently been characterized through studies of the protein from Streptococcus pneumoniae as dedicated to genetic transformation [6] , [7] . RecBCD is an enzyme complex best characterized in Escherichia coli ( reviewed in [8] , although studied homologues are present in Bacillus subtilis ( AddAB , [9] ) and S . pneumoniae ( RexAB , [10] ) amongst others . The RecBCD loader substrate is dsDNA and as transforming DNA is internalized as ssDNA , a RecBCD-like complex was not expected to be involved in chromosomal transformation [11] , which turned out to be true for S . pneumoniae where RexAB proteins are dispensable for transformation [10] . Mutants lacking the RecFOR proteins have shown increased sensitivity to DNA-damaging agents in various bacterial species including E . coli [12] , B . subtilis [13] and Deinococcus radiodurans [14] . Unlike RecBCD , there is no evidence that the RecFOR proteins form a tricomponent enzyme complex , although RecR does interact directly with both RecO and RecF [15] , [16] . However , all three proteins were shown to be able to access ssDNA coated in the single-stranded DNA binding protein ( SSB ) from ssDNA gaps in the chromosome and facilitating the loading of RecA to this ssDNA [17] . Indeed , the ability to displace SSBs from target ssDNA is a key activity of recombinase loaders that target ssDNA . The third loader , DprA , appears to be conserved in all transformable species [2] . In S . pneumoniae , as a loader dedicated to the transformation process , DprA can presumably displace SsbB , a competence-induced SSB [18] , [19] , and load RecA onto the transforming ssDNA , prompting RecA filamentation , search for homology [6] within the recipient chromosome and subsequent integration . Inactivation of dprA almost completely abolished transformation efficiency in pneumococci [20] , which is likely due partly to its role as a transformation-dedicated RecA loader [6] and partly due to its role in protecting internalized ssDNA , which is rapidly degraded in its absence [20] . This reduction was observed for both chromosomal and plasmid transformation . DprA was recently shown to interact directly with RecA , a property which is presumably necessary for loading of RecA onto transforming ssDNA and as such was found to be crucial for transformation [7] . A similar role for DprA as a transformation-dedicated RecA loader was recently established in B . subtilis [21] although cells lacking DprA were less severely impacted , with a 2-log deficit of transformation observed [22] , [23] . Despite the presence of DprA , studies in B . subtilis showed that inactivation of recO reduced efficiency of chromosomal transformation 2-fold [21] , suggesting that RecFOR might be involved in loading RecA onto ssDNA during both genome maintenance and transformation . For the transformation of replicative plasmids , no loss of efficiency was observed in B . subtilis recF or recR mutants , while recO mutants were 25-fold less efficient [24] . It was suggested that RecO was crucial for reconstruction of an intact plasmid molecule , allowing strand annealing from two internalized ssDNA fragments [25] . Interestingly , DprA was also important for plasmid transformation in B . subtilis , with a 50-fold reduction in efficiency observed in a dprA- mutant [25] . Crucially , replicative plasmid transformation is RecA-independent in B . subtilis [24] but not in S . pneumoniae [26] , possibly due to the degradation of internalized ssDNA in the absence of pneumococcal RecA . Here , we assess the importance of RecFOR for transformation in S . pneumoniae . We show that pneumococcal RecFOR proteins play no role in chromosomal or plasmid transformation but are required for resolution of chromosome dimers occurring as intermediates in the formation of merodiploids by transformation [27] . We report that chromosome dimers are generated at an unexpected high frequency by self-transformation , as well as interspecies transformation , and that their proper resolution also requires XerS [28] , a tyrosine recombinase related the XerCD tyrosine recombinase of Escherichia coli which catalyzes chromosome dimer resolution by site-specific recombination at dif sites [29] . We provide evidence that in the absence of RecFOR and/or XerS , dimers persist within a transformed population and can limit the efficiency of transformation , since resulting in the loss of transformant chromosomes .
Each recFOR gene was inactivated by mariner mutagenesis , as previously described [30] and the effect of mutating these genes on pneumococcal cells was investigated ( S1–S2 Text; S1–S2 Fig . ; S1 Table ) . All mutants were found to have doubling times of between 42 and 47 min , slower than the wild type which doubled every 34 min ( S1D Fig . ) . RecFOR proteins appeared involved in genome maintenance in S . pneumoniae as deduced from the extreme sensitivity of recFOR mutants to the alkylating agent methyl methanesulfonate and the DNA crosslinking agent mitomycin C ( S2 Fig . ) . To establish whether RecFOR played any role in transformation in pneumococci , we tested the ability of recFOR mutants to respond to signals inducing competence for genetic transformation . We analyzed the expression of a gene specifically induced during competence , ssbB , after addition of the competence-stimulating peptide ( CSP ) ( S2 Text ) . Results show that single and double mutant cells respond to CSP with the same kinetics as wildtype cells ( S1E Fig . ) . We first determined that RecFOR are not involved in replicative plasmid transformation ( S1 Text; S3 Fig . ; S3 Table ) . We previously suggested that antagonization of plasmid transformation by SsbB could be due to direct inhibition of plasmid strand annealing by RecO [19] . However , inactivation of ssbB had the same effect in wt and recO- cells whether the concentration of donor DNA was high or low ( S3A–B Fig . ) . These results confirm that RecO is not involved in annealing of internalized plasmid strands , a crucial step in reconstitution of intact plasmid molecules . To establish whether loss of recO impacted chromosomal transformation in pneumococci , we first compared transformation frequency of recO and dprA mutant cells . Transformation experiments with chromosomal DNA carrying the rpsL41 point mutation conferring streptomycin resistance ( SmR ) confirmed a major impact of dprA inactivation on chromosomal transformation , resulting in a ∼10 , 000-fold drop , while recO inactivation resulted in a limited deficit ( ∼1 . 6-fold drop; Fig . 1A ) . A further ∼40-fold drop was observed in two independent recO dprA double mutants ( Fig . 1A ) , suggesting that the RecO protein can contribute to the processing of internalized ssDNA but mainly when DprA is absent . To confirm that recFOR inactivation had only a minor impact on chromosomal transformation , point mutations carried on chromosomal DNA were transformed into recipient cells either wildtype or lacking recF , recO or recR . Selection for integration of rpsL41 and rif23 , a point mutation conferring rifampicin resistance ( RifR ) , showed that both point mutations were accepted by recFOR mutants with efficiencies ∼70% of wildtype ( 1 . 45-fold average drop; Fig . 1B ) . Though reproducible , this difference appeared limited , suggesting that the RecFOR proteins play no major role in pneumococcal chromosomal transformation . This point is further examined and clarified in next sections of Results . It is of note that the results obtained with rif23 , a mutation known to be targeted by the Hex mismatch repair system during pneumococcal transformaton [31] , [32] allowed us to conclude that loss of RecFOR proteins does not affect mismatch repair ( S1 Text; S2 Table ) . The presence of a few residual transformants in the recO dprA double mutant ( Fig . 1A ) suggested that in the absence of both DprA and RecFOR loaders , RecA is sometimes capable of self-loading on internalized ssDNA . To confirm this conclusion , the transformation experiment was repeated but using as donor a PCR fragment carrying the rpsL41 mutation to increase transformation efficiencies as a result of the use of a homogenous DNA preparation in which every fragment carries the genetic information under selection ( Fig . 1C ) . The results paralleled those obtained with total chromosomal DNA , confirming a ∼40-fold drop in two independent recO dprA double mutants compared to the dprA single mutant . However , the >10-fold increase in transformants compared to chromosomal DNA in the double mutant relieved any ambiguity , confirming the appearance of transformants in the absence of both RecA loaders . Surprisingly , the comparison of transformation frequencies for the rpsL41 mutation revealed no difference between wildtype and recO- cells with a PCR fragment as donor ( Fig . 1C ) , in contrast to the results when the donor was chromosomal DNA ( Fig . 1A–B ) , leading us to conclude that RecO is not involved in the process of chromosomal transformation itself in S . pneumoniae . While our results indicated that RecO was required neither for chromosomal nor for plasmid transformation , we remained intrigued by the ∼1 . 5-fold drop in transformation frequency observed in a recO mutant transformed with chromosomal DNA ( Fig . 1A–B ) . We recently established that transformation stimulates the formation of partial chromosomal duplications , or merodiploids [27] . A single ∼3-kb DNA fragment partly repeated in the chromosome ( see below ) was sufficient to trigger formation of tandem-duplications ranging from ∼100 to ∼900 kb in size at various chromosomal locations . A mechanistic model for the formation of merodiploids was proposed and validated in that study [27] . Key to this model is the creation of a chromosome dimer as an intermediate , resolution of which generates one merodiploid chromosome and another chromosome lacking this region ( Fig . 2A–B ) . We hypothesized that RecO was crucial for dimer resolution and that persistence of dimers in the transformed population could lead to chromosome or cell death , thus accounting for the observed drop in transformation of recO- cells . To check this hypothesis , we investigated the formation of merodiploids induced by transformation in parallel in wildtype and recO mutant cells . The experimental set up for measurement of transformation-induced merodiploids involves the use a donor DNA mixture containing a merodiploid trigger fragment and a merodiploid scoring cassette . The merodiploid trigger , R1A or R2Z , is a ∼3-kb DNA fragment in which R1 and R2 correspond to repeats in the chromosome , and A and Z their respective non-repeated flanks . Once R1A is internalized , ‘alternative pairing’ of R1 , defined as pairing with R2 on one arm of a partially replicated recipient chromosome , coupled with ‘normal pairing’ of A on the other chromosome arm ( i . e . pairing with its couterpart next to R1 in the recipient chromosome ) bridges the two chromosome arms ( Fig . 2A ) creating a dimer and , after resolution , a merodiploid ( Fig . 2B ) . A 107 kb region originally flanked by R1 and R2 repeats is duplicated ( referred to as duplication #1 [27] ) and roughly represents minutes 39–43 if the chromosome is represented as a clock face . It includes the codY gene , which has been shown to be essential in S . pneumoniae [33] . The merodiploid scoring cassette , conferring resistance to trimethoprim ( TrimR ) , is inserted in codY and thus can give rise to viable transformants only if this locus is duplicated , therefore selecting directly for merodiploid clones . A parallel control transformation with only the merodiploid scoring cassette allows measurement of background merodiploids spontaneously formed in the recipient culture . Using this experimental set up , similar factors of stimulation of merodiploid formation by transformation were observed for each donor DNA set ( codY::trim plus R1-A , R2-Z , or a mixture of R1-A and R2-Z ) in wildtype and recO mutant cells ( Fig . 2C–D ) . These results were consistent with RecO playing no role in the steps leading to formation of a chromosome dimer by transformation , which was not surprising since these steps are essentially identical to those involved in ‘classical’ chromosomal transformation , for which RecO is not required ( Fig . 1C ) . While inactivation of recO did not affect the stimulation factor , the absolute frequency of merodiploids , either spontaneously present or triggered by partly repeated donor fragment ( s ) was reduced by 3 . 4 to 6 . 3-fold in recO- cells ( Fig . 3A ) . This finding provided support to the hypothesis that RecO is involved at a later stage in the formation of merodiploids , namely the resolution of chromosome dimers required to generate a merodiploid chromosome ( Fig . 2B ) . Two mechanisms are envisioned for resolution , one involving HR via RecA and potentially requiring the assistance of RecO for loading , and the other mediated by the site-specific recombinase XerS acting at dif sites [28] . To further document the resolution of chromosome dimers in S . pneumoniae , we repeated the experiment in xerS- and recO- xerS- cells . The xerS mutant displayed a general loss of merodiploid formation of ∼4-fold ( Fig . 3B ) , similar to a recO mutant . Interestingly , the recO xerS double mutant showed an even greater loss , of around 35-fold ( Fig . 3B ) , suggesting that >95% of chromosome dimers could not be resolved in the absence of both RecO and XerS proteins . The similar effect of xerS and recO inactivation , and the cumulative effect of recO and xerS mutations on merodiploid formation provided strong support to our hypothesis that RecO is involved in chromosome dimer resolution and indicated that RecO and XerS are both required for proper resolution . To obtain physical evidence in support of our conclusions , we firstly confirmed that a wildtype level of dimeric chromosomes was indeed produced in the absence of RecO ( and XerS ) , by monitoring the presence of the novel junctions predicted in the chromosome dimer by PCR ( i . e . both tandem-duplication and deletion junctions; Fig . 3C–E ) on wildtype and recO- xerS- populations transformed with R1-A PCR fragment . The tandem-duplication junction was detected similarly in transformed wildtype and double mutant cells , at different time points after addition of transforming DNA ( 10–160 min; Fig . 3D ) . Note that weak bands due to low level spontaneous merodiploid formation were detected in the no-DNA controls , at the same time points . This result indicated that chromosome dimers are formed with similar efficiency in wildtype and recO- xerS- cells , suggesting no role for RecO in the formation of dimeric chromosomes both during normal growth and by transformation . Secondly , we confirmed the disappearance of the deletion junction at 100 min in wildtype cells ( Fig . 3E ) , which is consistent with our previous report [27] and readily explained by resolution of the chromosome dimer in the wildtype background and loss of cells with the abortive chromosome missing a large region of 107 kb , including the essential codY gene ( Fig . 2B; referred to as deletion #1 hereafter ) . In contrast , persistence of deletion #1 junction in recO- xerS- cells ( Fig . 3E ) provided direct evidence that as suggested above on the basis of genetic data ( Fig . 3B ) , these mutant cells did not properly resolve >90% of chromosome dimers . This supports our conclusion that RecO is involved at a late stage in merodiploid formation , rather than in transformation itself , which is dependent on DprA . Further physical evidence for the persistence of deletion junctions and therefore of chromosome dimers in recO- xerS- cells was obtained by investigating another two merodiploid chromosomes previously shown to form during transformation with total genomic DNA ( 31 ) . These merodiploid chromosomes contained a duplication of 144 kb ( site #2 , 10–15 minutes on the chromosome ) and 210 kb ( site#3 , 23–29 minutes on the chromosome ) respectively . The deletion junctions ( #2 and #3 ) of the reciprocal products generated through resolution of the parental dimeric chromosome were found to disappear with similar kinetics in wildtype cells , while persisting in recO- xerS- cells ( S5 Fig . ) . We previously observed that the deletion junction #1 had disappeared at 100 min post DNA addition in wildtype cells , as confirmed in this study ( Fig . 3E and S5 Fig . ) , but was still readily detectable after 70 min [27] . We were intrigued by the finding that even the basal level of deletion junctions produced by spontaneous merodiploid formation disappeared at 100 min ( Fig . 3E and S5 Fig . ) , suggesting a general phenomenon leading to death of all cells harboring abortive chromosomes in the culture . To determine whether chromosome dimer resolution was impacted by growth phase , we repeated the experiment with deletion #1 junction , determining more precisely when deletion junctions could no longer be detected by focusing on time-points between 70 and 100 min post-DNA addition , while monitoring in parallel the growth of the culture . Results show that the both spontaneous and transformation-induced deletion junctions disappear between 80 and 90 min ( Fig . 4A ) , which correlates with entry to stationary phase ( Fig . 4B ) . Note that although recO- xerS- cells display slightly delayed entry to stationary phase , this can by no means explain the failure to detect junction loss after 160 min in Fig . 3E . To confirm that entry to stationary phase was somehow dictating deletion junction loss , we changed the experimental set up used in this and a previous [27] study so as to delay entry of transformed cells into the stationary phase . Cells were diluted 20 min after DNA addition , and the presence of the deletion junction was investigated at later time points in wildtype , recO- , xerS- and recO- xerS- cells . Loss of transformation-induced deletion junction was not detected prior to 130 min , indicating that the growth phase had a net impact on the process ( Fig . 4C–D ) . Note that basal level spontaneous duplications still occurred , explaining the weak bands observed at 130 and 145 min . Identical kinetics were observed for single recO- and xerS- mutants suggesting that dimers can be resolved in these mutants . However , the deletion junction persisted in recO- xerS- cells , showing again that these are unable to resolve chromosome dimers . These results were consistent with our working hypothesis that RecO is crucial for dimer resolution and that persistence of dimers in the transformed population leads to chromosome or cell death . However , to account for the observed ∼1 . 5-drop in transformation frequency of a point mutation in recO- cells , our hypothesis necessarily implied that formation of chromosome dimers during transformation with total genomic DNA occurred at unexpectedly high frequency . To result in such a drop , this type of event should occur in 30–35% of the cells , being efficiently resolved and therefore remaining ignored in wild type but not in recO- cells . In the latter , co-transformation of the fragment carrying the selected marker ( e . g . the rpsL41 mutation ) and a merodiploid trigger fragment would result in the loss of the potential SmR transformant . On the basis of this explanation , we predicted that any transforming DNA capable of inducing chromosome dimerization at higher rate than pneumococcal DNA should further decrease the loss of transformants in recO mutant cells . Streptococcus mitis B6 chromosomal DNA represented an ideal tool to check our prediction as this species is a close relative of S . pneumoniae , therefore allowing homologous exchanges , but harboring an overall genome arrangement with a striking X-alignment when compared to pneumococcal genomes , indicative of many symmetrical inversions [34] . Every fragment overlapping an inversion site is predicted to generate a chromosome dimer upon integration into the pneumococcal chromosome . We therefore used as donor in transformation of wildtype and recO- cells a mixture of the rpsL41 PCR fragment and S . mitis B6 chromosomal DNA . Results show that while co-transformation with S . mitis DNA had no effect on the frequency of transformants for the rpsL41 PCR fragment in wildtype cells , it reduced this frequency by 6 . 7-fold in recO mutant cells ( Fig . 5A ) . To establish that this reduction was only due to the failure to properly resolve the chromosome dimers formed by co-transforming S . mitis DNA , this experiment was reproduced with xerS mutant cells . The results revealed a 4 . 4-fold reduction ( Fig . 5A ) providing strong support to our interpretation since XerS as a site-specific recombinase is not expected to have any effect on chromosomal transformation , except if chromosome dimers are generated in the process , requiring its action . A similar reduction , 5 . 8-fold , was observed in a control experiment with recO xerS double mutant cells ( Fig . 5A ) . Considering a 5 . 6-fold average reduction in SmR transformants , we conclude that chromosome dimers are formed in ∼80% of the cells upon co-transformation with S . mitis chromosomal DNA . It is of note that if chromosome dimers do not form in ∼1 cell out of 5 , this would readily account for the fact that no greater decrease in SmR transformants is observed in the recO xerS double mutant compared to the single mutants . In other words , if 20% of SmR transformants are chromosome-dimer free , this would de facto limit the maximum drop in transformation frequency observable in the recO xerS double mutant to ∼5-fold , as dimer-free transformants would obviously survive . While these results were consistent with a high incidence of chromosome dimers in the transformed population , none of the above experiments , including those monitoring the presence of the novel junctions predicted in the chromosome dimer by PCR , detected the presence of a dimer per se . In an attempt to obtain more direct evidence for the presence of chromosome dimers , we stained cells transformed with S . mitis DNA with DAPI ( 4' , 6-diamidino-2-phenylindole ) to quantify their DNA content . We tested whether we could detect a difference in cell fluorescence between wild type and recO- xerS- cells in stationary phase , when unresolved dimers should be present in the latter ( Fig . 4A–B ) . While there was no difference in fluorescence in wild type cells whether they had been transformed with S . mitis DNA or not ( Fig . 5B ) , a clear increase in intensity was observed in transformed recO- xerS- cells ( Fig . 5C–D ) , fully consistent with persistence of chromosome dimers within these cells . These results provide strong support to our hypothesis that a high frequency of chromosome dimers are formed by transformation , with RecFOR and XerS required for proper resolution .
As expected from studies in other species , the RecFOR proteins appear important for pneumococcal genome maintenance , as deduced from the impact of inactivation of the corresponding genes on growth rate ( S1 Fig . ) and sensitivity to both methyl methanesulfonate and mitomycin C ( S2 Fig . ) . This extreme sensitivity is reminiscent of that of a pneumococcal recA mutant [35] . On the other hand , quite unexpectedly , the RecFOR proteins appear involved in another facet of pneumococcal genome maintenance revealed by investigation of the impact of recO inactivation on the formation of merodiploids triggered by transformation . This analysis established a requirement for RecO for proper resolution of chromosome dimers ( see below ) , which to the best of our knowledge had so far not been documented in any bacterial species . The pneumococcal RecFOR proteins appear not to be required for plasmid transfer by transformation ( S1 Text ) . This contrasts with the situation in another model transformable species , B . subtilis , in which plasmid transformation was reduced by almost 2-logs in a recO mutant [21] . It was suggested that RecO was crucial for reconstruction of an intact plasmid molecule , allowing strand annealing from two internalized ssDNA fragments in this species [25] . In S . pneumoniae , we previously observed that the competence-induced single-stranded DNA-binding protein SsbB , which is dedicated to chromosomal transformation , antagonized plasmid transformation at a high concentration of donor plasmid DNA [19] . We tentatively attributed the inhibitory effect of SsbB to antagonization of RecO annealing of plasmid single strands [19] . However , plasmid transformation carried out in this study revealed that inactivation of ssbB resulted in a similar increase in transformation in both wildtype and recO mutant cells ( S3A Fig . ) . We conclude that the increase in plasmid transformation in the absence of SsbB is not due to a relief of inhibition of RecO-dependent annealing of internalized plasmid single-strands and that RecO is not involved in plasmid strand annealing in pneumococcal transformation . We suggest DprA as an obvious candidate based on the stimulation of the annealing of complementary strands by purified DprA previously observed in vitro [6] . Though the observation that plasmid transformation is abolished in dprA mutant cells [20] would be consistent with this proposal , it is inconclusive because in S . pneumoniae internalized ssDNA is very rapidly degraded in cells lacking DprA [20] . On the other hand , the finding that plasmid transformation is reduced by 2-logs in a dprA mutant of B . subtilis [21] , where DprA is not required for protection of internalized ssDNA , is fully consistent with a role of DprA in plasmid strand annealing . Furthermore , the observation of an only 2-fold reduction in plasmid transformation in a recO-recA double mutant [21] , implying efficient plasmid strand annealing , makes DprA an obvious candidate to promote annealing , at least in B . subtilis cells lacking RecO and RecA . As concerns chromosomal transformation , we could not detect any effect of the inactivation of recFOR on the efficiency of Hex-dependent mismatch repair of point mutations , occurring at the heteroduplex stage during integration of transforming DNA ( S1 Text ) . Chromosomal integration of a heterologous cassette was also unaffected in a recO mutant ( S4 Fig . ) . On the other hand , we repeatedly observed a limited ( ∼1 . 45-fold ) but significant reduction in the frequency of transformation of a point mutation when genomic DNA was used as donor in transformation of recFOR mutant cells ( Fig . 1B ) . However , the direct comparison of transformation frequencies of wildtype and recO- cells for a point mutation but using a PCR fragment as donor revealed no difference ( Fig . 1C ) . The latter finding leads us to conclude that RecO is not involved in the processing of transforming DNA in wildtype pneumococci . A number of studies have previously explored the role of RecO in chromosomal transformation in other naturally transformable species . Mirroring our results , RecO was shown to play no role in transformation in Neisseria gonnorhoeae [36] and Helicobacter pylori [37] , [38] . A slight reduction in transformation efficiency was observed in Deinococcus radiodurans recO- cells , and authors concluded that RecFOR was the major loader of RecA onto ssDNA during transformation [39] . Nevertheless , as DprA is present in D . radiodurans and though its role in transformation has not been determined , we suggest that this protein , and not RecFOR , is the main loader of RecA during transformation in this species as well . In any case , the finding that RecO is also not required for chromosomal transformation in wildtype pneumococci and most other tested species is fully consistent with the view that the transformation-dedicated loader DprA ensures the loading of RecA onto internalized ssDNA . Altogether , these results provide further support to the notion that different machineries exist in S . pneumoniae , as well as presumably in many other transformable species , to promote genetic transformation on one hand and ensure genome maintenance on the other . We were intrigued by the ∼1 . 5-fold drop compared to wildtype observed when a point mutation was transferred from chromosomal DNA but not if the same mutation was transferred on a short PCR fragment ( Fig . 1 ) . As we previously showed that transformation with pneumococcal chromosomal DNA generates a variety of merodiploids , proceeding through the creation of a chromosome dimer which is subsequently resolved [27] , we hypothesized that recO- cells were unable to properly resolve dimers , resulting in a loss of transformants in cells integrating both the fragment carrying the point mutation and a DNA fragment triggering chromosome dimerization . This hypothesis is supported by the observation of a reduction in merodiploid formation in recO mutant cells compared to wild type ( Fig . 3A ) , as well as of a similar reduction in xerS mutant cells ( Fig . 3B ) . The latter represents the first in vivo evidence of a function for pneumococcal XerS , even if this function was assumed due to identification of XerS as a tyrosine recombinase with a function potentially similar to the XerCD proteins in Escherichia coli [28] . Genetic evidence that both RecO and XerS are required for proper dimer resolution was provided by the >95% reduction in merodiploids in double mutant cells ( Fig . 3B ) . This finding was corroborated by the observation that two PCR fragments detecting the new junctions ( i . e . tandem-duplication and its accompanying deletion counterpart; Fig . 3C ) present in the chromosome dimer persist in cells lacking both XerS and RecO ( Fig . 3DE , 4A , 4C and S5 Fig . ) . This persistence is indicative of poor dimer resolution and provides physical evidence for the maintenance of unresolved chromosome dimers in the double mutant . It is unclear what the mechanistic significance of the genetic requirement for both recO and xerS for chromosome dimer resolution is . Owing to the atypical nature of the pneumococcal site-specific recombination machinery , comprising a single component instead of two as in the E . coli paradigm , RecO might directly assist XerS for recombination at dif , possibly to help stabilize the synapsis complex . Alternatively , RecO could for example be needed because the RecFOR machinery for HR contributes to proper chromosome segregation after XerS-catalyzed site-specific recombination has taken place at the dif site . It is of note that despite a>95% reduction in merodiploid formation in recO- xerS- cells , a few bona fide merodiploids were obtained ( Fig . 3B ) , indicating that chromosome dimers could still be resolved , though with poor efficiency , in the absence of both RecO and XerS . This result implies the existence of another resolution pathway , which could possibly rely on RecA-driven HR , involving the RexAB loader ( the pneumococcal counterpart of RecBCD [10] ) to load RecA . Interestingly , resolution of chromosome dimers as deduced from the loss of deletion junctions appeared faster in cells entering stationary phase compared to those remaining in exponential phase ( Fig . 4 ) . This loss occurred with similar kinetics irrespective of the nature of the deletion for transformation-induced merodiploids ( Fig . 4 and S5 Fig . ) . It also concerned the deletion junctions produced by spontaneous merodiploid formation which completely disappeared in stationary phase ( e . g . 100 min time point in Fig . 3E , 4A and S5 Fig . ) , suggesting a general phenomenon leading to death of all cells harboring abortive chromosomes . This 35–40 min delay in resolution ( 115–130 min compared to 80–90 min ) in exponentially growing cells , which roughly corresponds to one generation , may not indicate that resolution occurs earlier in stationary phase cells but simply that dimer resolution occurred at the same time in both populations of cells , and that cells with an abortive chromosome survive longer in exponential conditions . Alternatively , it is possible that entry to stationary phase forces resolution of chromosome dimers so that none remain in quiescent stationary phase cells . As a consequence , cells with a deleted chromosome would be generated and die rapidly , explaining the complete loss of deletion junction in wildtype cells entering stationary phase . Whatever the underlying mechanism , this phenomenon potentially ‘cleanses’ a pneumococcal population of cells from any rearrangements leading to major deletions . It can therefore be predicted that a pneumococcal population coming out of stationary phase ( or , more generally , non-dividing cells in nature ) thus contains a minority of non-healthy cells . The documented defect in resolution of chromosome dimers in recO- cells could account for the ∼1 . 5-fold drop in transformation of a point mutation only if dimers formed in 30–35% of cells transformed with S . pneumoniae genomic DNA , leading to loss of the transformed chromosome . In support of this interpretation , we predicted that any transforming DNA inducing a higher rate of chromosome dimerization than pneumococcal DNA should further decrease the loss of transformants in recO- cells . This prediction was verified using S . mitis B6 chromosomal DNA which harbors many symmetrical inversions compared to the S . pneumoniae chromosome [34] , with every fragment overlapping an inversion site potentially generating a chromosome dimer upon integration into the pneumococcal chromosome . Co-transformation of S . mitis chromosomal DNA with a PCR fragment containing a point mutation caused a loss of ∼80% of potential transformants in recO- cells ( Fig . 5A ) . Similar reductions were observed in xerS- cells ( Fig . 5A ) . Since the only suggested role of XerS is in site-specific recombination to resolve chromosome dimers , this provides strong support to our hypothesis that a high frequency of chromosome dimers are formed by transformation , with RecFOR and XerS required for proper resolution . Furthermore , the use of DAPI staining to quantify DNA content revealed a clear increase in intensity in recO- xerS- cells transformed with S . mitis DNA ( Fig . 5CD ) , fully consistent with a high incidence of chromosome dimers persisting within these cells . Overall , these results establish that an inability to resolve chromosome dimers efficiently , and not a specific role in the transformation process , accounts for the decreased transformation efficiency observed in recO- cells when chromosomal DNA is used as donor . While we attribute the decrease in transformation frequency in S . pneumoniae recO- cells to the defective resolution of chromosome dimers frequently formed during the process , it would be interesting to clarify whether a similar explanation accounts for the ∼2-fold loss of transformants in a B . subtilis recO mutant [21] . Interestingly , in B . subtilis , both recO and addAB ( B . subtilis RecBCD homologue ) mutant cells showed similar reductions in chromosomal transformation efficiency , while a double mutant showed a 10-fold reduction [24] , [40] . This can be readily explained by high dimer formation during transformation with chromosomal DNA , which are poorly resolved in the absence of both loaders involved in genome maintenance . Furthermore , since merodiploid formation relies on classic recombination mechanisms and may be stimulated by transformation in a wide range of transformable species , it is likely that dimer formation is a common occurrence in cells transformed with chromosomal DNA . Our observation that chromosome dimers frequently form via transformation is of practical importance when analyzing genetic transformation in S . pneumoniae by cell imaging . It is crucial to realize that a significant fraction of the population is engaged in the process of resolving chromosome dimers , via RecFOR , RecA and XerS proteins , and therefore to set up experimental conditions allowing a clear distinction between images corresponding to chromosomal transformation per se and those resulting from resolution of chromosome dimers subsequent to transformation . This cautionary note may also apply to B . subtilis if chromosomal transformation frequently produces dimers in this species as well ( which is not unlikely since repeated sequences are also present in the chromosome , e . g . ribosomal operons ) . It would be interesting to clarify this point , as it may lead to reinterpretation of previous analyses . For example , a study showed the formation of so called ‘threads’ of RecA in cells transformed with chromosomal DNA , concluding that these structures may mediate homology search and strand invasion for genetic transformation [41] . Since such structures were seen in a fraction of transformed cells ( 24% ) , it is possible that dimer resolution mediated by HR ( catalyzed by RecFOR and RecA ) was being observed . Future work will determine whether our conclusion that RecFOR proteins are involved in genome maintenance but play no role in either chromosomal or plasmid transformation in S . pneumoniae also applies to the RecFOR proteins of other transformable species .
All the strains and plasmids used in this work are listed , together with primers , in S1 Table . Standard procedures for transformation and growth media were used [42] . Antibiotic concentrations ( µg mL−1 ) used for the selection of S . pneumoniae transformants were: chloramphenicol ( Cm ) , 4 . 5; erythromycin ( Ery ) , 0 . 05–0 . 2; kanamycin ( Kan ) , 250; rifampicin ( Rif ) , 2; spectinomycin ( Spc ) , 100; streptomycin ( Sm ) , 200; tetracycline ( Tet ) , 1 . For the monitoring of growth and luc expression , precultures were gently thawed and aliquots were inoculated ( 1 in 100 ) in luciferin-containing [43] C+Y medium and distributed ( 300 ml per well ) into a 96-well white microplate with clear bottom . Relative luminescence unit ( RLU ) and OD values were recorded throughout incubation at 37°C in a LucyI luminometer ( Anthos ) . To detect novel junctions produced by merodiploidy during transformation , experiments were carried out as previously described [27] , with modifications . Briefly , recipient cells transformed with either pneumococcal chromosomal DNA or PCR amplifications of R1-A and R2-Z fragments , and 200 µL samples taken at varying time-points after DNA addition . Samples were centrifuged , and pellets resuspended in 10 µl C+Y medium with 15% glycerol . PCRs to detect various duplication and deletion junctions were done directly on 2 µL of these culture samples . Primers used for these PCRs are detailed in the appropriate Figure Legends . To quantify the DNA content of cells , DAPI staining was used . Wild-type and recO- xerS- cellswere grown to OD 0 . 1 in C+Y medium , and 25 ng mL−1 added . After 10 min , 1 µg mL−1 S . mitis B6 chromosomal DNA was added to the cultures ( except non-transformed controls ) . Cells were harvested upon entry to stationary phase , and resuspended 1/5 in C+Y+2 µg mL−1 DAPI . 2 µL of these cell cultures was spotted onto a microscope slide containing a pad of 1 . 2% C+Y agarose as described previously [44] before imaging . Images were captured and processed using the Nis-Elements AR software ( Nikon ) . Analysis of cell dimensions was carried out using the MATLAB-based open source software MicrobeTracker [45] . Cell contours were obtained using the alg4 spneumoniae3 algorithm implemented in MicrobeTracker , a derivative of alg4 ecoli2 with parameters spliltTreshold , joindist and joinangle refined to fit the shape of S . pneumoniae . Relative DAPI intensity was calculated by dividing the overall intensity of each cell by the area .
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Homologous recombination ( HR ) is a widespread process which maintains genome integrity and promotes diversity . In bacteria , HR mends damaged DNA to ensure genome integrity and is also involved in transformation , a mechanism of horizontal gene transfer allowing acquisition of new genetic traits . HR is driven by recombinases , which are loaded onto single-stranded DNA by the recombinase loaders RecBCD and RecFOR for genome maintenance . DprA was recently proposed as another loader dedicated to transformation . During transformation , foreign DNA is taken up as single strands and integrated into the chromosome by HR . In this study , we show that RecFOR is not involved in transformation in Streptococcus pneumoniae . These results provide further support to the existence of different HR machineries dedicated to genetic transformation and genome maintenance in this pathogen . In addition , we show that transformation with chromosomal DNA generates chromosome dimers with unexpectedly high frequency , and that their resolution requires RecFOR and the site-specific recombinase XerS . In cells lacking these proteins , dimers persist and have a detrimental effect on the efficiency of transformation . Since the HR mechanisms leading to dimer formation are most likely conserved , this effect is presumably general to naturally transformable species .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biochemistry",
"horizontal",
"gene",
"transfer",
"genetics",
"microbial",
"genetics",
"biology",
"and",
"life",
"sciences",
"dna",
"repair",
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2015
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RecFOR Is Not Required for Pneumococcal Transformation but Together with XerS for Resolution of Chromosome Dimers Frequently Formed in the Process
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Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization . Despite its importance in the sexual reproduction of plants , little is known about the role of gene regulation in this process . We report here that the pollen-expressed transcription factors MYB97 , MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception . They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes . None of the single or double mutants of these three genes exhibited any visible defective phenotype . Although the myb97 myb101 myb120 triple mutant was not defective in pollen development , pollen germination , pollen tube growth or tube guidance , the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac . In addition , the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains . All these results indicate that MYB97 , MYB101 and MYB120 participate in pollen tube reception , possibly by controlling the expression of downstream genes .
In flowering plants , the proper development of male gametophytes is essential for successful fertilization during reproduction [1] , [2] . The male gametophyte life cycle can be divided into two distinct phases: ( 1 ) a developmental phase , also known as the early developmental phase , which takes place in anther locules and leads to the formation of mature pollen grains; and ( 2 ) a functional or progamic phase , also called the later developmental phase , which begins when the pollen grains contact the stigma surface , continues with pollen tube growth and ends at double fertilization [1] . To date , many transcription factors involved in the early development of male gametophytes have been identified in efforts to understand the gene regulatory network involved in this process [1] , [2] . However , the gene regulatory network that controls later development remains poorly understood . The later developmental phase is delimited by pollination and fertilization . This process involves pollen hydration , germination , tube growth through the transmitting tissue , tube guidance , sperm cell discharge into the embryo sac and finally the fusion of male and female gametes . To date , four groups of transcription factors involved in this process have been identified . The AtMIKC* subgroup is composed of six members that belong to the MADS-box transcription factor family . Five of the six members in this subgroup ( AGL30 , AGL65 , AGL66 , AGL94 and AGL104 ) are expressed mainly in pollen and are expected to regulate the transcription associated with the later development of male gametophytes [3]–[6] . The AtMIKC* proteins interact with each other , forming five heterodimers that bind to the DNA motifs of their targets , namely AGL66-AGL30 , AGL66-AGL65 , AGL66-AGL94 , AGL30-AGL104 and AGL104-AGL65 . In the agl66-1 agl104-2 double mutant , in which all the five heterodimers are absent , pollen viability was decreased . In vivo , pollen germination and pollen tube growth are also affected [7] . Microarray analyses demonstrated that this double mutation has a significant impact on pollen gene expression , indicating that the AtMIKC* proteins may function as upstream transcription factors during the later development of male gametophytes [7] . Another transcription factor found to be involved in the later development of male gametophytes is AtbZIP34 , whose expression is detected in both male and female gametophytes during flower development [8] . A mutation in AtbZIP34 caused defects in the exine , resulting in a lower pollen germination rate and slower pollen tube growth in vitro and in vivo [8] . The transcription factor AtWRKY34 is specifically expressed in pollen . Under cold treatment conditions , the wrky34 pollen exhibited increased viability , germination efficiency and pollen tube growth rate in vivo relative to wild type pollen , indicating that WRKY34 might function as a negative regulator of the cold response in mature pollen . Further analyses suggested that WRKY34 acts downstream of the AtMIKC* genes in the cold stress response program [9] . In addition , the study shows that several MYB transcription factors are expressed in pollen tubes . Mutation in one ( MYB120 , At5g55020 ) of these genes caused defective pollen germination and tube growth in vitro . These data indicate that the pollen-expressed MYB transcription factors are also involved in the later development of male gametophyte [10] . Pollen tube reception is the final stage of male gametophyte development , which involves the bursting of the pollen tube to release sperm cells and the death of a synergid cell [11] , [12] . However , our knowledge of the molecular and genetic mechanisms of pollen tube reception remains very limited . The first studies of these mechanisms focused on two female gametophytic mutants , feronia ( fer ) and sirène ( srn ) . The pollen tubes of both fer and srn overgrow in the embryo sac and are unable to rupture ( overgrowth phenotype ) [13] , [14] . Later , fer and srn were found to be alleles of the same gene , FER [15] . FER encodes a receptor-like protein kinase that localizes to the filiform apparatus of synergid cells . It has been proposed that the undefined signal produced from the pollen tube activates the FER receptor and then triggers a signal transduction cascade in the synergid that feeds back to the pollen tube and cause pollen tube rupture [11] , [12] , [15] . Three additional female gametophytic mutants , lorelei ( lre ) , scylla ( syl ) and nortia ( nta ) , were also found to exhibit fer-like phenotypes . The LRE gene encodes a putative glycosylphosphatidylinositol-anchored protein [16] , [17] . The gene responsible for the syl phenotype has not been identified [18] . NTA encodes a Mildew Resistance Locus O family protein [19] . Recent studies showed that the FER pathway is important for the re-localization of NTA in the synergid cell upon the arrival of the pollen tube [19] , [20] . Apart from the four female gametophytic mutants , a mutation in ABSTINENCE BY MUTUAL CONSENT ( AMC ) also causes a defect in pollen tube reception , but this defect only occurs when both the male and female gametophytes carry the mutant amc allele [21] . Maize ( Zea mays ) has also emerged as a model system for studying pollen tube reception in grasses . Zea mays embryo sac 4 ( ZmES4 ) , a defensin-like protein , is exclusively expressed in female gametophytes . Knocking down the expression of ZmES4 by RNA-interference resulted in the fer-like phenotype [22] . However , the male components controlling pollen tube reception are still poorly understood , although they are expected to exist , and only a few candidate genes have been identified to date . ANXUR1 ( ANX1 ) and ANXUR2 ( ANX2 ) are close homologues of FER , exhibit high-sequence homology to one another and are expressed in pollen . The anx1 anx2 double-mutant pollen grains are able to germinate , but the pollen tubes rupture in vitro and in the transmitting tract in vivo and fail to reach the female gametophytes , indicating that ANX1/ANX2 might function to ensure the proper timing of sperm discharge [23] , [24] . That is , ANX1/ANX2 maintain pollen tube growth in the transmitting tract until the tube reaches the female gametophyte; then , they are deactivated by an undefined signal from the synergid cell , which triggers the rupture of the pollen tubes [23] . In addition , Autoinhibited Ca2+ ATPase 9 ( ACA9 ) encodes a Ca2+ pump in Arabidopsis and is expressed in pollen tubes . The aca9 mutant pollen tubes extend to the synergid cell and stop growing , but they do not rupture and discharge their sperm cells in vivo [25] . This finding indicates that the ion gradients in pollen tubes also play an essential role in sperm discharge . K+ channel Zea mays 1 ( KZM1 ) , a pollen tube-expressed shaker K+ channel , was discovered as a direct target of ZmES4 in maize . The interaction between ZmES4 and KZM1 triggers channel opening and a subsequent K+ influx that leads to pollen tube bursting [22] . Nevertheless , it remains unclear how these genes coordinate with each other to control pollen tube reception . Here , we report the characterization of the novel male components MYB97 , MYB101 and MYB120 that are involved in pollen tube reception . These proteins share a high amino acid sequence identity and are mainly expressed in mature pollen grains and pollen tubes . The myb97 myb101 myb120 triple mutation causes the overgrowth of the mutant pollen tubes in embryo sac and disrupts the discharge of sperm cells into the embryo sac , leading to a significant reduction in fertility . Microarray analysis showed that the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains . All these results indicate that the MYB transcription factors play important roles in pollen tube reception , possibly by controlling the expression of downstream genes .
MYB transcription factors play regulatory roles in diverse developmental processes in Arabidopsis . Several MYB genes have been identified as involved in the early development of male gametophytes and filament development ( Table S1 and Text S1 ) . Therefore , we speculate that the MYB genes might also function in the later development of male gametophytes . To study the gene regulation network that controls pollen tube growth , we manually searched the Arabidopsis gene expression profile data available in the TAIR databases ( http://www . arabidopsis . org ) for pollen-expressed transcription factor ( PTF ) genes [26] , [27] . Seven MYB transcription factors were identified as expressed in mature pollen and pollen tubes , namely MYB33 , MYB65 , MYB81 , MYB97 , MYB101 , MYB104 and MYB120 ( Figure 1A and also see Table S2 ) , which belong to the same subclass of the R2R3-MYB family [28] . Their expression patterns were first verified by RT-PCR of RNAs isolated from different tissues of wild type plants , including roots , stems , leaves , inflorescences , mature pollen grains , siliques and seedlings . The MYB97 , MYB101 and MYB120 transcripts were detected mostly in mature pollen grains , weakly detected in inflorescences and siliques and not detected in the other tissues , suggesting that these three genes were expressed mainly in mature pollen grains . MYB33 and MYB65 were expressed in all the tissues tested , with significantly different levels in different tissues , including mature pollen grains . In particular , MYB33 was expressed mostly in leaves , whereas MYB65 was detected mainly in roots , leaves and pollen grains . The expression of MYB81 was detected mainly in inflorescences and only weakly in mature pollen grains and siliques . MYB104 was expressed mainly in the inflorescences and siliques ( Figure 1B ) . Based on these results , the seven MYB proteins were classified into three subgroups: MYB97 , MYB101 and MYB120 comprised the first subgroup , MYB33 and MYB65 comprised the second , and MYB81 and MYB104 comprised the third . Further quantitative reverse transcription-PCR ( qRT-PCR ) assays also confirmed that MYB97 , MYB101 and MYB120 were expressed more highly in mature pollen , whereas the other four MYB genes were weakly expressed in pollen ( Figure 1C ) . Therefore , the three genes in the first subgroup were considered the strongest candidate MYB members to have possible roles in pollen development and tube growth and were selected for further functional characterization . To investigate the expression patterns of these genes in detail , the promoters of MYB97 , MYB101 and MYB120 ( the genes in the first subgroup ) were fused to a GUS reporter gene and introduced into wild type plants , respectively . GUS activity was detected mainly in the mature pollen grains and pollen tubes in the transgenic lines ( Figure 2 ) , consistent with the results of the RT-PCR and qRT-PCR assays . These results further demonstrated that the three MYB transcription factors are expressed mostly in mature pollen grains and pollen tubes , indicating that they could regulate transcription in the later development of male gametophytes . To investigate the phylogenetic relationships among the seven MYB proteins , we generated a phylogenetic tree using DNAMAN software to analyze the amino acid sequences of the seven proteins . As shown in Figure 1A , the seven proteins were assigned into three main branches , consistent with their expression pattern ( RT-PCR ) -based classification . In particular , MYB97 , MYB101 and MYB120 , which share 32% amino sequence identity , comprised one branch; MYB33 and MYB65 , which share a 62% amino acid identity , comprised the second branch; and MYB81 and MYB104 , which share 53% amino acid identity , comprised the third branch ( Figure 1A ) . Recently , MYB33 and MYB65 , which have similar expression patterns , have been reported to be functionally redundant in anther development [29] . MYB97 , MYB101 and MYB120 also have similar expression patterns in mature pollen grains and pollen tubes , implying that they may also be functionally redundant with each other . Therefore , this finding prompted us to study further their roles in pollen and pollen tubes by characterizing the myb97 myb101 myb120 triple mutant . To investigate their roles in the later development of male gametophytes , T-DNA insertion mutants of MYB97 , MYB101 and MYB120 were purchased from the Arabidopsis Biological Resource Center ( ABRC , www . arabidopsis . org ) and backcrossed with wild type Col plants . Finally , we obtained single T-DNA insertion mutants for MYB97 and MYB120 ( myb97-1 and myb120-3 , respectively ) . In the myb97-1 mutant , the T-DNA insertion is located in the second exon of MYB97 , 1096 bp downstream of the ATG start codon ( Figure 3A ) . In the myb120-3 mutant , the T-DNA is inserted in the second exon of MYB120 , 1099 bp downstream of the ATG start codon ( Figure 3A ) . Three single T-DNA insertion alleles were obtained for MYB101; the T-DNAs were inserted in the second and third exons , 841 bp , 1408 bp and 1788 bp downstream of the ATG start codon , respectively . These alleles were named myb101-1 , myb101-2 and myb101-3 , respectively ( Figure 3A ) . For the further phenotypic characterization of these mutations , single homozygous mutant plants were generated by self-pollination . RT-PCR assays using mature pollen cDNA pools showed that the RNAs corresponding to MYB97 , MYB101 and MYB120 were not detected in the corresponding single mutants . Further qRT-PCR assays also showed that the expression levels of the three genes were significantly decreased in the corresponding mutants compared with wild type plants ( Figure S1 ) . These results indicated that the T-DNA insertions had a strong impact on the expression of the corresponding genes . However , none of the single homozygous mutants exhibited any phenotypic defects ( Table S3 ) . To investigate whether MYB97 , MYB101 and MYB120 are functionally redundant with each other , we first generated double mutants of different allele combinations , namely myb97-1 myb120-3 , myb97-1 myb101-1 , myb97-1 myb101-2 , myb97-1 myb101-3 , myb101-1 myb120-3 , myb101-2 myb120-3 and myb101-3 myb120-3 . The individual double homozygous mutants were identified by PCR-aided genotyping in the F2 generations of the crosses . None of the double mutants exhibited any defective phenotype of either the pollen or the vegetative parts , indicating that mutations in any two members of the subgroup did not affect male gametophyte development or fertility , similar to the phenotypes of the single mutations ( Table S3 ) . Then , we further generated triple mutants using these double mutant lines . Three triple-mutant lines with different allele combinations , namely myb97-1 myb101-1 myb120-3 , myb97-1 myb101-2 myb120-3 and myb97-1 myb101-3 myb120-3 , were isolated . All of these triple mutants produced shorter siliques with lower seed sets compared to those of wild-type plants , whereas their vegetative parts appeared indistinguishable from those of wild type plants ( Figure S2A ) . In the shorter siliques of the triple mutants , the seed set was drastically reduced , and many unfertilized ovules were found ( Figure S3 ) . The seed sets in the single and double mutants were almost identical to those of the wild type , i . e . , nearly 100% . By contrast , the seed sets in the triple mutants were 38 . 93±6 . 13% , 36 . 61±6 . 13% and 72 . 75±6 . 13% of wild type , respectively ( Table S3 ) . These data suggested that MYB97 , MYB101 and MYB120 are functionally redundant and are required for fertility in Arabidopsis . To confirm that the phenotype of the myb97 myb101 myb120 triple mutants was caused by the combination of mutations in MYB97 , MYB101 and MYB120 , we investigated the expression levels of the three genes in the triple homozygous mutants and wild type by RT-PCR and qRT-PCR of the RNAs extracted from mature pollen grains of the triple homozygous mutants and wild type plants . The results showed that the expression of the three genes was significantly reduced in the triple mutant pollen grains compared to the wild type pollen grains ( Figure 3B and S2B ) , suggesting that the triple mutant is a loss-of-function mutant . Then , complementation experiments were performed using individual wild type genomic DNA fragments of MYB97 , MYB101 and MYB120 ( i . e . , members of the first subgroup ) . The full-length genomic DNA fragments , including the predicted promoters , transcribed regions and 3′-end non-transcribed regions , were cloned and introduced into two independent triple homozygous mutant lines ( myb97-1 myb101-1 myb120-3 and myb97-1 myb101-2 myb120-3 ) . Complementation screens in the myb97-1 myb101-1 myb120-3 background yielded a total of 103 independent T1 transgenic lines ( 35 with the MYB97 complementation construct , 23 with the MYB101 complementation construct and 45 with the MYB120 complementation construct ) . Preliminary observation showed that the seed set of the T1 plants was partially restored . Twenty T1 transgenic lines from each of the three different gene complementation constructs were selected for detailed examination . The quantitative data further confirmed that the phenotype of the siliques of myb97-1 myb101-1 myb120-3 could be partially restored in the heterozygous transgenic MYB lines ( data not shown ) . Then , homozygous transgenic MYB lines were generated by self-pollination for five randomly selected independent T1 lines for each complementation construct . In these T3 homozygous lines , the seed set was restored completely ( Figure 4; Table 1 ) . These results demonstrated that the fertility-defective phenotype of myb97-1 myb101-1 myb120-3 could be restored by each of the single genes from the first subgroup ( MYB97 , MYB101 and MYB120 ) . Furthermore , MYB97 , MYB101 and MYB120 also completely rescue the fertility-defective phenotype of myb97-1 myb101-2 myb120-3 ( Figure S4; Table S4 ) . In addition , we also performed an allelic analysis of the myb101 alleles by generating three hybrid myb101 allele combinations of the triple mutants , namely myb97-1/−;myb101-1/myb101-2;myb120-3/− , myb97-1/−;myb101-1/myb101-3;myb120-3/− and myb97-1/−;myb101-2/myb101-3;myb120-3/− . All three of these myb101 allele hybrid triple mutants exhibited a fertility-defective phenotype similar to that of the triple mutants described above ( Figure S3 ) . The seed sets in the triple mutant siliques were 39 . 03±5 . 38% , 55 . 39±6 . 31% and 54 . 68±6 . 46% , respectively , of the wild type set ( Table S3 ) . These results demonstrated that the combination of mutations in MYB97 , MYB101 and MYB120 caused a drastic reduction in fertility . To investigate whether the MYB proteins in the other two subgroups had similar effects on fertility , the MYB101 promoter was used to drive the expression of MYB33 , from the second subgroup ( MYB33 and MYB65 ) , and MYB81 , from the third subgroup ( MYB81 and MYB104 ) , in myb97-1 myb101-1 myb120-3 homozygous mutants . The results showed that pMYB101::MYB33 and pMYB101::MYB81 were able to completely rescue the fertility-defective phenotype of myb97-1 myb101-1 myb120-3 homozygous mutant ( Figure 4; Table 1 ) . Therefore , both MYB33 and MYB81 were able to perform the functions of MYB97 , MYB101 and MYB120 in the first subgroup , indicating that the MYB proteins may have the same biochemical function with respect to fertility . To carry out further genetic analyses , we generated triple mutants that were homozygous for two mutations and heterozygous for the third mutation . For myb97-1 myb101-1 myb120-3 , three independent combinations were obtained , namely myb97-1/+;myb101-1/−;myb120-3/− , myb97-1/−;myb101-1/−;myb120-3/+ and myb97-1/−;myb101-1/+;myb120-3/− . Because no obvious kanamycin-resistant selection marker was available for scoring the segregation ratio , PCR-aided genotyping was applied to identify the different genotypes of the progeny from the crosses using the triple mutants as male or female parents . In the self-pollination of myb97-1 myb101-1 myb120-3 , the progeny of the mutant genotypes displayed a distorted segregation ratio of individuals with insertion and without insertion ( Table 2 ) . The segregation ratios were much lower than the typical Mendelian segregation ratio of 3 to 1 , suggesting that the mutants were defective in gametophyte function . To determine the sex-related transmission efficiency ( TE ) of the mutations , reciprocal crosses between the mutants and wild type were performed . When the triple mutants were used as recipients ( female ) in crosses with wild-type pollen , approximately 50% of the resulting progeny contained the T-DNA insertions . When wild type plants were used as recipients in crosses with the triple mutant pollen grains , only a small number of the resulting progeny had the T-DNA insertions ( Table 2 ) . Furthermore , the analysis of myb97-1 myb101-2 myb120-3 crosses also produced a similar result ( Table S5 ) . Reciprocal crosses were also performed between the myb97-1 myb101-1 myb120-3 homozygous triple mutant and wild type plants . When wild type plants were manually self-pollinated or used as males in crosses with the myb97-1 myb101-1 myb120-3 homozygous mutant , the resulting siliques were fully fertile with full seed sets . In contrast , when myb97-1 myb101-1 myb120-3 homozygous mutants were manually self-pollinated or used as males in crosses with wild type plants , the resulting siliques were shorter and produced much lower seed sets ( Table 3 ) . In addition , for myb97-1 myb101-2 myb120-3 and myb97-1 myb101-3 myb120-3 homozygous mutants , the reciprocal crosses with wild type also produced similar results ( Table 3 ) . In summary , the triple mutants exhibited normal transmission through the female gametophytes but significantly reduced transmission through the male gametophytes . These results demonstrated that the myb97 myb101 myb120 triple mutants are male gametophyte-defective . To investigate the causes of the lower transmission of triple mutants through male gametophytes , we first examined the mature pollen grains . Alexander staining showed that all pollen grains from the triple mutants were indistinguishable from wild type pollen grains ( Figure 5A , 5D , 5G and 5J ) . DAPI staining showed that the pollen grains from the triple mutant had two sperm cell nuclei and a vegetative nucleus , similar to the wild type pollen grains ( Figure 5B , 5E , 5H and 5K ) . Scanning electron microscope ( SEM ) observations revealed no obvious morphological defects in the triple mutant pollen grains compared with wild type pollen grains ( Figure 5C , 5F , 5I and 5L ) . Therefore , the triple mutations did not affect pollen formation . Because the triple mutants could produce normal mature pollen grains , we next examined the germination of the triple-mutant pollen grains in vivo . The germination rates of the pollen grains from the three allelic triple mutants described above were 83 . 00±2 . 69% , 86 . 29±1 . 75% and 84 . 58±4 . 99% , respectively , similar to the wild type value of 88 . 59±4 . 95% . Therefore , the triple mutations also did not affect pollen germination . We then investigated growth of the triple mutant pollen tubes in pistils . At 4 , 8 and 12 h after pollination ( HAP ) , the pistils were harvested separately and examined by aniline blue staining to evaluate the pollen tube growth pattern . The results showed similar growth in the triple mutant and wild type pollen tubes , indicating that the triple mutations did not have a significant impact on growth of the pollen tubes prior to their encountering the female gametophyte . Surprisingly , however , unlike wild type , approximately 60% of the triple-mutant pollen tubes did not stop growing and failed to release the sperm cells into the embryo sacs at 48 HAP ( Figure 6M–6P ) . The overgrowth of pollen tubes in the embryo sac resembled the phenotype of the fer mutant [13]–[15] , which suggested that MYB97 , MYB101 and MYB120 might take part in pollen tube reception . If this phenotype was the reason that the ovule could not be fertilized , then the percentage of the overgrowing pollen tubes must be consistent with that of the unfertilized ovule in the triple-mutant siliques . Then , we compared the rates of unfertilized ovules with the rate of pollen tube overgrowth in the triple mutant siliques . The results supported our hypothesis ( Table 4 ) . To confirm whether MYB97 , MYB101 and MYB120 served as male components in pollen tube reception , reciprocal crosses were performed between the triple homozygous mutants and wild type to examine the growth of the triple-mutant pollen tubes in the wild type embryo sac . In the case when the wild type ovules were crossed with the triple mutant pollen , the percentage of overgrowing pollen tubes in the resulting pistils was close to that of the unfertilized ovules , similar to the data from the manual self-pollination of the triple mutants ( Table 4; Figure 6 ) . By contrast , very few overgrowing pollen tubes were observed in the crosses between triple mutant ovules and wild-type pollen tubes , similar to the data obtained in the manual self-pollination of wild type plant ( Table 4; Figure 6 ) . Therefore , the triple mutations affect the pollen tube reception through male gametophyte . The MYB proteins belong to the R2R3 MYB transcription factor superfamily . They are expected to localize to the nucleus . To confirm the subcellular localization of MYB97 , MYB101 and MYB120 , N-terminal GFP fusion constructs of each of the MYB proteins driven by the 35S promoter were introduced into onion epidermal cells using particle bombardment . The observation showed that the fusion proteins were specifically localized to the nuclei ( Figure 7D–7L ) , whereas the GFP control protein was found throughout the onion epidermal cells ( Figure 7A–7C ) . These results were consistent with the predicted functions of the three MYB proteins as transcription factors . The transcriptional activation ability of the three MYB proteins was investigated using a yeast assay system . Each of the three MYB proteins was fused to the GAL4 DNA binding domain in the pGBKT7 vector to generate the respective pMYB constructs ( Figure 8A ) . Then , each of these plasmids , as well as the positive control pAD and negative control pGBKT7 , were transformed into the yeast strain AH109 . The growth patterns of the transformants were then examined ( Figure 8B ) . The results showed that all transformants grew well on SD/-Trp medium . However , only the positive control pAD , pMYB97 and pMYB101 were able to grow on the selective SD/-Trp-His-Ade medium , whereas pMYB120 was not , indicating that MYB97 and MYB101 may function as transcription activators . As the first step to identify the downstream genes that are involved in pollen tube reception , we compared the gene transcription profile of myb97-1 myb101-1 myb120-3 homozygous triple mutant pollen grains to that of wild type pollen grains by the microarray analysis using Affymetrix ATH1 Genome Arrays . Three biological replicates were applied in this study . The results of six chips were compared using the GeneSpring GX software ( downloaded from http://www . genomics . agilent . com ) . Genes whose expression levels changed more than two-fold in mutant pollen grains compared with wild type pollen grains were selected as candidate target genes regulated by the three MYB transcription factors . Ultimately , 24 genes were selected based on this criterion; eight genes were obviously down-regulated , and the remaining 16 genes were significantly up-regulated in the triple-mutant pollen grains ( Table S6 ) . All 24 genes are expressed in pollen based on the data from TAIR ( www . arabidopsis . org ) . To verify the results of the microarray data , we examined the expression levels of the eight down-regulated genes ( DG ) in the triple mutant and wild type further by RT-PCR and qRT-PCR . Three genes ( DG1 , DG2 and DG3 ) were significantly down-regulated in the triple mutant relative to the wild type ( Figure 9A and 9B ) , and selected for further analysis . It has been reported that the MYB gene from barley ( Hordeum vulgare ) , HvGAMYB , a gene homologous to the MYB family , could bind to the TAACAAA motif of the barley high-pl α-amylase promoter and regulate the expression of α-amylase [30]–[32] . Therefore , the promoters of the three candidate genes were analyzed using the PLACE technique ( http://www . dna . affrc . go . jp/PLACE/ ) for potential MYB-binding sites [33] . The results showed that TAACAAA motifs are present in the promoter regions of the three selected genes , implying that they might be direct targets of MYB97 , MYB101 and MYB120 . To investigate whether the MYB proteins were able to bind to the promoters of the three genes , electrophoretic mobility shift assays ( EMSAs ) were performed with the recombinant MYB domain of MYB101 and the three labeled oligonucleotides containing the TAACAAA sequence derived from the promoters of DG1 , DG2 and DG3 ( Figure 9C ) . As a control , the recombinant MYB101 protein and a labeled oligonucleotide were added alone to the native gel . The reaction of the two components caused a shift in the mobility of the labeled oligonucleotide , indicating that MYB101 was able to bind these oligonucleotides in vitro ( Figure 9D–9F ) . To further determine whether the interaction is specific for the TAACAAA motif , unlabeled wild type and mutated oligonucleotides were added to the reaction as competitors . The interaction between MYB101 and a labeled oligonucleotide was clearly disrupted by the addition of excess wild type competitor but only weakly disrupted by the mutated competitor in which the TAACAAA sequence was mutated ( Figure 9D–9F ) . These results demonstrated that the MYB101 protein could bind specifically to the TAACAAA-containing sequences derived from the promoters of DG1 , DG2 and DG3 , which were down-regulated in the myb97 myb101 myb120 triple mutants .
We report here the identification and characterization of a group of MYB transcription factors that function as male factors involved in pollen tube reception . The final stage of later development of male gametophytes is pollen tube reception . After the pollen tube enters the embryo sac , it encounters and interacts with the receptive synergid . The interactive signaling process triggers the rupture of the pollen tube and the release of the sperm cells into the embryo sac [11] , [12] . This interaction between male and female gametophytes is expected to involve components from both the male ( pollen tube ) and female ( synergid ) partners . To date , although several components in the female synergid have been identified to be involved in pollen tube reception in Arabidopsis and maize [15]–[17] , [19] , [22] , [34] , much less information about the male components involved in this process is available . AMC is expressed in both the pollen tube and synergid . The amc mutant also exhibits a pollen tube overgrowth phenotype when the mutant pollen tube enters the mutant embryo sac [21] . Therefore , it is unlikely to be a male-specific component of the pollen reception pathway . Plants with mutations in the pollen tube-expressed ANX1/ANX2 , ACA9 and KZM1 genes exhibit phenotypes related to pollen tube growth but not the typical phenotype of pollen tube overgrowth [22]–[25] . More evidence is required to clarify whether these genes are actually involved in the pollen tube reception process . In this study , we demonstrate that at least three MYB members , MYB97 , MYB101 and MYB120 , are expressed in pollen but not in female partner synergids . The myb97 myb101 myb120 triple mutants exhibited the typical pollen tube overgrowth phenotype , almost identical to those of fer , lre and nta mutants [11]–[20] . The overgrowth of the triple-mutant pollen tubes also occurred in a cross of wild type plants with the myb97 myb101 myb120 triple mutant pollen grains , as expected . Therefore , we conclude that the MYB97 , MYB101 and MYB120 proteins function as male factors to participate in pollen tube reception , which supports the previous hypothesis that the transcription factors play essential roles in pollen-pistil interactions [10] . This new finding will be useful in the further search for new components of the pollen tube reception pathway in the future . Our data demonstrated that MYB97 , MYB101 and MYB120 are localized to the nucleus of the onion epidermal cells and that at least MYB97 and MYB101 have transcription transactivation activity in a yeast assay system . In addition , the recent work showed that the three MYBs accumulated in the pollen tube nucleus during pollen tube growth through the pistil [35] . Therefore , these three proteins likely function as transcription factors and control pollen tube reception by regulating the expression of the downstream genes that are involved in the pollen tube reception process . Microarray analysis showed that the triple mutation affected the expression of at least 24 genes in mature pollen grains , among which eight were down-regulated and 16 were up-regulated , including transcription factor genes , secreted protein genes and the genes that may be involved in cell signaling . RT-PCR and qRT-PCR analysis further confirmed that at least DG1 , DG2 and DG3 were regulated by MYB97 , MYB101 and MYB120 . The EMSA assays further demonstrated that recombinant MYB101 protein expressed in an E . coli system could bind to the promoter regions of the three DG genes in vitro . These data indicate that the MYB proteins may regulate the expression of downstream genes by binding to their promoter regions , as is the case for other MYB transcription factors [30]–[32] . However , whether these MYB-regulated downstream genes actually function in pollen tube reception remains unclear . Further investigation is required to address this question . FER has been proposed to function as a signaling receptor in pollen tube reception [15] . This raises the important question of where the signal molecule or ligand comes from . The most likely answer is that this signal could come from the pollen tube . Namely , FER might be activated by a ligand or signaling molecule from the pollen tube and subsequently trigger a downstream signal transduction pathway to feed back to the pollen tube and cause pollen tube rupture [11] , [12] , [15] , [34] . However , this ligand or signaling molecule has not yet been identified . Our results showed that the myb97 myb101 myb120 triple mutation affected the expression of several pollen-expressed genes . This result raises another interesting question , i . e . , whether there are any candidate FER ligands among the genes affected by the myb97 myb101 myb120 triple mutation . We identified one gene , DG3 ( AT3G19690 ) , which encodes a defensin-like protein with homologues that have been proposed to function as a signaling molecule in pollen tube guidance [36] . Our microarray , RT-PCR and qRT-PCR analyses showed that the expression of DG3 at a transcriptional level was significantly reduced in myb97-1 myb101-1 myb120-3 homozygous mature pollen grains compared to wild type mature pollen grains . Additionally , the expression level of DG3 ( AT3G19690 ) was changed from low to high during pollen tube growth [10] . Furthermore , EMSA assays suggested that MYB101 could bind to the TAACAAA motif-containing sequence derived from the promoter region of AT3G19690 . However , we still lack direct experimental evidence that DG3 is a ligand of the FER receptor . Nevertheless , these findings will prompt future work toward the identification and characterization of potential FER receptor-associated signaling factors from the pollen tube . The functional redundancy of the genes involved in male gametophyte development has been noted in different transcription factor gene families , for example , between MYB33 and MYB65 in anther development and among the AtMIKC* genes in pollen germination and pollen tube growth [7] , [23] , [24] , [29] . In this study , the MYB97 , MYB101 and MYB120 proteins are highly similar to each other in amino acid sequence . Furthermore , only myb97 myb101 myb120 triple mutants exhibited a pollen tube reception-defective phenotype; the double and single mutants did not . Moreover , the expression of a single gene from the MYB family could complement the phenotype of the triple mutant . In addition , RT-PCR and GUS analyses showed that MYB97 , MYB101 and MYB120 are mainly expressed in mature pollen and pollen tubes , consistent with a role in pollen tube reception . These data demonstrate that the MYB proteins are functionally redundant as predicted previously [10] . In the myb97 myb101 myb120 triple mutants , approximately 40% of the mutant pollen tubes were able to complete the process of sperm discharge , leading to successful fertilization and seed development , indicating that the triple mutation does not completely block the discharge of sperm cells . The reason for this observation remains unclear . Our results showed that pMYB101::MYB33 and pMYB101::MYB81 constructs could complement the phenotype of myb97 myb101 myb120 triple mutants , suggesting that the MYB proteins function in pollen tube reception fertility . Thus , one possible explanation is that the other MYB members might contribute to this process . To address this question , we initiated the construction of higher-level multiple mutants or universal disruption of all the seven pollen-expressed MYB genes identified in this study . Because MYB33 and MYB65 were more closely related to MYB97 , MYB120 and MYB101 than MYB81 and MYB104 ( as shown in the phylogenetic tree in Figure 1A ) , mutants of these two genes were first used to construct quadruple mutations . The results showed that neither the myb33 myb97 myb101 myb120 nor the myb65 myb97 myb101 myb120 quadruple mutant exhibited a more severe phenotype than the triple mutant ( Figure S5; Table S7 ) , indicating that neither MYB33 nor MYB65 has a significant impact on pollen tube reception . This finding is consistent with their much lower expression in pollen ( Figure 1C ) . Furthermore , as shown in Figure 1C , the expression levels of MYB81 and MYB104 are much lower than even that of MYB33 . Thus , MYB81 and MYB104 are also unlikely to make significant contributions to pollen tube reception . Further investigation will be required to understand whether other MYB genes may be involved in pollen tube reception . It is also worth noting that qRT-PCR assays using primer pairs targeting the sequences upstream of the T-DNA insertion sites showed that truncated MYB101 and MYB120 transcription products were present in the myb97 myb101 myb120 triple mutants ( Figure 3B ) . To date , however , we have not determined whether these truncated transcripts are associated with the partial fertility of the myb97 myb101 myb120 triple mutant . Further investigation is required to address this question . While this manuscript was under review , Leydon and coworkers [35] reported the characterization of the myb97 myb101 myb120 triple mutant using a different approach . The study was more focused on cell biological characteristic of the triple mutant in pollen tube reception . The results clearly show that lacking expression of MYB97 , MYB101 and MYB120 caused defect in discharge of sperm cells and affected degeneration of the synergid cells . In comparison , our study had a more detailed genetic analysis . The results indicate that the MYB proteins from the MYB family ( including MYB33 , MYB65 , MYB81 , MYB97 , MYB101 , MYB104 and MYB120 ) are functionally redundant . Our results also demonstrate that the MYB proteins can bind to the promoter sequences of the genes whose expression was affected by the myb97 myb101 myb120 triple mutation . In addition , our microarray assay was different from the one reported by Leydon and coworkers [35] . Our microarray assay with mature pollen grains showed that the expression of 24 genes were significantly changed in the myb97 myb101 myb120 triple mutant pollen grains , among which eight were down-regulated and 16 were up-regulated . In the microarray analysis with the pistil 8 hr after pollination , reported by Leydon and coworkers [35] , 48 genes with significantly different transcript abundance were identified among which 45 were down-regulated and three were up-regulated [35] . Comparison of the data from the two assays showed that none of the above genes was found to be affected overlappingly in the both microarray assays , implying that the impact of the MYBs on gene expression in mature pollen grains could be different from that in the growing pollen tubes . Nevertheless , more studies are required to address whether these genes are actually involved in pollen tube reception . In conclusion , our results provide important new evidence to demonstrate that male components participate in pollen tube reception and help us to understand further the mechanism of male-female communication during pollination and fertilization .
All mutant seeds used in this study were obtained from the Arabidopsis Biological Resource Center ( ABRC , www . arabidopsis . org ) : myb97-1 ( Salk_112329C ) , myb101-1 ( Salk_061355 ) , myb101-2 ( Salk_149918C ) , myb101-3 ( Salk_039489C ) , and myb120-3 ( Salk_063698 ) . The mutant and wild type seeds were first plated on Murashige and Skoog ( MS ) [37] agar plates supplemented with or without 25 mg/L hygromycin to select for transgenic lines . After two days at 4°C , the plates were transferred to the growth chambers with a 16 h light/8 h dark cycle at 22°C . Then , the seedlings were transplanted into soil ten days after germination and grown under the same conditions as that used for seed germination . The T-DNA insertion sites were confirmed by PCR using the T-DNA–specific primer LBa1 paired with the gene-specific primers MYB97-1-RP , MYB101-1-RP , MYB101-2-RP , MYB101-3-RP and MYB120-3-RP . The homozygous mutant plants were selected by PCR using these RP primers paired with the following LP primers: MYB97-1-LP , MYB101-1-LP , MYB101-2-LP , MYB101-3-LP and MYB120-3-LP . All primers used in this work are listed in Table S8 . Total RNA samples were extracted from different Arabidopsis tissues using a total RNA extraction kit ( Bioteke , Beijing , China ) . First-strand cDNA was synthesized using the alfalfa mosaic virus reverse transcriptase kit ( TaKaRa , Dalian , China ) according to the supplier's instructions . Tubulin was used as an internal control for RT-PCR . The primer pairs used in the assays were TUB-RT-F/TUB-RT-R , MYB97-RT-F/MYB97-RT-R , MYB101-RT-F/MYB101-RT-R , MYB120-RT-F/MYB120-RT-R MYB33-RT-F/MYB33-RT-R , MYB65-RT-F/MYB65-RT-R , MYB81-RT-F/MYB81-RT-R and MYB104-RT-F/MYB104-RT-R . The cDNA pools from different tissues were used for the expression pattern analyses , while the cDNA pools from mature pollen were used for comparisons of the gene expression levels in the mutant and wild type plants . Amplifications were run for 35 cycles for TUB , MYB33 , MYB65 , MYB97 , MYB101 and MYB104 and for 45 cycles for MYB81 and MYB120 . The RNA samples used in the qRT-PCR analysis were extracted from mature pollen grains . Then , cDNAs were reverse transcribed using SuperScript III Reverse Transcriptase ( Invitrogen , 18064-014 , USA ) and random primers ( Promega , Madison , USA ) . qRT-PCR assays were performed using Power SYBR Green PCR Master Mix and the Applied Biosystems 7500 Real-Time PCR System ( Applied Biosystems , http://www . appliedbiosystems . com ) . The experiments were repeated three times . The ACTIN2 RNA levels were quantified as an internal control to normalize the RNA quantity . The primer pairs used were ACTIN2-rl-F/ACTIN2-rl-R , MYB97-rl-F/MYB97-rl-R , MYB101-rl-F/MYB101-rl-R , MYB120-rl-F/MYB120-rl-R , MYB33-rl-F/MYB33-rl-R , MYB65-rl-F/MYB65-rl-R , MYB81-rl-F/MYB81-rl-R , MYB104-rl-F/MYB104-rl-R , DG1-rl-F/DG1-rl-R , DG2-rl-F/DG2-rl-R , DG3-rl-F/DG3-rl-R and DG4-rl-F/DG4-rl-R . The thermocycling settings were as follows: 10 min at 95°C ( one cycle ) followed by 15 s at 95°C and 34 s at 60°C ( 40 cycles ) . After each run , a dissociation curve was acquired by heating the samples from 60 to 95°C to ensure amplification specificity . For observations of unfertilized ovules , we dissected the siliques with a needle and scored the unfertilized ovules under a dissecting microscope . Alexander staining of mature pollen grains was performed as described previously [38] . DAPI staining of pollen grains was performed as described by Xia et al . [39] . Morphological observations of pollen grains by SEM were performed as described by Jiang et al . [40] . Flowers were emasculated before anthesis and pollinated with the pollen grains from wild type and mutants . To score pollen germination rates , pistils that were pollinated with a limited number of pollen grains were harvested at 1–2 h after pollination , stained in aniline blue buffer for 15 min and examined under a fluorescence microscope , as described previously [40] . For the phenotypic analysis of pollen tubes , the pistils were harvested 48 h after pollination and fixed in an acetic acid/ethanol solution ( 1∶3 [v/v] ) for 2 h . The fixed pistils were then softened in an 8 M NaOH solution overnight . The pistils were washed several times with clean water and subsequently stained with aniline blue for 5 h in the dark . The aniline blue buffer contained 0 . 1% aniline blue in 0 . 1 M K2HPO4-KOH buffer , pH 11 . 0 . The pollen tubes in the stained pistils were observed using a Leica DM2500 microscope ( Leica , Wetzlar , Germany ) . Different transgenic plant tissues were stained in 100 mM NaPO4 ( pH 7 . 0 ) solution containing 0 . 5 mM potassium ferricyanide [K3Fe ( CN ) 6] , 0 . 5 mM potassium ferrocyanide [K4Fe ( CN ) 6] , 0 . 1% Triton X-100 , 10 mM EDTA and 0 . 5 mg/ml bromochloroindoyl-β-glucuronide [40] . The tissues were stained at 37°C for 2–3 h and then clarified in an acetic acid/ethanol solution ( 1∶3 [v/v] ) overnight . The GUS-stained tissues were then examined using a Leica DM2500 microscope equipped with DIC system and MZ10F stereo microscope ( Leica , Wetzlar , Germany ) . For the gene expression pattern assays , promoter fragments of the MYB97 , MYB101 and MYB120 genes were amplified by PCR using the following gene-specific primer pairs: MYB97-GUS-F/MYB97-GUS-R , MYB101-GUS-F/MYB101-GUS-R and MYB120-GUS-F/MYB120-GUS-R . The resulting fragments were subcloned upstream of the GUS reporter gene in the pCAMBIA1300 vector ( CAMBIA , Australia ) and introduced into wild type plants using the Agrobacterium-mediated infiltration method [41] . For complementation experiments , full-length genomic DNA fragments of MYB97 , MYB101 and MYB120 , including the predicted promoters , transcribed regions and 3′-end non-transcribed regions , were amplified by PCR using the following primer pairs: MYB97-F-1F/MYB97-F-1R and MYB97-F-2F/MYB97-F-2R for MYB97 , MYB101-F-1F/MYB101-F-1R and MYB101-F-2F/MYB101-F-2R for MYB101 , and MYB120-F-1F/MYB120-F-1R and MYB120-F-2F/MYB120-F-2R for MYB120 . The resulting fragments were cloned into a pMD-18 T vector for sequence validation . Then , the full-length genomic DNA fragments were subcloned into the pCAMBIA1300 vector and introduced into the myb97-1 myb101-1 myb120-3 and myb97-1 myb101-2 myb120-3 triple homozygous mutants as described above . For MYB33 and MYB81 complementation assays , the transcribed regions of MYB33 and MYB81 were amplified by PCR using the following primer pairs: MYB33-T-F/MYB33-T-R and MYB81-T-F/MYB81-T-R . The resulting fragments were subcloned into the pCAMBIA1300 vector to generate pMYB101::MYB33 and pMYB101::MYB81 constructs and then introduced into the myb97-1 myb101-1 myb120-3 triple homozygous mutant as described above . To evaluate the subcellular localization of the MYB97 , MYB101 and MYB120 proteins , the CDS fragments of MYB97 and MYB101 and the genomic transcribed region of MYB120 were amplified by PCR using the following primer pairs: MYB97-G-F/MYB97-G-R , MYB101-G-F/MYB101-G-R and MYB120-G-F/MYB120-G-R . Then , the resulting fragments were subcloned into the pCAMBIA1300 vector to generate N-terminal fusions of the genes with the GFP coding sequence driven by the 35S promoter . The fusion expression vectors were introduced into onion epidermal cells by particle bombardment as described by Zhu et al . [42] . For transactivational activity assays , the CDSs of the MYB97 , MYB101 and MYB120 genes were amplified by PCR using the following primer pairs: MYB97-BD-F/MYB97-G-R , MYB101-BD-F/MYB101-G-R and MYB120-BD-F/MYB120-G-R . Then , the fragments were fused to the GAL4 DNA binding domain in the pGBKT7 vector to generate pMYB constructs . For the positive control pAD , the AD fragment from the pGADT7 vector was amplified by PCR using the primer pair AD-F/AD-R and subcloned into the pGBKT7 vector . The resulting plasmids were transformed into the yeast strain AH109 , and their transactivational activities were examined . The RNA samples used for the microarray assays were extracted from mature pollen grains of wild type and myb97-1 myb101-1 myb120-3 homozygous mutants . ATH1 Genome Arrays were used to compare the transcriptomes of wild type and myb97-1 myb101-1 myb120-3 homozygous mutant pollen grains . Three biological replicates were performed . Raw data ( CEL files ) for six ATH1 chips were analyzed using the GeneSpring GX software ( downloaded from http://www . genomics . agilent . com/ ) . Genes that displayed greater than two-fold changes in expression were selected as candidates for regulation by the three transcription factors . The cDNA fragment of the MYB domain ( amino acid 1 to 133 ) of MYB101 was amplified using the primer pair MYB101-P-F/MYB101-P-R and cloned into the BamH I-Sal I site of pET-30a ( + ) ( Novagen , Madison , USA ) . The resulting construct was introduced into Escherichia coli strain BL21 ( DE3 ) pLysS ( TIANGEN , China ) to generate a 6×His-MYB1011–133 fusion protein . The expression of the fused protein was induced by 1 mM isopropyl β–D-1-thiogalactopyranoside ( IPTG ) at 18°C for 16 h . The recombinant protein was purified using PrepEase His-Tagged Protein Purification Kit ( USB , 78793 , Germany ) and dialyzed against storage buffer ( 20% glycerol , 10 mM Tris-Cl , 50 mM KCl , 1 mM DTT , pH 8 . 0 ) . The EMSA was performed using the LightShift Chemiluminescent EMSA Kit ( Pierce , 20148 , USA ) according to the instructions provided by the supplier . The 5′-biotin-modified oligonucleotides were synthesized by Invitrogen ( Invitrogen , China ) . To generate the double-stranded oligonucleotides , complementary pairs of oligonucleotides were annealed in a Tris buffer ( 10 mM Tris-Cl , 1 mM EDTA , 50 mM NaCl , pH 8 . 0 ) in a thermocycler ( step 1: 95°C for 5 min; step 2: 95°C ( −1°C/cycle ) for 1 min; 70 cycles ) . The notation “−1°C/cycle” indicates that the temperature of the heating block was decreased by 1°C per cycle . The binding reactions were carried out in binding buffer ( 10 mM Tris-Cl , 50 mM KCl , 1 mM DTT , pH7 . 5 ) at room temperature for 20 min . For the reactions shown in lanes 2 to 7 , 20 fmol of biotin-labeled oligonucleotides , 200 ng of recombinant protein , 50 ng poly ( dI·dC ) and an appropriate concentration of unlabeled oligonucleotides were mixed in a 20-µl binding reaction . The reaction shown in lane 1 contained no protein , as a control . The reaction products were separated in 1-mm-thick 6% native polyacrylamide gels in 0 . 5× TBE buffer ( 45 mM Tris base , 45 mM boric acid and 1 mM EDTA , pH 8 . 0 , precooled to 4°C ) at 100 V for 1 h . The oligonucleotides were transferred to Amersham Hybond-N+ nylon membranes ( GE , RPN303B , UK ) by electroblotting at 380 mA for 30 min in 0 . 5× TBE and then cross-linked with a BLX-254 UV crosslinker ( Vilber Lourmat , France ) . The biotin-labeled oligonucleotides were detected according to the instructions provided with the Chemiluminescent Nucleic Acid Detection Module ( Pierce , 89880 , USA ) .
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Pollen tube reception is an important step of fertilization and is controlled by interactions between the pollen tube and synergid . Components of both the pollen tube and synergid are believed to be involved in the process . Several proteins associated with this process have been identified in synergid cells . However , very little is known about the components contributed by the pollen tube . This work identified a group of Arabidopsis pollen-expressed MYB transcription factors , among which at least three members are involved in pollen tube reception . The myb97 myb101 myb120 triple mutation caused overgrowth of the pollen tube into the embryo sac and disrupted sperm cell discharge , leading to failed fertilization . This study provides novel evidence demonstrating that male factors are involved in pollen tube reception .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2013
|
MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization
|
Recent estimates on global morbidity and mortality caused by Leptospirosis point to one million cases and almost 60 , 000 deaths a year worldwide , especially in resource poor countries . We analyzed how a commensal probiotic immunomodulator , Lactobacillus plantarum , affects Leptospira interrogans pathogenesis in a murine model of sub-lethal leptospirosis . We found that repeated oral pre-treatment of mice with live L . plantarum restored body weight to normal levels in mice infected with L . interrogans . Pre-treatment did not prevent L . interrogans access to the kidney but it affected the inflammatory response and it reduced histopathological signs of disease . Analysis of the immune cell profiles in lymphoid tissues of mice pre-treated with L . plantarum showed increased numbers of B cells as well as naïve and memory CD4+ helper T cell populations in uninfected mice that shifted towards increased numbers of effector CD4+ helper T in infected mice . CD8+ cytotoxic T cell profiles in pre-treated uninfected and infected mice mirrored the switch observed for CD4+ except that CD8+ memory T cells were not affected . In addition , pre-treatment led to increased populations of monocytes in lymphoid tissues of uninfected mice and to increased populations of macrophages in the same tissues of infected mice . Immunohistochemistry of kidney sections of pre-treated infected mice showed an enrichment of neutrophils and macrophages and a reduction of total leucocytes and T cells . Our results suggest that complex myeloid and T cell responses orchestrate the deployment of monocytes and other cells from lymphoid tissue and the recruitment of neutrophils and macrophages to the kidney , and that , the presence of these cells in the target organ may be associated with reductions in pathogenesis observed in infected mice treated with L . plantarum .
A recent review on global morbidity and mortality caused by Leptospirosis estimates about 1 . 03 million cases and 58 , 900 deaths a year worldwide [1] , mostly in resource-poor countries [2][3] . Human leptospirosis is an acute febrile illness with a broad clinical spectrum ranging from mild influenza-like symptoms to severe disease forms characterized by bleeding , jaundice , renal failure , pulmonary hemorrhage and death [2 , 3] . Although most leptospirosis patients recover without treatment [3 , 4] , diagnosis of the disease is hindered by the complexity and insensitivity of serology by the microagglutination test ( MAT ) in acute infection [5] . Early initiation of antibiotic therapy may thwart disease progression [3] . Hence , practical strategies should prioritize early treatment and prevention to improve outcomes from this spirochaetal zoonosis [6] . Vaccines to prevent human disease exist in some countries and are based in killed whole cell Leptospira [3] . However , these vaccines provide only short-time protection , are serovar specific and mostly target leptospiral LPS [2] . Lactobacillus plantarum is a Gram-positive bacterium that is known to have immunomodulatory properties [7] and is used as a probiotic usually following high dose repetitive administration regimens [8] . With the long-term goal of using commensal probiotics as vehicles to express Leptospira immunogens , we analyzed how repeated pre-exposure treatment of mice with live L . plantarum affected dissemination of Leptospira interrogans to target tissues as well as the ensuing pathology . In the process , we evaluated the immunological mechanisms involved in Leptospira pathogenesis .
Female , 5 week old , C3H/HeJ mice were obtained from The Jackson Laboratory . This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the NIH . The protocol was approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee , Animal Care Protocol Application ( Permit Number: 14–018 ) . We used Lactobacillus plantarum strain 256 ( kindly gifted by Dr . Jos Seegers , Caelus Pharmaceuticals BV ) , a bacterium Generally Recognized As Safe , to perform oral treatments as described [9] , [10] prior to Leptospira infection . The strain used in this study ( 256 ) was selected from a wide panel of rifampicin-resistant wild-type lactobacilli that were amenable to transformation and persisted in the gut for up to 12 days [8] . Infections of mice were done using 2 . 5x107 Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 ( hamster passage 2 , passaged in culture once ) in 100-200ul of PBS . Infection dose , culture conditions and spirochete enumeration were described previously [11] . Groups of 5-week old mice received 1010 CFU of live L . plantarum strain 256 ( Lp ) in 100μl of PBS or PBS alone via oral gavage . Mice received treatments daily for five days and two additional boosters every other week for a total of 30 oral treatments over a period of 5 weeks . One week after the final treatment mice were infected intraperitoneally with a sublethal dose of L . interrogans . Control groups of treated mice were kept uninfected . Mice were monitored for two weeks: daily records were kept for changes in body weight , urine was collected every day and 50μl of blood was collected every other day . At termination , 8-weeks after the start of oral treatments and 2-weeks post infection , blood , kidney , spleen and lymph nodes were collected . Kidney tissue was used for quantification of spirochetes , quantification of immune marker transcripts , histopathology and culture at 30°C for 7 days for determination of spirochetal viability . Peripheral lymph nodes ( mandibular , axillary , brachial , inguinal ) and spleen were used for immunophenotyping ( Fig 1A ) . Quantification of total mouse immunoglobulin concentration IgM , IgG , IgG1 , IgG2a , IgG3 in mouse serum was done using Ready-Set-Go ELISA kits ( eBioscience ) . A panel of chemokines and cytokines , CxCL1 ( KC ) , CxCL2 ( MIP-2 ) , CCL5 ( RANTES ) , TNF-α , and IFN-γ were measured in mouse serum ( 1:2 dilution in assay buffer ) with ProcartaPlex Multiplex Immunoassays ( eBioscience ) according to the manufacturer’s instructions . Samples were processed with xMAP Technology by MagPix Luminex using 50-bead count and analyzed with eBioscience Procarta Plex Analyst software . DNA was extracted from blood , urine , and kidney using a NucleoSpin tissue kit . Leptospira was quantified using TAMRA probe and primers ( Eurofins ) to Leptospira 16s rRNA by qPCR . Total RNA was extracted from tissues using an RNeasy mini kit and reverse transcribed using a high-capacity cDNA reverse transcription kit . cDNA was subjected to real-time PCR using primer and TAMRA probes previously described [11] . PCR data are reported as the relative increase in mRNA transcript levels of CxCL1 ( KC ) , CxCL2 ( MIP-2 ) , CCL5 ( RANTES ) , TNF-α , IFN-γ , iNOS and ColA1 corrected for by the respective levels of β-actin or to GAPDH . A primer list is provided in supplemental material ( S1 Table ) . Single cell suspensions of lymphoid tissues were prepared as described [11] . Live/dead cell stain was used to eliminate dead cells . Cells were incubated in Fc blocking for 15 min at 4°C in staining buffer and incubated with the appropriate marker for surface staining in the dark for 30 min at 4°C . Surface markers for T cells ( CD3 , CD4 , CD8 , CD62L , CD44 ) were described previously [11] . The surface marker for B cells was CD-19 conjugated with PercP Cy5 . 5 . For myeloid cells the following lineage surface markers ( Tonbo ) were used ( 1:200 ) : CD3 conjugated with pacific blue , MHC-II conjugated with allophycocyanin , CD11b conjugated with phycoerythrin Cy7 , CD11c conjugated with allophycocyanin Cy7 , and Ly6C conjugated with FITC . Cells were acquired on a BD-LSR II flow cytometer and analyzed using Flow Jo software . Kidneys were fixed with 4% paraformaldehyde and processed for histology ( paraffin blocks ) or immunostaining ( 5-μm cryosections ) . For histopathology , tissues were stained by H&E and evaluated for interstitial inflammation and glomerular size and tubular damage under an Axio Zeiss Imager A1 light microscope . Glomeruli were scored by measuring size in 5 fields per sample and averaging groups . Nephritis scores were graded blindly on a scale of 0–5 in a longitudinal section of the organ following previously published criteria [12] . Silver stain of kidney sections were used to visualize Leptospira under a light microscope . Fibrosis was evaluated after Masson’s trichrome staining of kidney sections [13] in which 3 randomly chosen fields were digitally analyzed ( 40x , Photoshop ) as a percentage of pixels of the total area . Immunohistochemistry was done at the Translational Pathology Shared Resource , Vanderbilt University Medical Center . Briefly , slides were deparaffinized and heat induced antigen retrieval was performed on the Bond Max using Epitope Retrieval 2 solution for 20 minutes . Sections were blocked at room temperature using saline containing 0 . 1% BSA and 10% goat serum ( Jackson ImmunoResearch Laboratories ) . The following primary antibodies were used: anti-CD45 ( Abcam ) , anti-neutrophil antibody ( NIMP-R14 , Abcam ) , anti-F4/80 ( NB600-404 , Novus Biologicals ) , and anti-CD3 ( Abcam ) , and biotinylated secondary antibodies were developed with streptavidin HRP and diaminobenzidine ( Vector lab ) . Blinded quantification of cells expressing the specified marker was done by counting positive cells/total cells in 10 randomly chosen fields ( 400X ) from the cortex and medulla of kidney . At least two sections per kidney were counted for each experiment . Images were taken at 200X , or 400X using a Zeiss microscopy with ZEN software . Data analysis was done using GraphPad Prism software . Multiple comparisons between groups were done by Two-Way ANOVA . Single comparisons within uninfected and infected groups were analyzed with two-tailed paired t-test , with two-tailed unpaired Mann-Whitney U test ( interstitial nephritis ) , with unpaired t-test with Welch’s correction and with multiple t-tests . α = 0 . 05 for all tests .
Mice repeatedly treated with a live bacterium , L . plantarum ( Lp ) , as well as PBS treated controls were infected intraperitoneally with a sub-lethal dose of Leptospira interrogans serovar Copenhageni on day 0 and were monitored for 2 weeks . Two additional groups of control mice received oral treatments but were not infected . Body weight loss/gain was recorded daily , blood was collected on alternate days , and urine was collected daily for quantification of number of Leptospira 16s rRNA by qPCR ( Fig 1A ) . In the infected groups , PBS treated mice lost a maximum of ~9% weight in specific days ( 4 , 10 , 11 , 12 ) , whereas mice treated with L . plantarum lost a maximum of ~4% weight on days 13 and 14 . Differences in weight between infected mice treated with L . plantarum were not different from the uninfected controls . Significant differences were seen between all groups ( infected versus uninfected , Two-Way ANOVA p<0 . 0001 ) and between infected mice previously treated and untreated with L . plantarum ( t-test p<0 . 0001 ) ( Fig 1B ) . Quantification of the number of Leptospira in blood and urine from infected mice pre-treated with L . plantarum ( Lp/Lepto ) trended towards lower numbers of spirochetes compared to infected controls ( PBS/Lepto ) , although these differences were not statistically significant ( Fig 1C and 1D ) . Empirical examination of H&E sections of kidneys collected 2 weeks post infection showed that mice pre-treated with L . plantarum ( Lp/Lepto ) had less mononuclear lymphocyte infiltrates and less tubular damage than infected PBS treated controls ( PBS/Lepto ) . Silver stain analysis of the same kidney sections showed morphologically intact Leptospira in the tubules of both groups of infected mice pre-treated with PBS or with L . plantarum ( Fig 2A ) . Quantification of Leptospira by qPCR showed about the same numbers of Leptospira per ug of kidney tissue from PBS and L . plantarum treated infected mice ( Fig 2C ) . Culture of the same kidney tissues showed live mobile spirochetes under dark field microscopy and qPCR quantification showed an average of 3-8x106 Leptospira in 7-day cultures of kidney from treated mice . We measured the interstitial nephritis scores and the glomeruli size structures in kidney H&E . We found that infected mice pre-treated with L . plantarum ( Lp/Lepto ) had lower interstitial nephritis scores than PBS treated infected mice ( PBS/Lepto ) and that the glomeruli structures of infected mice pre-treated with L . plantarum ( Lp/Lepto ) were comparable in size to the uninfected controls . In contrast , infected mice ( PBS/Lepto ) had decreased glomeruli sizes ( Fig 2B ) . We evaluated Leptospira-induced renal inflammation by quantification of mRNA transcription of CxCL1 ( KC ) , CxCL2 ( MIP-2 ) , CCL5 ( RANTES ) , TNFα and IFNγ by qRT-PCR . Infected mice pre-treated with L . plantarum ( Lp/Lepto ) had lower levels of all three chemokines CxCL1 , CxCL2 and CCL5 in kidney than PBS treated infected controls ( PBS/Lepto ) , although only differences in CxCL1 were significant; transcription of cytokines TNFα and IFNγ were not different than controls ( Fig 2D ) . Interstitial collagen deposition ( fibrosis ) was evaluated using Masson’s trichrome staining of kidney sections from infected mice , which was quantified by digital image analysis ( Fig 3 ) . Furthermore , transcription of fibrosis markers ( inducible nitric oxide , iNOS and fibroblast activation marker collagen1 A1 , ColA1 ) was quantified by qPCR . Kidney sections from infected mice pre-treated with L . plantarum had significantly less septa ( ~23% , Lp/Lepto ) of blue-staining fibrillar extracellular matrix expanding tubulointerstitial spaces than infected mice pre-treated with PBS ( ~50% , PBS/Lepto ) , ( Fig 3A and 3B ) . Furthermore , infected mice pre-treated with L . plantarum ( Lp/Lepto ) had lower levels of fibrosis markers iNOS and ColA1 than infected controls , although only differences for ColA1 were statistically significant ( Fig 3C ) . We measured the amount of total antibody in pre-treated uninfected and infected mice ( Fig 4 ) . Uninfected mice pre-treated with L . plantarum ( Lp ) did not produce higher levels of total antibodies ( IgM or IgG , IgG1 , IgG2a , IgG3 ) than controls ( PBS ) . Infected mice pre-treated with L . plantarum ( Lp/Lepto ) produced higher amounts of total IgM , IgG , IgG1 , IgG2a and IgG3 than the respective infected PBS treated controls ( PBS/Lepto ) ( Fig 4A ) . Measurements of the concentration of chemokines and cytokines in serum of infected mice pre-treated with L . plantarum showed significant decreases in chemokines CxCL1 ( KC ) and CCL5 ( RANTES ) whereas the concentration of CxCL2 ( MIP-2 ) , TNFa and IFNg did not differ from the PBS treated infected controls ( Fig 4B ) . We analyzed B and T cell populations in spleen and peripheral lymph nodes of uninfected and infected mice pre-treated with L . plantarum , by flow cytometric analysis ( Fig 5 ) . Uninfected mice pre-treated with L . plantarum ( Lp ) had significant increases of the percentages of B cells in spleen and in lymph nodes , and of CD4+ T cells in spleen ( Fig 5A and 5C ) . Infected mice pre-treated with L . plantarum ( Lp/Lepto ) responded with a significant decrease of CD4+ T cells in spleen and in lymph nodes , an increase of CD8+ T cells in spleen and a decrease of Double Negative ( DN ) T cells in spleen , whereas B cell populations were not affected in both tissues ( Fig 5B and 5D ) . We further analyzed CD4+ T helper and CD8+ cytotoxic T cell subsets by labeling naïve ( CD62L+ ) , early effector ( CD62L−/CD44− ) , effector ( CD44+ ) and memory T cells ( CD62L+/CD44+ ) in uninfected and infected mice pre-treated with L . plantarum ( Fig 6 ) . Analysis of helper cells revealed that treatment with L . plantarum ( Lp ) led to an increase of naïve and memory T cells and to a decrease of early effector/effector in spleen and lymph nodes of uninfected mice ( Fig 6A and 6C ) , whereas infected mice pre-treated with L . plantarum ( Lp/Lepto ) shifted CD4+ T cell responses toward an increase in early effector/effector and a decrease of naïve and memory T cells in both tissues ( Fig 6B and 6D ) . Analysis of CD8+ cytotoxic T cell profiles in pre-treated uninfected and infected mice mirrored the switch observed for CD4+ except that CD8+ memory T cell populations were not affected . We further evaluated inflammatory immune responses by profiling four subsets of myeloid cell populations in spleen and in peripheral lymph nodes of uninfected and infected mice pre-treated with L . plantarum , by flow cytometric analysis using MHC II , CD11b , CD11c , Ly6C markers ( Fig 7 ) . Uninfected mice treated with L . plantarum ( Lp ) had marked increases of neutrophils ( MHC II− CD11b+ ) in spleen and monocytes ( MHC II− CD11b+ Ly6C+ ) in peripheral lymph nodes , and decreased numbers of macrophages ( MHC II+ CD11b+ ) and dendritic cells ( MHC II+ CD11c+ ) in spleen ( Fig 7A and 7C ) . Infected mice pre-treated with L . plantarum ( Lp/Lepto ) responded with a significant decrease of monocytes and an increase of macrophages in both tissues ( Fig 7B and 7D ) . We analyzed whether L . plantarum treatment promotes recruitment of distinct leucocyte populations to the target organ in the presence and absence of Leptospira infection by immunohistochemistry of kidney sections using CD45+ , NIMP-R14+ , F4/80+ and CD3+ to stain total leucocytes , neutrophils , macrophages and T cells , respectively ( Fig 8 ) . In the absence of infection , kidneys of mice pre-treated with L . plantarum ( Lp ) had moderately increased numbers of myeloid cells compared to PBS treated controls ( PBS ) . However , in the presence of Leptospira infection numbers of CD45+ total leucocytes and CD3+ T cells increased by 10 fold ( PBS/Lepto ) ; these numbers were reduced by a third in kidneys of infected mice pre-treated with L . plantarum ( Lp/Lepto ) , ( Fig 8A and 8D ) . A striking observation was that infected mice pre-treated with L . plantarum ( Lp/Lepto ) , which we had previously determined to have less kidney damage ( Figs 2 and 3 ) , had a marked increased in NIMP-R14+ neutrophils and F4/80+ macrophages ( at least 2 fold higher ) as compared to infected PBS-treated controls ( PBS/Lepto ) , ( Fig 8B and 8C ) . In the kidney of these mice ( PBS/Lepto ) , which we previously determined to have more damage than L . plantarum treated infected mice , we detected a large number of infiltrating leucocytes looking like PMN that are not positive for NIMP-R14 .
New technologies are being used to advance knowledge of host immunity to pathogenic Leptospira species [14–21] . One aspect of the work done in our laboratory is the design of molecular tools and development of oral delivery vehicles for prevention or therapeutic management of infectious diseases [9 , 10 , 22–24] . There is scientific evidence supporting the health effects generally attributed to consumption of commensal probiotics with a number of studies discussing application to diseases caused by gastrointestinal , respiratory and urogenital bacterial infections [1 , 25 , 26] . In this study , we evaluated if a commensal probiotic immunomodulator , Lactobacillus plantarum , can be used for prevention of leptospirosis . Analysis of Leptospira burden and disease scores using the sublethal C3H/HeJ mouse model of infection [11] showed that repeated oral pre-treatments with live L . plantarum restored weight gain and affected L . interrogans burdens in blood and urine , but it did not prevent colonization of the kidney as demonstrated by the detection of Leptospira DNA by qPCR , visualization of morphologically intact spirochetes in silver stains and culture of live L . interrogans from kidney tissues in infected mice pre-treated with L . plantarum . Kidney H&E histopathology data showed that infected mice pre-treated with L . plantarum ( Lp/Lepto ) had less mononuclear lymphocyte infiltrates , less tubular damage and less interstitial nephritis scores than infected controls pre-treated with PBS ( PBS/Lepto ) . Glomeruli size in kidney of infected mice pre-treated with L . plantarum ( Lp/Lepto ) were about the same size as Lp and PBS uninfected controls [27][11 , 28] . Further analysis of immune marker transcripts in kidney ( CxCL1/KC , CXCL2/MIP-2 , CCL5/RANTES , TNFα and IFNγ ) showed that infected mice pre-treated with L . plantarum produced less pro-inflammatory chemokines KC , MIP-2 and RANTES known to be upregulated during infection [13 , 29–31][11] although transcription of cytokines TNFα and IFNγ was not affected . A decrease in the same chemokine markers was also observed in blood . Differences in KC were significant in kidney and blood , and differences in RANTES were significant only in blood . Furthermore , renal fibrosis was analyzed by staining of interstitial collagen deposition of kidney sections and by molecular quantification of mRNA transcripts of fibroblast activation marker ( collagen1 A1 , ColA1 ) and inducible nitric oxide ( iNOS ) which play a role in Leptospira-induced interstitial nephritis [13] and induction of kidney fibrosis [16] . Infected mice pre-treated with L . plantarum ( Lp/Lepto ) had ~50% less fibrosis and produced less transcripts of ColA1 than infected mice pre-treated with PBS ( PBS/Lepto ) . These results suggest that repeated oral treatment with live L . plantarum prevented upregulation of collagen 1A1 mRNA and transcription of pro-inflammatory chemokines which may have reduced accumulation of collagen in the tubulointerstitial spaces , thereby rescuing severe kidney pathology . Moreover , since the bacterial load in kidney is equivalent in L . plantarum ( Lp/Lepto ) and PBS treated infected mice ( PBS/Lepto ) , these data suggest that renal lesions may be triggered by the host inflammatory immune response rather than by direct presence of Leptospira toxins or metabolism . However , we previously showed that renal fibrotic lesions occur only when live Leptospira , not remnant antigens , are present in the kidney and that the fibrosis is not proportional to the bacterial load [16] . We also showed that fibrosis could occur in absence of B or T cells [16] . Thus , the question remains about the factors leading to renal lesions in infected mice , that we show here to be reduced by oral treatments with L . plantarum . It is generally understood that protection against Leptospira infection is most likely driven by humoral antibody responses . We analyzed the contribution of antibody responses to Leptospira infection in mice pre-treated with L . plantarum and with PBS . We detected significant increases in total IgM , IgG , IgG1 , IgG2a and IgG3 only in infected mice pre-treated with L . plantarum . High amounts of IgG1 and IgG3 suggest that initial neutralization of Leptospira may be mediated by IgM with help of IgG1 and IgG3 which may have affected bacterial loads in blood and urine . However , this response was either late or not robust enough to prevent access of Leptospira to the kidney , since equivalent loads of Leptospira were measured in kidney of L . plantarum and PBS treated infected mice . It is known that pathogenic Leptospira reach the kidney in the very first hours post intraperitoneal infection to escape blood defenses [14 , 15] . Analysis of the effect of L . plantarum treatments in adaptive immune response shows that uninfected mice had increased numbers of B cells , increased numbers of naïve and memory CD4+ T cells and lower numbers of effector CD4+ cells in spleen and lymph nodes . In contrast , the immune cell profiles of infected mice pre-treated with L . plantarum shifted toward a decreased number naïve and memory CD4+ helper T cells and to an increased number of effector CD4+ T cells in both tissues , whereas B cell populations remained the same as infected PBS treated controls . Changes in populations of CD8+ cytotoxic T cells mimicked CD4+ helper T cells except that CD8+ memory was not affected . These results suggest that infection with Leptospira engages a systemic immune response spearheaded by effector T helper and cytotoxic T cells in mice pretreated with L . plantarum and that memory T cells do not appear to be engaged . These results are consistent with findings in other studies in which depletion or absence of T cells led to severe pathological lesions in kidney in C3H/HeJ mice [32] and worse kidney lesions in C57BL/6J [14] . A striking result observed was that , in the absence of infection , treatments with L . plantarum led to an increase of neutrophils and a decrease of macrophages and dendritic cells in spleen , and to an increase of monocytes in peripheral lymph nodes , whereas Leptospira infection of mice pre-treated with L . plantarum led to decreased populations of monocytes and increased populations of macrophages in both tissues . Immunohistochemistry of sections of kidney of infected mice pre-treated with L . plantarum showed an overall decrease of CD45+ total leucocytes and CD3+ T cells which is consistent with the lower numbers of mononuclear lymphocyte infiltrates observed using H&E staining . Furthermore , we observed an enrichment of neutrophils and macrophages in this target tissue . We speculate that the enrichment of populations of monocytes in peripheral lymphoid tissue after treatment with L . plantarum may result in deployment of larger numbers of these cells to target organs after Leptospira infection , which could explain the higher numbers of macrophages we measured in spleen , lymph nodes and in the kidney . In kidney of infected mice treated with PBS we detected a number of leucocytes that failed to stain for the NIMP-R14 marker suggesting that Leptospira infection does not trigger recruitment of PMNs , which were abundantly found in kidneys of mice pretreated with L . plantarum . If these PMN were phagocytic , we would have expected a decrease in the number of Leptospira in the kidney of L . plantarum treated group , which was not the case . Neutrophil enrichment in lymphoid tissue after L . plantarum treatment could potentially explain homing of these cells to the kidney after Leptospira infection that could result in entrapment of Leptospira via NETosis . Neutrophil extracellular traps have been shown to be involved in the innate immune response to infection with Leptospira interrogans Fiocruz L1-130 [19] also used in our study . However , in Scharrig et al . depletion of PMN resulted in a decrease of the number of Leptospira in blood 3 days post infection and in kidney 15 days post infection , even though nephritis remained the same in non depleted mice , suggesting that PMN do not play a role in nephritis [19] . In our study , the presence of both macrophages and neutrophils in kidney appears to be associated with the decrease of pathogenic activity of Leptospira in this target tissue . This is paradoxical considering the common notion that both macrophages and neutrophils are supposed to be the cells producing nitric oxide and fibrogenic components , which have negative effects on host cells [13] . A reduction of T cells in kidney could imply that T cell signaling is important to orchestrate systemic immune responses to infection with Leptospira but these cells may not be directly involved in resolving infection in the kidney . Our results suggest that in mice infected with L . interrogans , pre-treatment with L . plantarum triggers a complex myeloid and T cell response that orchestrates the deployment of monocytes from lymphoid tissue and the recruitment of neutrophils and macrophages to kidney . Further , the presence of these myeloid cells in kidney may be associated with the reductions in pathogenesis observed . Future studies to analyze the effect of L . plantarum in post-exposure treatments could also shed light on the possible use of commonly available probiotics as alternative palliative care for leptospirosis .
|
Leptospirosis is an emerging neglected zoonotic disease with worldwide distribution that affects nearly all vertebrates and causes infection in ~1 million people on a yearly basis . Effective cross-protective vaccines are not available and antibiotic treatment is only effective if used early in the course of infection . In this study we describe how repeated oral treatment of mice with a commonly used probiotic , Lactobacillus plantarum , did not completely prevent colonization of the kidney by Leptospira interrogans but it did reduce signs and symptoms of leptospirosis . We also analyzed a number of immune cell types in spleen , lymph nodes and kidney after treatment and found that complex responses orchestrate the deployment of phagocytes to the kidney in infected mice . Our results suggest that pre-treatment with L . plantarum modulates systemic immune responses in a beneficial way in a mammalian host later exposed to L . interrogans infection .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"methods",
"Results",
"Discussion"
] |
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] |
2017
|
Pre-treatment with Lactobacillus plantarum prevents severe pathogenesis in mice infected with Leptospira interrogans and may be associated with recruitment of myeloid cells
|
Dengue imposes a substantial economic and disease burden in most tropical and subtropical countries . Dengue incidence and severity have dramatically increased in Mexico during the past decades . Having objective and comparable estimates of the economic burden of dengue is essential to inform health policy , increase disease awareness , and assess the impact of dengue prevention and control technologies . We estimated the annual economic and disease burden of dengue in Mexico for the years 2010–2011 . We merged multiple data sources , including a prospective cohort study; patient interviews and macro-costing from major hospitals; surveillance , budget , and health data from the Ministry of Health; WHO cost estimates; and available literature . We conducted a probabilistic sensitivity analysis using Monte Carlo simulations to derive 95% certainty levels ( CL ) for our estimates . Results suggest that Mexico had about 139 , 000 ( 95%CL: 128 , 000–253 , 000 ) symptomatic and 119 ( 95%CL: 75–171 ) fatal dengue episodes annually on average ( 2010–2011 ) , compared to an average of 30 , 941 symptomatic and 59 fatal dengue episodes reported . The annual cost , including surveillance and vector control , was US$170 ( 95%CL: 151–292 ) million , or $1 . 56 ( 95%CL: 1 . 38–2 . 68 ) per capita , comparable to other countries in the region . Of this , $87 ( 95%CL: 87–209 ) million or $0 . 80 per capita ( 95%CL: 0 . 62–1 . 12 ) corresponds to illness . Annual disease burden averaged 65 ( 95%CL: 36–99 ) disability-adjusted life years ( DALYs ) per million population . Inclusion of long-term sequelae , co-morbidities , impact on tourism , and health system disruption during outbreaks would further increase estimated economic and disease burden . With this study , Mexico joins Panama , Puerto Rico , Nicaragua , and Thailand as the only countries or areas worldwide with comprehensive ( illness and preventive ) empirical estimates of dengue burden . Burden varies annually; during an outbreak , dengue burden may be significantly higher than that of the pre-vaccine level of rotavirus diarrhea . In sum , Mexico’s potential economic benefits from dengue control would be substantial .
Dengue fever is the most important arthropod-borne viral disease affecting humans , with about half the world’s population estimated to be at risk of infection , and epidemics increasing in frequency , magnitude , and geographical reach [1–4] . Dengue imposes a substantial economic and disease burden in most tropical and subtropical countries . Mexico is no exception [5] . Dengue is hyperendemic in Mexico , with all four dengue virus ( DENV ) serotypes isolated in the country , high levels of disease and an increasing impact during the last decades [5–8] . Transmission of dengue is regularly reported in 28 of the 32 Mexican states; the main mosquito vector , Aedes aegypti , has been reported in 30 states [6 , 8 , 9] . The severity of dengue episodes has also steadily increased , with a substantial increase in severe dengue episodes since 1995 , although case fatality rate has remained relatively low compared to other Latin American countries [7 , 10 , 11] . Objective , comparable measures of the burden of dengue are important to inform decisions about health policy , research , and health service priorities and to increase scientific and social awareness of the disease [12–15] . Despite the need for timely and reliable epidemiological data , dengue burden estimates are sparse . The total burden imposed by a disease includes the illness or disease burden , which measures the impact of a disease on morbidity and mortality in a specific population , and the economic burden [16 , 17] , which includes the cost of illness , prevention and monitoring or surveillance strategies , and other economic impacts ( e . g . , decrease in travel , seasonal overload of health systems ) [18 , 19] . Because dengue is a reportable illness in most endemic countries , an initial approximation of the total number of dengue episodes in a year is simply the total episodes reported to the country’s Ministry of Health ( MoH ) through surveillance systems . Dengue is a reportable disease in Mexico; the MoH has promulgated protocols for laboratory confirmation and collects and disseminates weekly surveillance data [20] . The MoH is responsible for setting national guidelines , rules , and procedures that the 32 state health departments need to follow , although state and local health services are responsible for daily operations . Vector control and dengue surveillance systems guidelines are defined by the MoH at the federal level , although it collaborates with the 32 state health services and other health organizations including Mexican Institute of Social Security ( IMSS ) , Institute of Social Security and Services for State Workers ( ISSSTE ) , Mexican Petroleum ( PEMEX ) , and the Armed Forces medical services [9 , 20] . A sample of patients with suspected DENV infection is diagnosed by a public health laboratory network ( all probable patients in areas with no recent dengue episodes or during low transmission periods and about 30% of patients when there is evidence of transmission and during outbreaks ) using confirmatory assays ( NS1 , IgM , or IgG ELISA ) , and a subset of these samples is analyzed for virus isolation ( 10% of the positive samples ) [8 , 9 , 21] . Patients with probable and confirmed dengue have to be reported weekly , while probable or confirmed DHF and dengue-related deaths must be reported within 24 hours [20] . The MoH estimates the number of dengue episodes in two steps: probable cases are first multiplied by the proportion of positive cases from the lab-diagnosed sample ( called possible cases ) , and then added to the total lab-confirmed cases . The MoH assumes that all episodes are notified [8] . However , passive surveillance systems have limitations . Passive surveillance systems are adequate for monitoring general trends in DENV infections; however , they usually underreport the total episodes of symptomatic dengue [22–26] . Febrile DENV infections with relatively mild symptoms have very low reporting ratios ( number of reported dengue episodes / total dengue episodes in the population ) , and reporting increases with severity [27 , 28] . Other limitations in passive surveillance systems , even in well-funded systems such as Mexico or Puerto Rico , include misdiagnosis due to limited sensitivity of diagnostic tests , cost constraints , unrecognized dengue symptoms , variation in reporting ratios by severity of symptoms , and differences in diagnosis between epidemic and non-epidemic years [22 , 23 , 29–32] . Some health-seeking behaviors also reduce reporting ratios , such as symptomatic patients visiting alternative health providers , including pharmacies or local healers , or simply staying at home . In Mexico , there is little or no reporting from the private sector [33] , and there is wide variation in the quality of reporting of notified cases . Limited reporting of symptomatic DENV infections leads to conservative estimates of economic and disease burden [24–26 , 34] , which may affect health policy decisions . Many dengue-endemic countries are transitioning to the revised WHO dengue case classification [35]; however , while the new WHO classification is used in some clinical settings in Mexico , surveillance data are still compiled as DF and DHF [36] . The new web-based Epidemiological Surveillance Platform ( EPS ) was implemented in 2008 [8] in which health care workers enter cases directly into the national data base . It provides real-time data to support public health decisions . Before the EPS , dengue was reported in paper forms to state epidemiological departments , entered into a local electronic system , and then emailed to the federal health authority , which many times resulted in fragmented , non-compatible data [9] . Although the EPS has improved the quality of reporting , there is still substantial room for improvement: about 40% of the reports of dengue episodes in 2009 were still considered of bad or very bad quality [8] . We overcame this limitation adjusting officially reported dengue episodes , based on reporting ratios from a prospective cohort study in Morelos , Mexico , to obtain the overall number of symptomatic DENV infections . In addition to surveillance strategies , prevention and control are a substantial part of the economic burden of dengue . While there are various promising dengue prevention and control technologies under development [37–40] , currently the only way to prevent DENV transmission is to control the vector population [41] . Vector control , prevention , and surveillance are financed through the MoH at the federal level , including the design and maintenance of the EPS . Prevention and control activities include entomological surveillance and risk assessment through mosquito ovitraps and larval indices , as well as vector control activities such as insecticide nebulization , indoor spraying , and use of larvicides . Other activities include educational and awareness campaigns , training health and vector control personnel , and community-based participatory control programs [9 , 33 , 42 , 43] . The objective of this study was to measure the economic and disease burden of symptomatic DENV infections in Mexico . We estimated the economic costs of dengue using a societal perspective , including vector control and surveillance costs , and the disease burden of dengue in disability-adjusted life-years ( DALYs ) . Previous studies have estimated the economic and disease burden of dengue illness in countries from the Americas [44–52] , including Mexico [44] . However , these estimates for Mexico are limited due to incomplete data and extrapolation from neighboring countries [53] . Here we addressed these limitations by combining data from multiple sources and refined estimates of the economic and disease burden of dengue in Mexico . Specifically , we estimated ( i ) total average annual number of dengue episodes , ( ii ) unit costs per episode , ( iii ) vector control and surveillance costs , and ( iv ) disease burden using DALYs . With this study , Mexico joins Panama [45] , Puerto Rico [47] , Nicaragua [49] , and Thailand [54] as the only countries or areas worldwide with comprehensive ( illness and preventive ) peer-reviewed empirical estimates of the cost of dengue .
We estimated the economic burden of dengue from a societal perspective and the disease burden of dengue in DALYs , using the WHO methodology [55 , 56] . Specifically , we used the following equations: Economic burden of dengue ( US dollars ) = total episodes x costs per episode + dengue prevention and surveillance activities + other economic impacts Disease burden of dengue ( DALYs ) = years of life lost ( YLL ) due to premature death + years lived with disability ( YLD ) An accurate estimate of the total number of dengue episodes is critical to obtain the economic and disease burden of dengue , and previous studies have found that uncertainty in the total number of dengue episodes is the main source of variability [44 , 57] . The costs per dengue episode include direct medical and non-medical costs and indirect costs per non-fatal and fatal case . The burden of disease was measured in DALYs , a summary measure of population health that combines information on mortality and non-fatal disease outcomes [16] . We based our burden estimates on the years 2010 and 2011 because we had access to detailed surveillance data in those years from the EPS [8 , 9] . The years 2010 and 2011 are , on average , relatively close to historical averages in reported cases . The average annual reported episodes were 30 , 941 in 2010–2011 , 58 , 688 in 2007–2011 ( which includes the 2009 outbreak ) , 35 , 091 in 2002–2011 , and 32 , 886 in 1995–2011 [8] . Thus , if anything , our burden estimates are slightly conservative considering long-term patterns . Last , we performed a probabilistic sensitivity analysis of the economic and disease burden estimates using Monte-Carlo simulations . Monte Carlo simulations are commonly used to model phenomena with substantial uncertainty in its parameters . The method relies on running repeated trials based on random sampling from the probability distribution of each parameter in the model , and recording the results of each simulation . The results from the repeated trials were used to describe the uncertainty in the model . We report our results in 2012 US dollars ( USD ) using the 2012 exchange rate ( USD1 = 12 . 88 Mexican pesos , MXN ) , and GDP deflators [58] . To refine the estimates of the total number of dengue episodes , officially reported dengue episodes can be adjusted for underreporting using an expansion factor ( EF ) . An EF can be calculated as the analyst’s best estimate of the total number of dengue cases in a population divided by the number of reported cases considered dengue ( EF = 1/reporting ratio ) [59] . We estimated total episodes of dengue by multiplying reported episodes ( 41 , 333 episodes in 2010 , and 20 , 548 in 2011 ) by an empirical EF derived from a prospective cohort study in Morelos [60 , 61] . The prospective cohort study was conducted in a dengue-endemic urban area in Morelos , Mexico , to assess the rate of DENV infections among the neighbors of reported dengue cases [61] . Set in the towns of Tepalcingo and Axochiapan during the 2011–2012 dengue season ( June 2011-March , 2012 ) , the study contained 1 , 172 participants aged 5 years and above . All participants or the parent or legal guardian of minors ( 5–17 years of age ) gave written informed consent . The Morelos study was approved by the Ethics Commission of the National Institute of Public Health , Mexico and the Brandeis University Committee for Protection of Human Subjects . Researchers collected 10-ml blood samples ( 6-ml for serological diagnosis and 4-ml for DNA extraction [62] ) from all participants at baseline and 6-ml in a follow-up 3–4 months later , in addition to demographic , environmental , health-seeking behavior ( e . g . , number of visits to health care facilities , type of facility , private or public ) , and socio-cultural and entomological data . Passive and active monitoring occurred between the two rounds of data collection , including phone calls or house visits at least once a month . All dengue episodes were laboratory-confirmed by means of a paired DENV-specific IgM and IgG Capture ELISA ( PanBio ) at baseline and follow-up . Recent DENV infections were defined as: ( i ) IgM or IgG positive by capture assay , which measures recent dengue infection ( 2–3 months ) in the baseline sample ( pre-enrollment infections ) [63 , 64] , ( ii ) IgM or IgG positive in the follow-up sample where IgM and IgG were negative in the baseline sample ( post-enrollment infections ) , and ( iii ) availability of RT-PCR/NS1/IgM/IgG positive during the follow-up months from a visit to the local health service . We used the blood samples collected at baseline or follow-up to confirm DENV infection; 12 patients were also diagnosed during the febrile episode by the state of Morelos health services ( Servicio de Salud de Morelos ) using NS1 or IgM/IgG capture assays . Symptomatic dengue episodes were defined as lab-confirmed dengue and reported fever . Morelos provides a good reference value of reporting ratios of dengue episodes in Mexico . Morelos has strengthened its epidemiological surveillance in recent years , there is high level of dengue awareness and willingness to participate in dengue surveillance among the population and clinicians in the public health sector [33] . A recent study of benchmarking of effective healthcare coverage ( “the proportion of potential health gain that could be delivered by the health system to that which is actually delivered” , p . 1729 ) in Mexico based on 14 healthcare interventions [65] , suggests that Morelos’ quality of healthcare provision is not too different from the country’s average . Specifically , compared to other states Morelos’ measure of effective coverage was 0 . 54 standard deviations below the national mean or at the 30th percentile nationally . Recent studies have used healthcare indicators to estimate reporting ratios of dengue , based on access [16] and quality [34] of healthcare , with the latter probably better reflecting the idiosyncrasies of the system that may lead to underreporting . For these reasons , we consider that using the Morelos prospective cohort study to obtain point estimates of dengue burden is reasonable , and if anything , slightly conservative . To adjust for variation in reporting ratios , we used empirical estimates of EFs from a previous study of dengue in the Americas [44] in the sensitivity analysis . We derived costs per episode by combining patient interviews in four major hospitals in the states of Quintana Roo , Morelos , and Tabasco , macro-costing data from two major public hospitals in Tabasco , MoH health and surveillance data [66] , WHO-CHOICE [67] estimates for Mexico , and previous literature on dengue burden . Indirect costs were obtained based on productivity losses by age , considering both the patient and the patient’s caregivers . We estimated vector control and surveillance costs based on MoH data . Direct costs per episode . We estimated unit costs per dengue bed-day ( inpatient episodes ) and per visit ( outpatient episodes ) by combining macro-costing data from two major public hospitals , MoH surveillance data [66] , WHO-Choice estimates [67] , data from the Morelos cohort study [60] , and national health statistics [66 , 68] . Direct medical unit costs were obtained using a macro-costing technique based on data reported by two tertiary public hospitals . To derive direct medical inpatient and outpatient unit costs that were representative of the country , we derived cost ratios for the treatment of dengue in various settings from WHO-CHOICE costs estimates for Mexico [67] . For hospitalized patients , we estimated the relative weight of treated episodes in each type of hospital based on its share total hospital beds ( obtained from national health statistics ) assuming that the proportion of patients who are treated in each type of hospital is equal to its fraction of total hospital beds . For ambulatory episodes , we obtained the relative weights of episodes treated in each type of setting by combining data from the Morelos cohort study ( share of patients who did not visit a private or public health facility ) , average annual outpatients visits from health statistics and WHO-CHOICE estimates ( used a proxy for its relative utilization ) [67 , 68] . As the study did not provide any treatment to participants , it was unlikely to have any major effect on health care utilization . While receiving regular questions about febrile illness may have sensitized participants through a Hawthorne effect [69] , we expect the effect to be small , if any , since there was already substantial awareness of dengue in the area [33] . The costs for homecare ( including pharmacy visits ) were derived from combining data sources . The share of patients with apparent dengue who did not visit a hospital or health center ( about 30% , largely consistent with a previous study of healthcare use [70] ) were obtained from the Morelos cohort study . Of those patients who did not visit a health center , about 37% visited a pharmacy at the onset of their febrile illness . We derived the average expenditures on medications , transport , and diagnostic tests of these early pharmacy visits from36 interviews of hospitalized dengue patients ( S1 Table ) . We assumed that the patients who stayed at home had similar costs in medications as those who visited a pharmacy at the onset of their illness , but no transport or diagnostic costs associated with their dengue episode . The hospitalized patient interviews were also used to obtain non-medical direct costs , including transport , food , and hotel expenditures for dengue patients who visited a healthcare facility and their caregivers ( S1 Text ) . Indirect costs per episode . We used the human capital approach , based on work-time loss caused by dengue , to derive indirect costs per fatal and non-fatal episode [71] . Productivity loss estimates included days of work or school lost by the patient as well as relatives’ time spent caring for the patient . The breakdown by age and occupation at onset of dengue illness affect the estimates of productivity loss . Fig . 1 shows the breakdown by age of the reported cases in years 2010 and 2011 . The breakdown by occupation was derived assuming that all patients aged 5–15 years old were enrolled in school , patients aged 16–17 were divided between school and work based on empirical data from school enrollment [72] . We derived the average economic value of a work day lost for economically active adults ( employed or actively looking for employment ) based on Mexico’s wage distribution and employment rate from the Mexican National Institute of Statistics and Geography for patients aged over 17 years old [73] . For non-economically active adults ( unemployed and not actively looking for employment ) , the estimate was based on their reported main activity ( students , household chores , retired , disabled , and non-active ) . Individual and societal costs of school absence are difficult to value , but , being conservative , are at least equal to the cost of providing a day of school . We derived unit cost per-day of school lost using data on total educational expenditure at the federal , state , and municipal levels , and from the private sector , for years 2010 and 2011 . A school year averages 200 schools days [74] . Economic loss from days lost was valued as the number of days lost to dengue illness times the average value per day . We took the length of hospitalization from the Mexican MoH surveillance data . We estimated the durations of illness for ambulatory and hospitalized patients as the average values from surveys of dengue patients in the corresponding setting ( 326 hospitalized and 834 ambulatory ) across five countries in the Americas ( Brazil , El Salvador , Guatemala , Panama , and Venezuela ) [46] . We obtained the number ambulatory visits of hospitalized patients from interviews with them or their caregivers in Mexico . For ambulatory episodes , we used the same surveys from the Americas to derive the average duration of illness and total healthcare visits [46] . We based indirect costs of fatal cases on productivity losses by age using the age distribution of reported deaths from MoH surveillance data , the average economic value of a work day ( see above ) , and a 3% discount rate ( for consistency with international recommendations and previous studies ) [71] . We estimated the years of premature life lost based on life expectancy using WHO life tables [75] . Due to the paucity of data , we assumed that the rate of reporting of deaths attributed to dengue was equal to the rate of reporting in hospitalized episodes . We relaxed this assumption in the sensitivity analysis . Dengue prevention and surveillance . We estimated vector control and surveillance costs based on the Mexican MoH annual budget for dengue . The available data included only the years 2009 and 2010 , so we imputed the vector and surveillance budget in 2011 , using the average budget of the two previous years with adjustment for inflation . While vector control and dengue surveillance systems are managed by the MoH at the federal level , our estimates are conservative as they do not include possible additional spending on surveillance and vector control by state level agencies and municipalities ( mainly nebulization , larvae control , and patio clean-up campaigns ) . Other economic impacts of dengue . While important , data limitations did not allow us to reasonably estimate other economic costs associated to symptomatic DENV infections . These impacts include the detrimental effect of dengue outbreaks on tourism and travel [76–79] , co-morbidities and complications associated with dengue infection [80–85] , or the effects of health system overload [86] . When dengue outbreaks are clustered in time or location [87–90] , they may worsen treatment quality and decisions or degrade performance of clinical laboratories . The burden of disease was measured in DALYs , and is composed of the person’s years of life lost ( YLL ) due to premature death—based on incidence , fatality rate , and life expectancy , and a measure of the time the person lives in less than full health ( years lived with disability , YLD ) —based on incidence , length of illness , and impact on quality of life [14] . We estimated the burden of disease using the WHO methodology [55 , 56] , for comparability with previous studies , and expressed burden in DALYs per million population . We obtained the age distribution of non-fatal dengue episodes and deaths from surveillance data ( Fig . 1 ) and used model parameters ( age weights , disability weight , and discount rate ) based on previous studies [44 , 52 , 91] . Because the 2010 estimates of global burden of disease [16] changed their definition of DALYs ( dropping age weighting and time discounting ) , we also provide these numbers in the results section for comparability with future estimates . Under this new definition , a child death converts to a larger number of DALYs than previously . We used a probabilistic sensitivity analysis to address the uncertainty in our estimates of the disease and economic burden of dengue . We computed 10 , 000 Monte Carlo simulations simultaneously varying our parameter estimates for EFs , unit costs , days lost per episode , health service utilization , and household impact using RiskAMP [92] ( iterations drew random values from the distribution of each input using the Mersenne Twister random number generator ) . Our results include a base-case scenario , using our best estimates for each parameter , the uncertainty around these estimates based on the sensitivity analyses , and 95% certainty level ( CL ) bounds . To model variation in reporting ratios , we used a beta-PERT distribution ( hereafter PERT ) with the Morelos cohort empirically-derived EF as our best estimates . The range of variation in the distribution was based on a recent study of dengue in the Americas [44] , which identified five field studies that included reporting ratios . We used conservative estimates , including an EF of 1 . 0 as the lower bound in hospitalized cases . We also used a PERT distribution for direct medical costs , with the minimum and maximum values obtained in primary and tertiary hospitals for hospitalized cases and homecare and tertiary hospitals for ambulatory episodes from combining WHO-CHOICE [67] estimates , health statistics data [66 , 68] , macro-costing estimates , expert opinion , and patient interviews . We derived direct non-medical costs from patient interviews in four major hospitals . The variation in duration of dengue episodes and health service utilization was estimated using a normal distribution with parameters based on detailed MoH surveillance data , hospital interviews , and empirical estimates from a previous study in five countries in the Americas [46] . Last , we used a normal distribution of household impact based on weighted averages from Suaya et al . [46]
Expansion factors to adjust reported episodes . Table 1 shows a summary of the results from the prospective cohort study in Morelos , Mexico . We found a total of 253 DENV infections . Most of these infections were asymptomatic ( 61% ) , consistent with previous studies [93–96] . Only 67% of the participants with symptomatic infections visited a doctor , and most of them ( 74% ) sought care at least once in the public sector . Of all symptomatic dengue episodes that were attended by a doctor either as outpatient or inpatient , 32% were reported to the State of Morelos surveillance system [60] . In other words , for every symptomatic episode of dengue that was treated by a health professional and reported to the surveillance system , 3 . 1 episodes occurred . If we considered all cases of symptomatic dengue , irrespective of whether they were attended by a healthcare professional or not , only 21% were reported to the surveillance system . Thus , there were 4 . 7 symptomatic dengue episodes for every reported symptomatic episode . Reporting ratios vary considerably between the public and private sectors . In the public sector , 43% of the dengue episodes were reported ( reporting ratio = 0 . 43 ) , whereas no dengue episode was reported by the private sector . Limited or no reporting from the private sector has also been noted elsewhere [27 , 33 , 97] . Based on these results , Table 2 shows a summary of the expansion factors for overall ( EFT ) , hospitalized ( EFH ) , and ambulatory ( EFA ) dengue episodes needed to estimate the total cases of symptomatic dengue . Direct medical and non-medical unit costs . Table 3 shows the estimation procedure and main data used to obtain direct medical unit costs using macro-costing [98] . Combining these data with the distribution of cases and the cost ratio relative to a tertiary hospital , we derived an average cost estimate per bed-day ( $240 . 04 ) and per outpatient visit ( $65 . 53 ) , as shown in Table 4 . Non-medical direct costs were obtained from patient interviews . For hospitalized patients , daily non-medical costs were $25 . 16 for adults and $27 . 85 for children , and daily non-medical costs for ambulatory patients were $11 . 96 for adults and $9 . 09 for children , on average . For hospitalized patients , additional daily non-medical costs for other household members were $8 . 39 for adults and $6 . 56 for children . For ambulatory patients , the additional daily non-medical costs for other household members were $3 . 00 for adults and $6 . 00 for children . Indirect unit costs . We estimated indirect costs based on productivity loss from the number of school-days and work-days lost . The estimated average daily unit costs for elementary education ( 5–14 year olds ) were $7 . 32 in 2010 , and $7 . 59 in 2011 , and for high school education ( 15–18 year olds ) were $9 . 05 in 2010 and $9 . 14 in 2011 [72 , 99] . A work-day lost for economically active adults was estimated at $10 . 93/day in 2010 and $11 . 06/day in 2011 and for non-economically active adults at $4 . 26 in 2010 and $4 . 22 in 2011 . Overall , the economic value of the average work day lost was $8 . 20 in 2010 and $8 . 22 in 2011; about 1 . 7 times the minimum wage , which is consistent with estimates from previous studies [46 , 97] . Duration of dengue episodes and productivity loss . We estimated the duration of hospitalized dengue episodes at 13 . 9 days , including both the acute and the convalescent phases ( 7 . 4 days acute phase , 6 . 5 days convalescent phase ) . Based on hospital interviews in Mexico , we estimated that an adult had 2 . 4 ambulatory visits on average before being hospitalized , and a child had an average of 3 . 7 ambulatory visits prior to hospitalization . Ambulatory patients had a total of 3 . 9 healthcare visits , and illness had a total duration of 12 . 0 days . From the interviews , we obtained that each hospitalized patient affected on average 1 . 7 adults and 0 . 6 children in the household , and each ambulatory patient affected 2 . 2 adults and 0 . 4 children in the household on average . At the household level , school-days lost were 3 . 7 days for inpatients and 2 . 2 days for outpatients , and 6 . 1 work-days were lost for inpatients and 3 . 8 work-days were lost for outpatients . Summary of parameters and probability distributions for sensitivity analysis . Table 5 shows a summary of the main parameters used in the analysis , assumed probability distributions , and sources . The parameters described above were used to derive base case point estimates , and the distributions and range were used in the sensitivity analysis to obtain 95% certainty levels of economic and disease burden ( show in parentheses in the tables henceforth ) . Total adjusted symptomatic DENV infections . Table 6 shows a summary of reported cases by setting for years 2010 and 2011 , and the total estimated cases using EFs . MoH reported episodes of dengue include lab-confirmed episodes plus the proportion of positive cases from the lab-diagnosed sample multiplied by the probable cases reported ( probable dengue are suspected episodes of dengue with specific clinical symptoms ) . Overall , we estimated a total of 195 , 154 ( 95%CL: 180 , 459–355 , 343 ) non-fatal and 126 ( 95%CL: 80–180 ) fatal episodes of symptomatic dengue in 2010 , and 82 , 429 ( 95%CL: 75 , 203–142 , 041 ) non-fatal and 112 ( 95%CL: 75–170 ) fatal episodes of dengue in 2011 . Economic burden of dengue . The average cost per non-fatal dengue episode was $1 , 327 for hospitalized patients ( direct medical: $1 , 010; direct non-medical: $174; indirect: $143 ) and $451 for ambulatory patients ( direct medical: $253; direct non-medical: $92; indirect: $106 ) . The average indirect cost per fatal dengue episode was $63 , 817 . Altogether , the aggregate economic cost of dengue was $190 ( 95% CL: $165-$357 ) million in 2010 , with a per capita costs of $1 . 76 ( 95% CL: $1 . 52-$3 . 29 ) , and $149 ( 95% CL: $136-$231 ) million in 2011 with $1 . 36 ( 95% CL: $1 . 24-$2 . 11 ) per capita ( Table 7 ) . These results amount to an average economic burden of dengue of $170 ( 95% CL: $151-$292 ) million , or $1 . 56 ( 95% CL: $1 . 38-$2 . 68 ) per capita . The fatal episodes of dengue represent a relatively small share of the total economic burden ( 4 . 5% on average for years 2010 and 2011 ) . Surveillance and vector control cost about $0 . 76 per capita ( $0 . 71 in 2010 and $0 . 81 in 2011 ) , and this represents about 48 . 9% of the total economic burden of dengue in Mexico ( Fig . 2 ) . Fig . 2 shows the distribution of the economic burden of dengue in Mexico . Direct medical costs represent ~29% of the total average economic costs of dengue ( 34% in 2010; 23% in 2011 ) , and direct non-medical costs sum ~8% of the total costs ( 10% in 2010; 6% in 2011 ) . Fatal and non-fatal indirect costs , due to productivity loss , represent ~14% of the total economic costs of dengue ( 15% in 2010; 11% in 2011 ) . The main sources of variation for the economic burden of dengue estimates are shown in the tornado plot in Fig . 3 . The vertical line shows the point estimate for the average total economic burden of dengue ( $170 million ) . The variation for each parameter corresponds to the 95% certainty level obtained through the computation of 10 , 000 Monte Carlo simulations for each parameter , and for the simultaneous variation of all parameters ( top bar ) . The diagram shows that health service utilization represents the biggest source of variation among the parameters considered in the sensitivity analysis in this study , closely followed by EFs to refine estimates of reported dengue episodes . Disease burden of dengue . The total disease burden for the adjusted average of dengue episodes was 65 . 1 ( 95%CL: 36 . 0–98 . 7 ) DALYs per million population ( 83 . 5 in 2010; 46 . 7 in 2011 ) . Fatal episodes represented about 27% of the disease burden of dengue ( DALYs ) in 2010 and 45% of the disease burden in 2011 ( 2010: 22 . 3 YLL; 2011: 20 . 8 YLL ) . The Institute of Health Metrics and Evaluation’s 2010 global disease burden study ( GBD 2010 ) [16] dropped age weighting and time discounting from the original 1994 definition of DALYs [55 , 56] , which results in a higher relative weight of young children compared to adults . Table 8 shows a summary of DALYs estimated for Mexico using the original definition of DALYs ( WHO method ) [55] for comparison with past estimates , and the new GBD 2010 method [16] . The latter method results in less conservative estimates of disease burden , and higher relative weights of fatal cases ( YLL ) in the total DALY estimates ( YLL represented about 57% on average of total DALYs; 50% in 2010 and about 68% in 2011 ) . Most of the years lost to disability ( 85% ) , YLD , were due to ambulatory episodes of dengue . The numbers in parentheses indicate the region of uncertainty around base-case estimates ( 95% certainty levels ) . Uncertainty in DALYs is driven by the probabilistic distribution of EFs and the duration of hospitalized and ambulatory dengue episodes ( Table 5 ) . Extrapolation of dengue burden using historical data . If we assume that the age distribution of dengue episodes , proportion of ambulatory and hospitalized patients , overall fatality rates , and the reporting ratios of ambulatory and hospitalized cases in 2010–2011 are on average representative of the situation of dengue in Mexico in the previous years , we can estimate approximate economic and disease burden for those years . While these assumptions might be strong , the objective of this exercise is not to give precise estimates of dengue burden in previous years , but to assess how comparable are 2010–2011 data to historical data . Fig . 4 shows the total estimated number of dengue episodes and economic and disease burden for the previous 5 ( 2007–2011 ) , 10 ( 2002–2011 ) , and 17 ( 1995–2011 ) years . While the 5-year estimates ( 2007–2011 ) were heavily affected by the 2009 outbreak , the average of the annual burden of dengue in 2010–2011 seems a reasonable estimate of the 10-year and 17-year averages .
Dengue imposes a substantial economic and disease burden in Mexico . Because of limited data , combining multiple data sources is a key factor in achieving reliable estimates of dengue burden . Our 2010–2011 average economic and disease burden estimates ( $0 . 80 per capita excluding costs of surveillance and vector control , and 65 DALYs per million population ) are below the previous 95% confidence intervals of US$1 . 5–4 . 3 per capita and 82–147 DALYs per million population found for Central America and Mexico [44] . Reasons for our lower estimates include the use of refined , Mexico-specific reporting ratios based on the prospective cohort from Morelos , and dengue’s clustering in coastal and tropical areas [5] . Our estimates for the burden of dengue in 2010–2011 were similar to those obtained for the previous 10 and 17 years , but conservative compared to the average burden of disease in the past 5 years , driven partly by the 2009 outbreak ( Fig . 4 ) . In DALYs per million population , during this outbreak year dengue imposed a greater disease burden in Mexico ( 203 ) than pre-vaccination rotavirus diarrhea ( 174 ) [100] . Other studies have found comparable estimates of the economic and disease burden of dengue in the region . Suaya et al . [46] estimated economic burden of dengue per capita for Brazil ( $0 . 85 ) , Venezuela ( $0 . 71 ) , El Salvador ( $0 . 30 ) , Guatemala ( $0 . 10 ) , and Panama ( $0 . 31 ) . These numbers are underestimated since they were not adjusted for the underreporting of dengue episodes , and did not include vector control and surveillance costs . Halasa et al . [47] estimated an economic burden in Puerto Rico of $3 . 01 per capita without adjusting for EF , $10 . 84 using a refined estimate of dengue episodes , and $13 . 00 per capita including prevention activities , vector control , and surveillance costs . Explanations of the higher burden for Puerto Rico compared to Mexico include the island’s higher GDP per capita ( $27 , 678 ) compared to Mexico ( $9 , 747 ) , the island-wide distribution of dengue in Puerto Rico , and higher EFs for Puerto Rico ( EFH: 2 . 4; EFA:10 ) . Armien et al . [45] estimated a per capita economic burden of $6 . 49 , including surveillance and vector control costs , during the 2005 outbreak in Panama ( EFT:6 . 0 ) . If we assumed that the characteristics of dengue ( e . g . distribution , share of hospitalized cases ) during the 2009 dengue outbreak were similar to 2010–2011 , our per capita cost estimate of dengue in Mexico would have been $3 . 99 ( 95%CL: $3 . 14-$8 . 72 ) . Our estimates of the economic burden of dengue per capita in Mexico are within the range of comprehensive cost estimates for Nicaragua [49] ( $0 . 97-$2 . 49; up to $5 . 44 in an epidemic year ) . The study in Nicaragua estimated that disease burden ranged from 99–805 DALYs per million population , which is comparable to our estimates for Mexico . Other estimates of disease burden in the region include Puerto Rico [52] ( annual average 1984–1994: 658 DALYs per million population; range 145–1 , 519 ) , and Brazil [91] ( annual average 1986–2006: 22 DALYs per million population; range 14–30 ) . We found no notification of dengue episodes from the private sector in the cohort study in Morelos , a finding that has been confirmed by local public health officers [27] . The paucity of data from the private sector has been found elsewhere [97] , and is possibly among the most critical gaps in estimating the true number of symptomatic DENV infections . One interesting finding in this research relates to the population’s health-seeking behavior . The cohort study in Morelos showed that one third of the participants had not visited a private or public healthcare facility , despite having a symptomatic DENV infection . The questionnaire for dengue patients in 4 hospitals suggests that about 11% of patients had visited a pharmacy seeking treatment at the onset of their dengue episode . These results suggest that milder symptoms of dengue go underreported , which is consistent with previous findings [27 , 28] . Our refinement of reported dengue episodes using EFs include unreported cases from the private sector , as well as patients with symptomatic episodes who did not seek healthcare . Overall , we found an EF for all symptomatic episodes of 4 . 7 or a reporting ratio of 0 . 21 . To check the representativeness of our estimated reporting ratio , we compared it with findings from elsewhere in Mexico and neighboring countries . A study in two cities in the state of Tamaulipas [101] , near the Texas-Mexico border , suggests that the number of DENV infections represent about 20 times the number of notified cases between 1980 and 2007 . Considering that 39% of the DENV infections in the Morelos cohort were symptomatic , using these numbers from Tamaulipas , we would obtain an overall reporting ratio for symptomatic cases of 0 . 13 ( EFT = 7 . 8 ) . Although the quality of the health system for Morelos is not too different from that for Mexico overall [65] , a dengue awareness and education campaign in Morelos may have increased its reporting ratios compared to the rest of the country [33] . Findings from other countries in the Americas [44] found a reporting ratio of 0 . 08 ( EFT = 11 . 9 ) for total , 0 . 43 for hospitalized ( EFH = 2 . 3 ) , and 0 . 07 for ambulatory ( EFA = 15 ) cases . As the Mexican data are recent and reflect the EPS , which facilitated reporting and increased the quality of data [8 , 9] , the higher reporting ratios are also considered reasonable . Reporting ratios vary in time and by region [26]; hence , our estimate was based on the two years of the cohort study to provide a more stable estimate . Had we considered only post-enrollment infections , we would have obtained a reporting ratio of 0 . 13 ( EF = 8 . 0 , derived from Table 1 ) and a point estimate of the economic burden of dengue of $253 million or $2 . 32 per capita ( which is included within our 95% certainty level ) . We think our current estimate of $170 million ( $1 . 56 per capita ) is statistically more stable and accurate , although our expansion factor may be an underestimate in relation with other parts of the country with less dengue awareness , or lower overall quality of the health system . Our estimates suggest that at least 48 . 9% of the economic burden of dengue corresponds to surveillance and vector control . This share of total costs is higher than those of previous estimates of vector control in other countries . For example , the surveillance and vector control shares of estimated annual economic burden of dengue were 17% in Puerto Rico [47] , 30% in Panama [45] , and 28% in Thailand [54] . However , the per capita costs of surveillance and vector control ( in 2012 US dollars ) were lower in Mexico ( $0 . 76 ) than these other countries ( $2 . 14 in Puerto Rico , $1 . 79 in Panama , and $1 . 15 in Thailand ) . This pattern is partly explained by Mexico’s lower share of the national population at risk of dengue , as the disease is clustered mainly in Mexico’s coastal and tropical regions [5] . Also , Mexico did not experience an outbreak during our study years . Reflecting these patterns , the number of dengue episodes per 1 , 000 population was lower in Mexico ( 1 . 29 ) than in the other three countries—2 . 87 in Puerto Rico [47] , 9 . 76 in Panama [45] , and 4 . 08 in Thailand [54] . Several areas of uncertainty in our estimates of disease and economic burden of dengue in Mexico deserve attention . First , estimating the total episodes dengue is difficult due to paucity of data . For example , the cohort study in Morelos showed that 26% of the participants sought care in the private sector; but this estimate may be low as data from the Mexican National Health and Nutrition Survey 2012 showed that about 39% of all outpatient visits ( for any illness ) were in the private sector [102] . Second , the Morelos cohort is limited in geographical range , calendar years , and age groups , and therefore not necessarily representative of all regions with dengue transmission in Mexico . Local variations in the quality of the health system [34] and accessibility to health services [16] may result in differences in dengue patients’ health-seeking behavior , thus affecting reporting rates of apparent DENV infections . Third , our direct medical costs for dengue episodes were based on macro-costing in two tertiary hospitals in Tabasco , which may not necessarily be representative of hospitals in Mexico . We partially addressed this by adjusting our estimates based on WHO-CHOICE data , and varying our estimates in the sensitivity analysis . Costs in the private sector are probably higher than the costs we used , which possibly makes our economic burden estimates conservative . Fourth , we only considered surveillance and vector control costs from the federal level . Due to data limitations , we could not distinguish operating and capital expenditures , and did not include allocated and donated resources such as the time allocated by field personnel or volunteers to surveillance and vector control activities , as has been done elsewhere [103] . These limitations make our estimates of costs of surveillance and vector control conservative . Fifth , despite having improved previous estimates of economic burden by including costs of illness and dengue prevention and control strategies , we did not include other impacts of dengue illness due to data limitations . Last , our estimates of the burden of dengue were based on the acute and convalescent phases of a dengue episode ( Table 5 ) . Recent studies suggest that dengue patients may present long-term symptoms [104–109] like fatigue syndrome or depression , a possibility acknowledged by the WHO since 1997 [110]; unfortunately , there is not enough evidence or agreement on the characteristics ( e . g . , frequency , intensity , duration ) of these persistent symptoms , and whether or not they are caused by dengue alone .
Dengue costs the Mexican economy an annual average of US$170 ( 95%CL: 151–292 ) million , or $1 . 56 ( 95%CL: 1 . 38–2 . 68 ) per capita . Of this , $87 ( 95%CL: 87–209 ) million or $0 . 80 per capita ( 95%CL: 0 . 62–1 . 12 ) corresponds to illness and $83 million or $0 . 76 per capita to vector control and surveillance . These estimates do not include other costs , such as long-term sequelae of dengue , comorbidities , impacts on travel and tourism , or the disruption of health services during epidemics . Mexico’s annual disease burden from dengue is 65 . 1 DALYs per million population . Having objective and comparable estimates of the economic and disease burden of dengue is essential to inform health policy , increase disease awareness , and assess the impact of dengue control technologies [12 , 111] . More so , considering that several vaccine candidates [112] and other prevention and control technologies [37 , 40 , 42 , 113] are currently under development , and that Mexico might be an early adopter [12 , 53 , 114] . Results from the phase III clinical efficacy multicenter trial of a dengue vaccine candidate in the Americas suggest an overall vaccine efficacy of 60 . 8% , and a reduction in the risk of hospitalization of 80 . 3% [115] . These recent results make burden estimates even more urgent as Mexico confronts real choices . Effective dengue prevention and control strategies will probably require a combination of approaches and the involvement of various stakeholders [116] . With this study , Mexico joins Panama [45] , Puerto Rico [47] , Nicaragua [49] , and Thailand [54] as the only countries or areas worldwide with comprehensive ( illness and preventive ) empirical estimates of the cost of dengue . The results from this study reaffirm that exploring approaches to control dengue further would be economically valuable .
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During the past decades , dengue fever has become the most common arthropod-borne viral disease , imposing a substantial economic and disease burden in most tropical and subtropical countries , including Mexico . Dengue incidence and severity have dramatically increased in Mexico , with transmission regularly reported in 28 of 32 states . Objective estimates of the burden of dengue are important to inform policy decisions and priorities . We merged multiple data sources to estimate ( i ) total episodes , ( ii ) costs per episode , ( iii ) surveillance and vector control costs , and ( iv ) disease burden ( 2010–2011 ) . Results suggest that Mexico had about 139 , 000 symptomatic and 119 fatal dengue episodes per year on average . The annual cost , including surveillance and vector control , was about US$170 million , or $1 . 56 per capita , comparable to other countries in the Americas . Annual disease burden averaged 65 disability-adjusted life years per million population , with most of the years lost to disability corresponding to ambulatory episodes . The results show a substantial burden of dengue on the health care system and economy of Mexico . This quantification of the economic burden should help public health officials make informed decisions about current and promising new preventive and control measures to reduce dengue infections .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion",
"Conclusions"
] |
[] |
2015
|
Economic and Disease Burden of Dengue in Mexico
|
RIG-I-like receptors ( RLRs: RIG-I , MDA5 and LGP2 ) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA . Upon RNA binding , RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines . In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands . In vitro and transfection studies identified 5′-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells . The nature of RNA ligands of MDA5 is less clear . Several studies suggest that double-stranded RNAs are the preferred agonists for the protein . However , the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated . In this work , we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus . Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene . Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro , suggesting that they result in more stable MDA5 filaments . These data provide a possible model of how AU-rich sequences could activate type I interferon signaling .
The retinoic acid inducible gene I ( RIG-I ) -like receptor ( RLR ) proteins are key players in innate immunity and act by recognizing viral RNA ( vRNA ) in the cytosol . The RLR family consists of the members retinoic acid inducible gene I ( RIG-I ) , melanoma differentiation associated protein 5 ( MDA5 ) , and laboratory of genetics and physiology 2 ( LGP2 ) [1]–[3] . In vitro studies have shown that RIG-I and MDA5 recognize the majority of viruses in a complementary manner . While many negative-strand RNA viruses like rabies and influenza viruses are predominantly sensed by RIG-I , picornaviruses are predominantly recognized by MDA5 . The observed preferences are , however , unlikely to be exclusive and the exact role of LGP2 still needs to be investigated [4]–[9] . In case of MDA5 , a minor contribution to recognition of measles , rabies , vesicular stomatitis and Sendai virus has been reported [10]–[13] . The RLR proteins belong to the DExD/H-box ATPases sharing a central ATP-dependent helicase domain and a C-terminal regulatory domain ( RD ) that is responsible for initial RNA binding . In addition , RIG-I and MDA5 possess N-terminal tandem caspase activation and recruitment domains ( CARDs ) that are responsible for downstream signaling transduction [2] , [14] , [15] . Several crystal structures of RIG-I have shown that , in the absence of virus , the protein exists in an auto-inhibited state where the RD domain folds back to the CARDs , thereby shielding them from the cytosol . Upon viral infection and initial vRNA binding , the protein undergoes large conformational changes leading to the interaction with the mitochondrial associated signaling protein ( MAVS ) [16]–[19] . This leads to the activation of a downstream signaling cascade and finally to the induction of type I interferon ( IFN ) expression and the establishment of an anti-viral state . Although the exact nature of RLR ligands is not yet fully understood , several studies report that RIG-I preferentially binds to relatively short ( between 25 to 1000–2000 bp ) 5′-triphosphate double-stranded RNAs ( 5′-triphosphate dsRNA ) like those of Sendai virus ( SeV ) defective interfering ( DI ) particles [20]–[23] . In contrast , MDA5 seems to have a preference for long ( more than 1000–2000 bp ) dsRNA stretches [24] , [25] . Upon binding to dsRNA , MDA5 is thought to cooperatively form polar helical filaments leading to association with MAVS and activation of the downstream signaling cascade [26]–[28] . Viruses have developed numerous strategies to evade the immune system . For instance , viruses of the paramyxovirus family ( e . g . measles , parainfluenza , Sendai and Nipah viruses ) encode V inhibitor proteins that specifically bind to MDA5 and LGP2 , but not always to RIG-I [29]–[31] . By determining the structure of MDA5 in complex with parainfluenza virus V-protein , we previously showed that the viral protein unfolds the ATPase domain of MDA5 . This leads to the disruption of the MDA5 ATP-hydrolysis site and prevents RNA bound MDA5 filament formation [32] . One of the remaining key questions in this field is how RLR proteins are able to distinguish between self and non-self RNA in the cytosol . Recently , several studies showed that 5′-triphosphate RNA is not the only RNA ligand for RIG-I . Specific poly U/C-rich regions within certain viral genomes seem to contribute to efficient recognition by the protein [33] , [34] . In case of MDA5 , it is not known which features of vRNA are required in order to induce an immune response . Expression of subgenomic and subgenic RNA from parainfluenza virus 5 ( PIV5 ) indicated that MDA5 recognizes a specific region within the L mRNA [35] . For picornaviruses , it is speculated that MDA5 binds to long dsRNA that represents replicative intermediates composed of the positive genome and the negative antigenome [36] . These studies were , however , based on in vitro transfection experiments and it has so far not been possible to isolate a natural RNA ligand for MDA5 directly from virus-infected cells . In this study we combined different methods , including RNA-protein crosslinking and deep sequencing , to investigate in vivo RNA ligands for RLR proteins from virus-infected cells . Based on the crosslinking we were able to co-purify immunostimulatory RNA in a RIG-I and MDA5 dependent manner from measles virus ( MeV ) -infected cells . Deep sequencing and bioinformatics analysis revealed that RIG-I and MDA5 bind RNA of positive polarity originating from the L gene of the MeV genome . In addition , RIG-I binds to the 5′ ends of genomic and antigenomic RNAs , which probably represent 5′-triphosphate RNA , and are therefore not recognized by MDA5 . Furthermore , we showed that RIG-I , but not MDA5 , binds RNA of negative polarity , indicating that MDA5 does not efficiently recognize the MeV genome . Based on bioinformatics analysis , we observed a correlation between MDA5-enriched RNA sequences and the AU content and this was confirmed by in vitro transcription assays . In summary , we report the isolation of MDA5-associated RNA from virus-infected cells and the discovery of in vivo occurring activating viral RNA ligands for MDA5 .
Several in vitro studies showed that MDA5 preferably recognizes long dsRNA stretches [24] , [25] . However , it is still unclear if the protein has a preference for specific RNA sequences . The main reason for this may lie in the weak interaction between the protein and its ligand resulting in very poor RNA levels that co-purify from MDA5 immunoprecipitates . In order to address this problem , we established an RNA-protein crosslinking approach adapted from the PAR-CLIP ( Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation ) methodology [37] . With this approach , we intended to improve RNA recovery from RLR immunoprecipitates in the context of a viral infection . For validation of the method , we compared the crosslinking approach with a conventional pull-down technique previously used for the identification of SeV DI particles as potent RIG-I inducers [20] . We infected A549 human lung carcinoma cells with SeV at a high multiplicity of infection ( MOI ) in the presence of 4-thiouridine ( 4SU ) and allowed infection to occur over 24 h . A part of the cells was then exposed to 365 nm UV light and endogenous RIG-I was immunopurified ( Figure 1a ) . The recovered RNA was isolated and subjected to quantitative PCR ( qPCR ) analysis and immunoactivity experiments . The data indicate that treatment of cells with 4SU and exposure to 365 nm UV light lead to a reduction of immunostimulatory activity of RIG-I-associated RNA to 50% ( Figure 1b ) . However , the results of qPCR analysis showed that the crosslinking approach yields a quantitatively improved RNA recovery , with an increase of 50% in SeV DI particles in comparison to the non-crosslinking approach ( Figure 1c and d ) . Furthermore , we confirmed that treatment of cells with the photoreactive nucleoside does not affect cell viability or virus replication ( data not shown ) . Taken together , our data indicate that the crosslinking technique is a promising tool to study in vivo occurring RNA ligands for RLR proteins . Next , we validated the crosslinking approach on cells that were infected with a variety of viruses , including negative-stranded ( − ) RNA viruses ( MeV [38] and rabies [39] ) and positive-stranded ( + ) RNA viruses ( Encephalomyocarditis virus ( EMCV [40] ) and Mengo virus [41] ) . In all cases , we infected A549 cells at an MOI of 1 . 0 in the presence of 4SU . Cells were crosslinked 24 h post infection ( hpi ) and RIG-I and MDA5 were immunopurified . The recovered RNA was subjected to immunoactivity experiments . Based on the data , we concluded that immunoactive RNA was co-purified in a RIG-I- and MDA5-dependent manner from MeV-infected cells . This induction was significant in comparison to the negative control ( Figure 2 ) . In the case of RIG-I-associated RNA , we obtained an immunostimulatory effect that was 2600-fold higher in comparison to the control . For MDA5 , we observed an 800-fold induction . The data show that the approach yields RIG-I- and MDA5-specific immunoactive RNA from MeV-infected cells in a RIG-I- and MDA5-dependent manner . Although we detected significant immunostimulatory activity for RLR-associated RNAs from MeV-infected cells , the experimental set up is currently unsuitable for the isolation of RLR RNA ligands from the other viruses ( Figure S1 ) . The reason for this may lie in the heterogeneity and the need for precise timing of viral replication cycles or in the efficiency of 4SU incorporation and crosslinking . Utilization of this technique for other viruses may require adjustment of parameters , such as the time points of 4SU addition , crosslinking and harvesting after infection . Based on the above-mentioned results , we focused our studies on MeV , which belongs to the order of Paramyxoviridae . MeV has a single-stranded RNA genome of negative polarity consisting of 15 , 894 nucleotides . It comprises six non-overlapping genes , which are flanked by small terminal non-coding regions known as leader ( le ) and trailer ( tr ) sequences . These sequences serve as promoter regions during viral replication and transcription [42] , [43] . While the replication of the genome and antigenome is performed in a continuous process , viral transcription is carried out in a sequential manner , giving rise to an mRNA gradient declining in the 3′ to 5′ direction ( Figure S2 ) , as previously published [44] . Since ( − ) RNA virus polymerases eventually fail in transcription termination , they generate , in addition to monocistronic mRNAs , numerous alternative RNA species including read-through transcripts , such as leader-N , bi- or tricistronic mRNAs [45] . Furthermore , replication can give rise to abortive replication products and DI RNA with large internal deletions or copy-back genomes [46] . Due to the complex RNA composition of a virus-infected cell , the analysis of specific RNA ligands for RLR proteins is challenging . In order to shed light on the exact nature of RIG-I and MDA5-associated RNAs derived from MeV-infected cells , we performed a deep sequencing analysis on isolated RNA species from co-immunopurifications with antibodies against endogenous RIG-I and MDA5 . As a control , we used an antibody against GFP ( GFP protein was not present ) . The MeV strain used for the studies presented here was a recombinant measles virus rescued from cDNA with the exact sequence of the Schwarz vaccine strain ( Genbank AF266291 . 1 ) [38] . Obtained sequences were mapped to the MeV antigenome and the relative abundances of these sequences between RIG-I pull-down , MDA5 pull-down , and GFP pull-down were compared . Analysis of the reads showed that RIG-I and MDA5 bind to similar regions within the L gene-derived RNAs . In addition , RIG-I , but not MDA5 , binds to RNAs derived from the 3′ and the 5′ ends of the MeV genome ( Figure 3a and b ) . These regions probably represent le or trRNA generated in the course of replication or transcription . Additionally , internal genomic and antigenomic sequences found in the pull-downs could potentially originate from MeV DI particles [46]–[51] . To address this question , we performed a PCR analysis of RLR libraries in which we specifically amplified copyback DI RNA of MeV [47]–[49] . Indeed , we detected copyback DI particles not only in the RIG-I pull-down but also within RNA recovered from MDA5 immunoprecipitates ( Figure S3 ) . We did not find DIs in the GFP control pull-downs . Consistent with previous work , the higher copy numbers of reads indicate that RIG-I binds MeV RNA with higher affinity than MDA5 [11] . This observation is in good agreement with the increased immunostimulatory activity of isolated RNA from RIG-I pull-down samples in comparison to MDA5 . Regarding the immunostimulatory activity , RIG-I-associated RNA gives a 4-fold higher induction in comparison to MDA5-associated RNA ( Figure 3d and e ) . Based on the protocol used for cDNA library preparation , sequencing reads could be separated according to their strand orientation . During cDNA synthesis , adaptors were specifically ligated to the 3′ or 5′ ends , thereby keeping the information of strand specificity during the deep sequencing run . Separation of sequences revealed remarkable differences between both protein immunoprecipitations . RIG-I associated RNA sequences of positive polarity , which represent either antigenomic RNA or mRNA transcripts , are enriched in regions close to the 5′ end of the viral antigenome ( leader ) but also in distinct regions within the L gene . In contrast , sequences of negative polarity , representing the viral genome , are exclusively enriched in the 5′ end of the genome ( trailer region ) and in regions of the L gene ( Figure 4a ) . Analysis of MDA5-associated RNA revealed that sequences of positive polarity were enriched within the L gene originating from similar regions as ( + ) RNA from the RIG-I library ( Figure 4b ) . In contrast to RIG-I , however , MDA5 did not bind to RNA sequences comprising the 5′ end of the antigenome or leader RNA . Comparison of ( − ) RNA from RIG-I and MDA5 libraries further revealed that , in contrast to RIG-I , MDA5 did not enrich sequences of negative polarity , including trailer sequences . According to the analysis of strand specific enrichment , it appears that MDA5 does not bind vRNA of negative polarity that represents the MeV genome . Furthermore , the data evidently rule out the possibility that MDA5 recognizes RNA duplexes of ( + ) and ( − ) RNA that might represent replication intermediates , as previously suggested for a positive-strand RNA virus [36] . In fact , the result suggests that MDA5 binds ( + ) RNA that could either represent mRNA or the MeV antigenome . To further validate the specificity of the accumulation of RIG-I and MDA5-associated RNA , we calculated specific read enrichments [52] of the RLR libraries compared to the control library ( Figure S4 ) . Enrichment ( greater than 2× compared to the control library ) of RIG-I-associated RNA of positive polarity can be found across the whole genome , whereas only few reads of negative polarity are enriched within the N and L segment . In contrast , enriched sequences of MDA5-associated RNA are exclusively present within the L segment of positive polarity , whereas no specific enrichment was observed for ( − ) RNA . Based on the data , we observed a good correlation between the deep sequencing analysis and enrichment calculations , indicating that distinct regions within the MeV genome are indeed specifically enriched in a RIG-I- and MDA5-dependent manner in comparison to the control . To independently validate the relative amount of RLR-associated RNA , qPCR amplification was performed . The obtained copy read numbers were normalized to the GFP negative control in order to compare the genomic segments in the RIG-I and MDA5 samples ( Figure 5a ) . Analysis of relative abundances confirmed that RIG-I specifically enriches sequences from the 3′ and 5′ regions of the MeV genome , representing either antigenome or viral mRNA . Interestingly , the analysis showed that RIG-I-associated RNA from the genomic 3′ end most likely represents leader read-through transcripts or abortive replication products and not N mRNA . In MDA5 pull-downs , RNA was enriched in the case of the L mRNAs and partly in the case of H mRNAs , while no relevant copy numbers were obtained at other genomic positions . This is in good agreement with the results of the deep sequencing analysis , indicating that MDA5 indeed recognizes RNA originating from the L gene of the MeV genome . Furthermore , comparison of the relative copy numbers between RIG-I and MDA5 revealed remarkable differences between both proteins . The relative abundances in the RIG-I sample were up to 40-fold higher in comparison to MDA5 . This observation again indicates that RIG-I has a higher affinity for MeV RNA sequences in comparison to MDA5 . Our conclusion is further supported by immunoactivity experiments , where the relative immunostimulatory activity of RIG-I-associated RNA was 20-fold higher in comparison to MDA5 ( Figure 5b and c ) . To elucidate the exact nature of sequences enriched by RIG-I and MDA5 immunoprecipitations , we conducted a bioinformatics analysis . For this , the complete genome was divided into fragments of size 201 nt with a shifting window of 5 nt . Each sequence was folded in silico ( RNAfold [53] ) and several RNA primary and secondary structure features were analyzed . The analyzed parameters were set in relation to the mean coverage of sequencing reads from RIG-I and MDA5 pull-down experiments . Heat scatter plots indicate that sequences rich in AU correlate with a high mean coverage of sequencing reads in both the RIG-I ( cor = 0 . 273 , cor = 0 . 334 ) and MDA5 ( cor = 0 . 358 , cor = 0 . 348 ) libraries ( Figure 6a and b ) . These data suggest that RIG-I and MDA5 preferably bind to AU-rich sequences originating from the viral genome . Although we further analyzed a variety of secondary structure parameters , including paired nucleotides and bulges , we did not see any other relevant correlation with the mean coverage of sequencing reads ( Figure S5 and Figure S6 ) . To further confirm the obtained sequencing data , we generated 17 single-stranded , 200 nucleotide long in vitro transcripts ( IVTs ) covering different regions of the MeV antigenome ( Table S1 ) . RNAs were double-dephosphorylated in order to ensure that 5′-triphosphate groups were removed . For immunoactivity experiments IVTs were transfected into 293T ISRE-FF reporter cells . The stimulatory effect revealed a correlation of high read numbers from deep sequencing analysis and high stimulatory activity of the IVT sequences ( Figure 7 ) . According to the immunostimulatory experiment , we observed increased immunostimulatory activities for transcripts 8 , 9 , and 12 ( Figure 7a ) . These transcripts correspond to regions at the 5′ end of the L gene , which is also the region with the highest copy numbers of reads ( Figure 3 ) . In general , IVTs representing regions within the L gene have higher immunostimulatory activity in comparison to the upstream genomic segments . This is in good agreement to the deep sequencing analysis . Furthermore , calculated Pearson correlations showed that the best correlation between maximal numbers of sequencing reads and the immunostimulatory activity of RNA transcripts can be found in the MDA5 sequencing data ( cor = 0 . 526 ) , while RIG-I and GFP samples showed less correlation ( cor = 0 . 369 and cor = 0 . 217 ) ( Figure 7b ) . In order to find a possible explanation for the different immunostimulatory potentials of IVTs , several characteristics of the transcripts were analyzed in silico . The obtained data revealed that the immunostimulatory potential correlates with the AU content of IVTs ( cor = 0 . 599 ) ( Figure 7d ) , which is consistent with the results from the deep sequencing analysis . Visualization of transcripts on an Agilent bioanalyzer RNA chip indicates that no higher-order structures due to the sequence composition were formed that might explain differences in immunostimulatory activity ( data not shown ) . In order to get a more general conclusion about the contribution of the AU content to the immunostimulatory potential of RNAs , in vitro transcripts from Mengo virus ( Table S3 ) were tested for their immunostimulatory activity . The transcripts were generated according to the protocol for MeV RNA sequences . We again observed a correlation ( cor = 0 . 583 ) of the AU content of the tested sequences and their immunostimulatory potential ( Figure S 7a and b ) . These data are consistent with the in vitro analysis of MeV RNA sequences indicating that the AU composition of RNA might play a general role in activating RLR signaling . Finally , we asked whether the ATP hydrolysis activity of MDA5 correlates with the immunostimulatory potential of the tested IVTs . We measured the ATP hydrolysis rate of recombinant mouse MDA5 in the presence of RNA transcripts ( Figure 7 and Figure S8 ) and observed a negative correlation between the maximum number of sequencing reads in the MDA5 library and the ATP hydrolysis rate ( cor = −0 . 414 , Figure 7c ) . Analysis of the in vitro data revealed that AU-rich sequences lead to a decrease in ATP hydrolysis activity of MDA5 ( cor = −0 . 445 ) . Furthermore , the ATP hydrolysis rate negatively correlates with the immunostimulatory potential of RNA transcripts ( cor = −0 . 426 ) ( Figure 7d ) . This result suggests that the ATPase hydrolysis activity of MDA5 is not correlated to the binding and the immunostimulatory potential of the RNA transcripts and could therefore provide a model of RNA recognition by the protein . The data are consistent with previous work on MDA5 filament formation upon dsRNA binding [26] , [27] . In structural and biophysical studies , Berke et al showed that ATP hydrolysis by MDA5 causes filaments to disassemble , perhaps by inducing translocation along the RNA or triggering a conformational change in the protein . According to our data , this may explain the observed inverse correlation between the immunostimulatory activity of IVTs and their potential to induce the ATPase activity of MDA5 .
Until now , in vivo RLR ligands were poorly understood and a naturally occurring MDA5 ligand could only be purified indirectly by immunoprecipitation of LGP2:RNA complexes from virus-infected cells overexpressing LGP2 [54] . By applying a combination of RNA-protein crosslinking , immunoprecipitation of endogenous proteins and RNA deep sequencing analysis , we were able to investigate RLR-associated RNA from MeV infected cells . We compared our results to the empty GFP antibody control resembling a previously published immunoprecipitation strategy [20] . Our approach reveals that MDA5 preferentially binds measles virus RNA of positive polarity , whereas RIG-I additionally binds to ( − ) sense RNA within the trailer region as well as in the adjacent L gene . We propose that enriched RNA of positive polarity most likely represents mRNA species , since antigenomic RNA is only generated during replication and is immediately packed into nucleocapsids [55]–[57] . For Mononegavirales , these RNA-protein complexes are considered inaccessible for cytoplasmic proteins [55] , [58] and might not be ligands for RLR proteins unless they become released . We show that , unlike MDA5 , RIG-I binds ( + ) sense RNA originating from not only the L genomic segment , but also from the 3′ end of the MeV genome , which could be either le-N read-through transcripts or abortive replication products comprising 5′-triphosphate ends [45] , [46] . Furthermore , we hypothesize that RIG-I specific enriched RNA of negative polarity represents abortive replication products also having 5′-triphosphate ends [20]–[23] . Additionally , 5′-copyback DI sequences combining vRNA of positive and negative polarity were found both in RIG-I and MDA5 immunoprecipitates and may contribute to recognition [49] . Bioinformatics analysis and in vitro transcription experiments revealed a correlation between AU content and read coverage of the obtained sequences or IVTs , respectively . As shown before [59] , this indicates that RNA rich in AU could serve as a putative ligand for RIG-I and MDA5 , or in a secondary manner lead to a specific structure that is recognized by both proteins . The slightly weaker correlation of RIG-I associated sequences with their AU content compared to MDA5 bound RNAs could be explained by additional sequences or triphosphate RNAs recognized by RIG-I that originate from regions less rich in AU . Interestingly , ATP hydrolysis assays performed with recombinant MDA5 and RNA transcripts indicate that the AU content of RNA negatively correlates with the ATP hydrolysis rate of the protein . This inverse correlation between the immunostimulatory potential of RNAs and their capability to stimulate ATP hydrolysis by MDA5 lets us speculate that the ATPase activity might not be necessary for , or even interfere with , the immunoactivity of RNA ligands . Although this observation disagrees with recent findings about the role of ATP hydrolysis in RIG-I oligomerization on 5′-triphosphate dsRNA [60] , we assume that MDA5 and RIG-I differ markedly in their mechanical activation and the role of ATP hydrolysis . Our data is supported by results suggesting that MDA5 filament formation is abrogated in an ATP-sensitive manner . By electron microscopy ( EM ) analysis it was shown that MDA5 filaments disassemble in the presence of ATP , indicating that ATP hydrolysis triggers the translocation of the protein along the dsRNA molecule or reduces the binding affinity , thereby interfering with downstream signaling [26] , [27] . In light of the available data in the literature we therefore hypothesize that the ATPase activity of the MDA5 helicase domain contributes to substrate specificity by detaching the protein from low affinity substrates . To further test this hypothesis we generated RIG-IE373Q and MDA5E444Q , which are mutated in the “Walker B” ATP hydrolysis motif [61] , slowing down or abrogating the ATP hydrolysis activity of the proteins , while preserving formation of ATP complexes . Overexpression of these mutant proteins from transfected plasmids showed a dramatic increase in their immunostimulatory potential in the absence of any viral ligands in comparison to expressed wild-type MDA5 ( Figure S9 ) . Furthermore , pull-down studies with the RIG-I Walker B mutant revealed an increase in the amount of recovered RNA while their immunostimulatory potential decreased ( data not shown ) . The increased immunostimulation of ATPase deficient RLRs is consistent with the model that RNAs that lead to a reduced ATP-hydrolysis rate are more proficient in immunostimulation , possibly by stabilizing RLR∶RNA complexes . The negative correlation between AU-rich sequences and the ATP hydrolysis rate suggests that MDA5 binds AU-rich RNA in preference to GC-rich RNA . This would lead to a stronger interaction between RNA and MDA5 and result in a higher immunostimulatory signal . In order to test this hypothesis , we performed binding assays with MDA5 and IVTs but we were not able to demonstrate differences in the binding affinities between the different transcripts that might support this theory ( data not shown ) . Finally , we speculate that RNA ligands for RLR proteins could be divided into two classes . The first class would comprise RNA molecules originating from the 5′-triphosphate ends of the genome or antigenome . These molecules could be generated in the course of read-through transcription and abortive replication [45] , [46] and could therefore represent preferred ligands of RIG-I , as shown previously [20] . The second class of RNA molecules could be recognized by both receptor proteins . Our data suggest that recognition of these RNAs might occur through the AU composition of sequences [34] . This second class might also prominently include defective interfering ( DI ) particles generated during MeV replication . For MDA5 , however , our deep sequencing data show that the ( − ) strand portion of the DIs is either relatively short or the fraction of DIs binding to MDA5 is magnitudes lower than the binding to L derived ( + ) sense RNAs and therefore not easily detectable during sequencing . A more detailed analysis of the deep sequencing data is currently ongoing in order to shed more light on the complex nature of the DIs involved . It will be interesting to see what types of RNA associate with RIG-I and MDA5 during infections with different viruses and to what extent the AU composition and DI generation contributes to RNA recognition in these types of viruses . In particular , the finding that both RIG-I and MDA5 localize to AU rich regions suggests partially overlapping roles in detection of different viruses . The specificity of RIG-I and MDA5 for certain viruses may lie not only in the detection of 5′-triphosphate by RIG-I , but also in the heterogeneity of viral evasion strategies [62] . Our findings support a model for the recognition of AU-rich sequences by RIG-I and MDA5 from MeV-infected cells . Consistently , we find a similar correlation for in vitro transcribed RNA from the Mengo virus genome . In general , the data support previous experiments indicating that MeV is mainly recognized by RIG-I , while MDA5 seems to play a minor role [4] , [5] , [13] , [63] . It could be possible that RIG-I initially recognizes le-N read-through transcripts or abortive replication products containing 5′-triphosphate ends , leading to the activation of the signaling cascade . In a second round of recognition , RIG-I and MDA5 then recognize viral transcripts that are rich in AU . To further test this hypothesis , time dependent experiments need to be carried out . One feature of the applied crosslinking technique is the introduction of specific T to C transitions at the interaction sites of 4SU-labeled RNA and the protein upon UV light exposure [37] . By identifying these point mutations in the deep sequencing data , one can exactly pinpoint the RNA sequences that interact with the protein of interest . However , our bioinformatics analysis did not reveal significant enrichment of T to C mutations , which could be explained by the rather low incorporation efficiency of the photoreactive nucleoside into viral RNA , consistent with the low incorporation level of 4SU into host RNA . Nevertheless , by increasing the incorporation efficiency in future studies , the identification of point mutants could further narrow down the precise binding sites of RLRs . In summary , our approach provides a first insight into the molecular basis of vRNA derived from MeV interaction with MDA5 in living cells and reveals a preference for binding of AU-rich regions originated from ( + ) -sense RNA of the L gene . In vitro , these RNA molecules appear to be a poorer stimulator of the ATPase activity of MDA5 , and result in more stable MDA5 filaments and support better downstream signaling .
Infection experiments were carried out in A459 human lung carcinoma cells . HEK 293T ISRE-FF reporter cells ( stable expression of firefly luciferase under the control of an interferon stimulated response element ) were used for interferon stimulation luciferase reporter gene assays . All cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 2 mM L-glutamine , 1% Penicillin-Streptomycin and 10% FBS ( all purchased from Invitrogen ) . Viruses used for infections were recombinant measles virus with a sequence identical to the vaccine strain Schwarz ( AF266291 . 1 . ) , Sendai virus , Sendai virus defective interfering particles H4 ( kindly provided by Dominique Garcin , Geneva , Switzerland ) , Mengo virus strain pMC0 ( kindly provided by Anne Krug , TU Munich , Germany ) and EMCV . Primary antibodies to human MDA5 ( AT113 ) and RIG-I ( Alme-1 ) were purchased from Enzo Life Science ( Loerrach , Germany ) . Antibody to GFP ( ab1218 ) was obtained from Abcam ( Cambridge , UK ) . Secondary antibodies were supplied by GE Healthcare ( Buckinghamshire , UK ) . A549 cells were infected with virus with an MOI of 1 . 0 in the presence of 400 µM 4SU . Infection was allowed to proceed for 24 h and living cells were washed with PBS ( 10 mM phosphate , 137 mM NaCl , 2 . 7 mM KCl , pH 7 . 5 ) and exposed to 1 J/cm2 365 nm UV light using a photocrosslinker ( Vilbert Lourmat ) . Cells were harvested and incubated in Nonidet P-40 lysis buffer ( 50 mM HEPES , 150 mM KCl , 1 mM NaF , 10 µM ZnCl2 , 0 . 5% NP-40 , 0 . 5 mM DTT , protease inhibitor , pH 7 . 5 ) for 10 min on ice . The lysate was cleared by centrifugation and endogenous proteins were immunoprecipitated for 4 h with the respective antibodies ( 1 µg/mL ) bound to protein G Dynabeads ( Life Technologies ) . The beads were washed five times with high-salt wash buffer ( 50 mM HEPES , 500 mM KCl , 0 . 05% NP-40 , 0 . 5 mM DTT , protease inhibitor , pH 7 . 5 ) and incubated with proteinase K ( Thermo Scientific ) for 30 min at 55°C . The RNA was isolated by phenol/chloroform/isoamylalcohol extraction and subjected to further analysis . A549 cells were infected with MeV with an MOI of 1 . Cells were harvested 24 hpi . Total RNA was isolated according to manufacturer's protocol of the RNeasy Protect Mini Kit ( Qiagen ) and subjected to Illumina deep sequencing . Immunoactivity experiments were carried out in 24-well plates . 2 . 5×105 HEK 293T ISRE-FF reporter cells were transfected with 250 ng of recovered RNA , 500 ng in vitro transcripts or 500 ng plasmid DNA using Lipofectamine 2000 ( Invitrogen ) according to manufacturer's protocol . After 24 h incubation , cells were subjected to immunoactivity experiments using the Dual-Glo luciferase assay system ( Promega ) according to manufacturer's instructions . The luciferase activity was determined in a 96-well plate reader . Significance of differences in luciferase activity between samples were determined via an unpaired t-test . Isolated RNA was prepared for Illumina sequencing using the mRNA-Seq library preparation kit ( Epicentre ) according to manufacturer's protocol . To remove ribosomal RNA species from the sequencing libraries a Ribo-Zero rRNA removal kit ( Epicentre ) was used . Quality of RNA-Seq libraries was validated on a DNA1500 chip for the Bioanalyzer 2100 ( Agilent ) . Sequencing was performed on the Illumina Genome Analyzer in the Gene Center sequencing facility ( LAFUGA ) . Obtained sequences were processed with the FASTX toolkit ( http://hannonlab . cshl . edu/fastx_toolkit/ ) in order to remove adapter sequences and reads with PHRED scores below 30 . Remaining sequences were mapped to human and viral genomes by utilization of the Bowtie algorithm [64] , allowing maximal one mismatch per unique read . The Bowtie sequence alignments were converted with SAMtools [65] to pileup format , which was subsequently used for further data analysis . Relative sequence abundances were analyzed between RLR pull-down samples and the GFP control . Specific read enrichments were calculated by determining the relative sequence abundance at each position on the genomic segment and calculating the average of the RLR/GFP ratios over a dynamic window of 200 reads . Relative sequence abundances with log2 ratios above +1 were defined as significantly enriched in the RLR library . RNA secondary structure prediction from measles virus genome or in vitro transcripts was performed by utilization of RNAfold from the ViennaRNA package [53] using standard parameter settings . For this purpose , the genome was divided into 201 nt fragments with a shifting window size of 5 nt . The sequences were folded in silico and the linear relationship between different data sets was quantified with the Pearson correlation coefficient . DNase treatment of the immunoprecipitated RNAs and qPCR was performed as previously described [66] . The primer pairs used for quantification were identical to those published [67] . For cDNA synthesis a random hexanucleotide mix was used ( Roche ) . Full length MeV vac2 cDNA with a known concentration was used for standard generation . Copy number values obtained for MDA5 and RIG-I were normalized to the control GFP . Specific primers for reverse transcription ( Roche transcriptor transcriptase ) and the subsequent PCR ( Biozym Phusion Polymerase ) were adapted from Calain et al [47] . PCR products were analyzed on agarose gels and stained with ethidium bromide . Templates were generated for in vitro transcription in a PCR adding the T7 promoter sequence ( TAATACGACTCACTATA GGG ) to the 5′ end of the desired MeV or Mengo virus genomic fragment , respectively ( for oligonucleotides see Tables S2 und S4 respectively ) . PCR products were subsequently purified on agarose gels . RNA was transcribed using the Ambion Megashortscript T7 Kit according to the manufacturer's protocols . The reaction was incubated overnight at 37°C and RNA was precipitated using LiCl at −20°C for 30 minutes . Afterwards , RNA was subjected to triphosphate digestion using FastAP ( Fermentas ) according to the manufacturer's instructions and purified on denaturing 8 M urea/10% polyacrylamide gels at 25 mA constant current . Gel slices containing RNA were incubated overnight with 450 µL probe elution buffer ( 0 . 5 M ammonium acetate , 1 mM EDTA , 0 . 2% SDS ) . Eluted RNA was isolated by phenol/chloroform/isoamylalcohol extraction and precipitated with ethanol . ATPase hydrolysis activity was determined using [γ-P32] ATP . Mouse MDA5 was purified as described previously [32] and 1 . 6 µM of protein was preincubated with 80 nM in vitro transcribed RNA for 10 min at room temperature . The reaction was initiated by addition of ATPase hydrolysis buffer ( 20 mM HEPES , pH 7 . 5 , 150 mM NaCl , 1 . 5 mM MgCl2 , and 2 mM DTT ) containing 2 mM ATP and 0 . 2 µCi [γ-P32] ATP . The hydrolysis rate was monitored over 1 h and analyzed by thin layer chromatography ( TLC ) . Sequences encoding full-length human RIG-I with N-terminal FLAG-tag and full-length human MDA5 with N-terminal FLAG-tag were cloned into pcDNA5 FRT/TO ( Invitrogen ) . Mutants ( FLAG-RIG-I E373Q and FLAG-MDA5 E444Q ) were generated by site directed mutagenesis with PfuUltra ( Agilent ) .
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RIG-I-like receptors ( RLRs ) are helicase-like molecules that detect cytosolic RNAs that are absent in the non-infected host . Upon binding to specific RNA patterns , RLRs elicit a signaling cascade that leads to host defense via the production of antiviral molecules . To understand how RLRs sense RNA , it is important to characterize the nature and origin of RLR-associated RNA from virus-infected cells . While it is well established that RIG-I binds 5′-triphosphate containing double-stranded RNA , the in vivo occurring ligand for MDA5 is poorly characterized . A major challenge in examining MDA5 agonists is the apparently transient interaction between the protein and its ligand . To improve the stability of interaction , we have used an approach to crosslink MDA5 to RNA in measles virus-infected cells . The virus-infected cells were treated with the photoactivatable nucleoside analog 4-thiouridine , which is incorporated in newly synthesized RNA . Upon 365 nm UV light exposure of living cells , a covalent linkage between the labeled RNA and the receptor protein is induced , resulting in a higher RNA recovery from RLR immunoprecipitates . Based on next generation sequencing , bioinformatics and in vitro approaches , we observed a correlation between the AU-composition of viral RNA and its ability to induce an MDA5-dependent immune response .
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2014
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In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells
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Varicella zoster virus ( VZV ) causes chickenpox in humans and , subsequently , establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster . Infection of rhesus macaques with simian varicella virus ( SVV ) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection . While the type I interferon ( IFN ) response has been shown to affect VZV replication , the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes ( ISG ) . Here , we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway . SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation . Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment . Interestingly , SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner , suggesting that SVV employs multiple mechanisms to counteract the effect of IFN . Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase ( DHFR ) -degradation domain additionally confirmed its requirement for viral replication . Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells . In addition , cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein . Taken together , our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation . SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses .
The alphaherpesvirus varicella zoster virus ( VZV ) is the causative agent of chickenpox . After primary infection , VZV establishes latency in sensory ganglia . Reactivation from latency , which typically occurs in elderly individuals , can cause shingles or herpes zoster that is associated with a number of debilitating complications , including postherpetic neuralgia [1] . In vivo research on VZV is limited because the virus does not produce varicella or zoster in animals [2 , 3] . Simian varicella virus ( SVV ) is closely related to VZV sharing about 75% DNA homology and exhibiting a highly similar genome organization [4] . Inoculation of nonhuman primates , including African green monkeys and Cynomolgus macaques , results in a persistent viremia [4] . In contrast , infection of rhesus macaques ( RM ) with SVV results in a primary infection followed by latency that is similar to VZV infection in humans . SVV-induced skin lesions are resolved by 21 days post infection which correlates with the absence of virus DNA in blood . Latent SVV can be detected in ganglia of infected RM [5] . Infection of RM with SVV thus represents a robust animal model that recapitulates most hallmarks of a primary human VZV infection . The innate host immune response to viral infection is dominated by interferons ( IFNs ) that are subdivided in three families , namely types I , II and III . In particular , several subtypes of IFNα and IFNβ that represent type I IFNs are key players in the anti-viral innate immune response [6] . Transcription of IFN is initiated by pattern recognition receptors ( PRRs ) engaging pathogen associated molecular patterns ( PAMPs ) such as double-stranded RNA , lipopolysaccharide and cytosolic DNA . Downstream signaling pathways lead to the activation of transcription factors such as IFN regulatory factor ( IRF ) 3 and nuclear factor κB ( NFκB ) that induce the transcription of IFNβ . Secreted IFNβ can signal in an autocrine and paracrine fashion by interacting with the type I IFN receptor complex ( consisting of IFNAR1 and IFNAR2 ) both on infected and neighboring uninfected cells [7] . Receptor binding activates the JAK-STAT signaling pathway , which results in the expression of hundreds of IFN-stimulated genes ( ISG ) and corresponding proteins that inhibit virus growth by counteracting multiple molecular steps of the replication cycle and by signaling to innate immune cells including natural killer cells [8 , 9] . In addition , type I IFNs have been shown to be involved in dendritic cell maturation and antigen presentation thereby stimulating the development of virus-specific adaptive immune responses [10 , 11] . The ability of VZV and SVV to spread and establish latency in the presence of these immediate immune responses implies that both viruses display evasion strategies that circumvent or counteract the induction or function of IFNs and ISGs . VZV infection of human skin xenografts in severe combined immunodeficiency ( SCIDhu ) mice showed that VZV-infected cells do not express type I IFN , while uninfected bystander cells stained positive for the cytokine [12] . Several reports have shown that the effect of IFN is counteracted by at least four different VZV-encoded proteins: both IE62 and ORF47 alter the phosphorylation of IRF3 and prevent gene activation [13 , 14] , whereas ORF61 inhibits pathogen-induced cytokine expression by degrading IRF3 [15] and by blocking the activation of NFκB via inhibiting IκBα [16] . In addition , Cohen et al . showed the deletion of ORF63 severely attenuates the growth of the virus in the presence of IFNα but not IFNγ , suggesting a possible involvement of ORF63 in regulating JAK-STAT signaling [17] . Expression of IFNα and IFNβ and subsequent binding to their receptor triggers the dimerization of IFNAR1 and IFNAR2 , which activates the Janus kinases JAK1 and TYK2 that are constitutively associated with the receptor . The kinases phosphorylate the receptor creating a docking site for the transcription factors STAT1 and STAT2 . Subsequent phosphorylation of the STAT proteins by the JAKs leads to conformational changes within the molecules that allow the formation of stable STAT1 , STAT2 and IFN regulatory factor 9 ( IRF9 ) complex termed ISGF3 . ISGF3 then shuttles to the nucleus where it activates the transcription of type I IFNs and ISGs [18] . VZV-infected cells in skin xenografts in the SCIDhu mice did not show nuclear localization or phosphorylation of STAT1 , in contrast to uninfected bystander cells [12] . Since IFNα produced by bystander cells can induce JAK-STAT signaling in VZV-infected cells , the absence of pSTAT1 in infected cells suggests that VZV interferes not only with IRF3-mediated activation of IFN transcription but also with JAK/STAT signaling . The VZV ORF63 protein is a 30 kDa immediate early protein that is phosphorylated by host and viral kinases [19 , 20] . The protein is abundantly present during lytic infection and its expression has also been observed in latently infected ganglia [21 , 22] . A duplicate of the ORF63 gene , designated ORF70 , is found in the terminal repeat region [19] . VZV ORF63/70 regulates viral gene expression and impairs the expression of certain cellular genes [23–26] . In addition , VZV ORF63/70 inhibits apoptosis in cultured primary human neurons [27] . SVV encodes both ORF63/70 orthologous proteins that share 52% amino acid identity with their VZV homologs [28] . These gene products are required for replication of SVV in cell culture [29] . In vivo studies using SVV-infected RM and African green monkeys confirmed expression of the ORF63 protein in ganglia during latent infection [5] . In this report , we show that both SVV and VZV interfere with type I IFN signaling . SVV inhibits type I IFN-induced ISG expression by downregulating the expression of IRF9 and prevents phosphorylation of STAT2 upon IFN stimulation . We also observed a minor decrease in STAT2 levels in SVV-infected cells . SVV ORF63 was found to be responsible for the reduction in IRF9 expression , but was not directly involved in downregulation of STAT2 expression or inhibition of STAT2 phosphorylation . These data suggest that multiple SVV proteins counteract type I IFN signaling . Similarly , we observed reduced levels of STAT2 and IRF9 proteins in VZV-infected cells and the cells were refractory to IFN-induced STAT2 phosphorylation . Ectopic expression of VZV ORF63 affected steady state levels of IRF9 , but did not block STAT2 expression or phosphorylation . Thus , the multi-level inhibition of JAK-STAT signaling seen in SVV-infected cells is conserved in VZV-infected cells .
Telomerized rhesus fibroblasts ( TRFs ) , TRF-ISRE cells [30] , the African green monkey kidney epithelial cell line Vero , Vero-CRE ( kindly provided by Dr . Linda van Dyk , University of Colorado , Denver ) [31] , telomerized human fibroblasts ( THF ) -ISRE [32] , human embryonic kidney ( HEK ) 293T cells ( ATCC ) , and the human fibroblast cell line MRC-5 ( ATCC ) were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum ( FBS ) , 140 IU of penicillin/ml and 140 μg of streptomycin/ml . TRFs and Vero cells were infected with a recombinant SVV Delta strain , in which eGFP was inserted between US2 and US3 through homologous recombination [33] . A confluent monolayer of TRF or Vero cells was infected by cocultivation of SVV . eGFP-infected cells with uninfected cells at the indicated ratios . Complete infection was confirmed by visualizing eGFP using fluorescence microscopy . Infected cells were maintained in DMEM supplemented with 2% FBS and harvested at 48 hours p . i . For VZV infections , we used the recombinant VZV Oka strain , in which eGFP was fused to the N-terminus of ORF66 ( generously provided by P . R . Kinchington , University of Pittsburgh , Pennsylvania ) [34] . VZV . eGFP-infected MRC-5 cells were cocultivated with uninfected MRC-5 cells at a 1:5 ratio . Rhesus IFNα2 was obtained from R&D systems . Human IFNα and universal type I IFN ( uIFN ) were obtained from PBL Assay Science . MG132 ( Fisher Scientific ) was dissolved in DMSO and used at the indicated concentrations for 16 hours . Control wells were treated with same concentration of DMSO without MG132 . Trimethoprim ( TMP; Sigma-Aldrich ) was dissolved in DMSO and used 10 μM or less where indicated . Viral cultures were supplemented with fresh TMP every 24 hours . The following antibodies were used for detection of endogenous and viral proteins in western blot: anti-ISG15 F-9 ( Santa Cruz ) , anti-ISG54/IFIT2 ( Abcam ) , anti-Mx-1 ( GeneTex ) , anti-STAT1 M22 ( Santa Cruz ) , anti-phosphorylated STAT1 Tyr701 ( Santa Cruz ) , anti-STAT2 C20 ( Santa Cruz ) , anti-phosphorylated STAT2 Tyr690 ( Cell Signaling Technology ) , anti-IRF9/ISGF3γ clone 6 ( BD Biosciences ) , anti-GAPDH 6C5 ( Santa Cruz ) , anti-p84 5E10 ( GeneTex ) , anti-IRF1 H-205 ( Santa Cruz ) , anti-IRF3 ( Santa Cruz ) and anti-FLAG M2 ( Sigma-Aldrich ) . The monoclonal antibodies specific for SVV and VZV ORF63 ( clone 63_6 ) , ORF62 ( clone 62_6 ) and ORF31 ( clone 31C_8 ) have been previously described [35] . STAT2 C20 ( Santa Cruz ) was also used for immunofluorescence microscopy . TRFs were transduced with a replication-defective lentivirus encoding firefly luciferase downstream of an IFN-stimulated response element ( ISRE ) and a lentivirus constitutively expressing renilla luciferase driven by a CMV promotor ( Qiagen ) . Transduced TRFs ( TRF-ISRE cells ) were selected by culturing in the presence of 4 μg/ml puromycin . TRF-ISRE cells were infected with SVV . eGFP as described above and 24 hours p . i . the cells were seeded in a black 96 well plate ( Corning Incorporated ) . At 42 hours p . i . , the cells were stimulated with rhesus or human IFNα to induce expression of ISRE-driven firefly luciferase . After 6 hours , expression of firefly and renilla luciferase was measured using the Dual-Glo luciferase assay system ( Promega ) . Luminescence was measured on a Veritas microplate luminometer ( Promega ) . Data are presented as the ratio between firefly luciferase expression and renilla luciferase expression . For the experiment described in Fig 1B the cells were sorted for high GFP expression at 40 hours p . i . using a FACS Aria II cytometer . We seeded 2000 of the sorted and mock-infected cells cells in black 96 well plate and stimulated with uIFN for 6 hours , after which luciferase expression was measured using the ONE-Glo luciferase assay system ( Promega ) . HEK 293T cells were co-transfected with an ISRE-firefly luciferase reporter plasmid ( pGL3-ISRE-Luc ) and the pcDNA3 . 1 expression vectors ( described below ) . At 24 and 42 hours post transfection , the cells were treated with 5000 U/ml uIFN for 6 hours . Cells were transferred to a black 96 wells plate before or right after treatment with uIFN treatment . Expression of firefly luciferase was measured using the ONE-Glo luciferase assay system ( Promega ) . To generate nuclear and cytoplasmic fractions , cells were resuspended in dounce buffer ( 100 mM KCl , 20 mM Hepes [pH 7 . 4] , 0 . 1 mM EDTA , 3% sucrose ) supplemented with HALT protease and phosphatase inhibitors ( Thermo Scientific ) . After 15 minutes 10% Nonidet P-40 was added to the lysate in a 1:20 ratio and lysates were vortexed for 10 seconds . The cytoplasmic fraction was removed immediately and nuclei were washed twice with D-PBS and lysed in RIPA buffer ( 10 mM Tris-HCl [pH 7 . 4] , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 1% Sodium Deoxycholate , 0 . 1% SDS ) supplemented with protease and phosphatase inhibitors . For all other experiments , cells were lysed directly in Laemmli sample buffer ( 100 mM Tris-HCL [pH 8 . 0] , 4% SDS , 20% glycerol , 10% 2-mercaptoethanol , Bromophenol blue ) . Proteins were separated by SDS-page and transferred to polyvinylidene difluoride membranes ( Thermo Scientific ) . Membranes were first incubated with the indicated antibodies , which was followed by incubation with horseradish peroxidase ( HRP ) -conjugated secondary antibodies specific for mouse ( Santa Cruz ) or rabbit ( Thermo Scientific ) IgG . Binding of secondary antibodies to the membranes was visualized by using Pierce ECL 2 ( Thermo Scientific ) . Mock- or SVV-infected TRFs were grown on cover slips , washed twice with PBS and fixed with 3 . 7% formaldehyde ( Fisher Scientific ) at room temperature ( RT ) for 40 minutes . After washing with PBS , residual formaldehyde was quenched with 50 mM Ammonium Chloride for 10 minutes and the cells were permeabilized with 0 . 1% Triton for 5 to 7 minutes . Non-specific protein binding sites were blocked with 2% bovine serum albumin ( BSA ) ( Fisher Scientific ) and cells were incubated with STAT2-specific antibody in 2% BSA for 1 hour at 37°C . Cells were washed with 2% BSA and incubated with the secondary antibody Alexa Fluor 594 Goat anti-Rabbit ( Life Technologies ) . Cells were then washed with 2% BSA , followed by one PBS wash . Cover slips were mounted on glass slides using Prolong Gold Anti-fade reagent ( Cell Signaling ) . Staining was visualized on a Zeiss Axioskop 2 Plus fluorescence microscope , and images were taken using AxioVision v4 . 6 software ( Zeiss ) . The recombinant adenoviruses expressing SVV ORF63 ( AdORF63 ) and VZV AdORF63 were produced as previously described [36] . The vector contains a tetracycline-responsive promoter and requires the addition of a tetracycline-regulated transactivator ( tTA ) [37] , which was provided by co-infecting with AdtTA . TRFs cultured in six-well clusters were co-transduced with the purified ORF63 adenoviruses and AdtTA at the indicated MOI in 0 . 5 ml of serum-free DMEM . After 1 . 5 hour of incubation at 37°C , 1 . 5 ml of DMEM supplemented with 10% FBS was added and incubation continued for a total of 48 hours . Where indicated , doxycycline was added to the infections to regulate tTA-dependent gene expression . Total RNA was extractedand treated with DNase using the NucleoSpin RNA isolation kit ( Machery Nagel ) according to the manufacturer's protocol . The concentration of the RNA samples was measured using the NanoDrop 1000 Spectrophotometer ( Thermo Scientific ) . Single-stranded cDNA was made from total RNA using random hexamers ( TaKaRa ) to prime first-strand synthesis by Maxima Reverse Transcriptase ( Thermo Scientific ) as recommended by the manufacturer . The induction of ISG54 and Mx-1 mRNA expression upon uIFN stimulation was determined using SYBR green-based semiquantitative real-time RT-PCR ( qPCR ) using the following primers: ISG54 Fw: 5’-gttactggaactaataggacac-3’ , ISG54 Rev: 5’-tggcaagaatggaaca-3’ , Mx-1 Fw: 5’-atgatcgtcaagtgccg-3’ , Mx-1 Rev: 5’-gccttgccttcctcca-3’ . SVV ORF63 expression was confirmed using the primers ORF63 Fw: 5’-CAGCGTCCTACAGTGAC-3’ and ORF63 Rev: 5’-GTTGCTGGTAGCATCATC-3’ . Levels of IRF9 mRNA were determined using the primers IRF9 Fw: 5’- TACCCGAAAACTCCGGAAC-3’ and IRF9 Rev: 5’-AAGAAGGCAGCATCCTGG-3’ . Levels of STAT2 mRNA were determined using the primers STAT2 Fw: 5’- ATGGCGCAGTGGGAAATG-3’ and STAT2 Rev: 5’-ctgccagttctggtcttc-3’ . Reactions were performed using SYBR green PCR core reagents and Platinum Taq DNA Polymerase ( Invitrogen ) . Relative expression of ISG54 , Mx-1 , and ORF63 was calculated using the method described by Livak and Schmittgen [38] . GAPDH was used as a housekeeping gene to establish a baseline against which target genes were compared ( Fw: 5’-GCACCACCAACTGCTTAGCAC-3’ , Rev: 5’- TCTTCTGGGTGGCAGTGATG-3’ ) . For IRF9 and STAT2 mRNA expression we calculated delta cycle threshold ( ΔCt ) by subtracting background Ct ( GAPDH ) from the Ct value for IRF9/STAT2 . Purified DNA from TRFs infected with SVV . eGFP was used as a template for PCR amplification of SVV ORF63 . PCR was performed using AccuPrime Taq DNA polymerase High Fidelity ( Life Technologies ) using the primers 5’-AATAAAGGATCCGCCACCATGCAGGCGCCCCGAG-3’ ( Fw ) and 5’-AATAAAGAATTCTTATGTATTGTGTACAGACTCTCGTAACTCCGTG-3’ ( Rev ) to amplify the coding sequence of the SVV ORF63 gene . The PCR-generated product was inserted into pcDNA3 . 1-IRES-nlsGFP using BamHI/EcoRI sites , creating pcDNA3 . 1 ORF63 . To create pcDNA3 . 1 FLAG-ORF32 we amplified ORF32 from the same template with the primers 5'- ( AATAAAGGATCCGCCACCATGGCATCATCTAATACTTGCGAAGAACAAAATAATTCTA ) -3' ( Fw ) and 5'- ( AATAAAGAATTCTTActtatcgtcgtcatccttgtagtcATCCGTTTCGCTCTCGCTAGATGAAGGTTG ) -3' ( Rev ) using the Expand High Fidelity PCR system ( Roche ) . The PCR-generated product was inserted into the pcDNA3 . 1-IRES-nlsGFP vector using BamHI/EcoRI sites . VZV ORF63 was amplified from DNA extracted from VZV . eGFP-infected MRC-5 cells using the Expand Expand High Fidelity PCR system ( Roche ) and the following primers: 5'- ( AATAAAGGATCCGCCACCATGTTTTGCACCTCACCGGC ) -3' ( Fw ) and 5'- ( AATAAAGAATTCCTACACGCCATGGGGGGGCGGTATATC ) -3' ( Rev ) . The resulting insert was cloned into pcDNA3 . 1-IRES-nlsGFP using BamHI/EcoRI sites . Rhesus IRF9 was synthesized and codon-optimized for expression in rhesus cell lines by GenScript . The insert was cloned from the pUC57 plasmid into pcDNA3 . 1-IRES-nlsGFP using BamHI/EcoRI restriction sites , creating pcDNA3 . 1 IRF9 . All ligations were performed using the Rapid DNA Dephos and Ligation kit ( Roche ) . The DNA sequences of all expression plasmids were verified . HEK 293T cells were transfected with the indicated plasmids and pGL3-ISRE-Luc ( a kind gift from Dr . John Hiscott , Vaccine and Gene Therapy Institute , Florida ) using the Lipofectamine 2000 reagent ( Life Technologies ) using the manufacturers protocol . For the IRF9 overexpression experiment we used 1 μg pGL3-ISRE-Luc and the indicated amounts pcDNA3 . 1 ( p ) ORF63 and pcDNA3 . 1 ( p ) IRF9 . Control pcDNA3 . 1 or pRetro-E2 expressing GFP was used to equalize all transfection samples to a total of 6 μg DNA . Transfection efficiency was confirmed measuring GFP expression in all samples using Synergy HTX Multi-Mode Reader ( Bio-Tek ) or by staining for specific proteins in western blot . To test whether VZV ORF63 inhibits IFN-signaling , we used 1 μg of pGL3-ISRE-Luc and 3 μg of the other indicated plasmids . 48 hours post transfection IFN-signaling was assessed using a luciferase assay , described below . GIPZ lentivirus constructs expressing shRNA specific for human IRF9 were obtained from Open Biosystems/GE Healthcare . The constructs used are V3LHS-322329 ( shRNA-1 ) , V3LHS-322332 ( shRNA-2 ) , and V2LHS-69847 ( shRNA-3 ) . Replication deficient lentiviruses were produced by transfecting the shRNA vectors into HEK 293T cells and providing the vesicular stomatitis virus G ( pMD2 . G VSV-G , Addgene ) protein and the packaging plasmid psPAX2 ( Addgene ) in trans . The plasmids were transfected using Lipofectamine LTX ( Life Technologies ) . 48 hours post transfection the supernatant containing lentivirus was harvested and transferred to target cells , which were transduced in the presence of 5 μg/ml Polybrene ( Hexadimethrine bromide; Sigma-Aldrich ) . After 24 hours , this process was repeated . The resulting cell lines were grown in the presence of 3 μg/ml Puromycin to select for shRNA expressing cells . P-values were determined using unpaired Student’s t-test . To prepare the SVV ORF63/70 mutant , we used an SVV BAC containing the complete SVV genome and eGFP driven by the CMV immediate-early promoter [39] . To introduce mutations into SVV ORF63/70 , we used the two-step red-mediated mutagenesis protocol [40] . Mutagenesis of SVV BAC using this protocol has been previously described [29] . Briefly , we used a recombinant plasmid encoding red fluorescent protein ( RFP ) interrupted by the kanamycin gene ( kindly provided by Dr . Benedikt Kaufer , Freie Universität Berlin , Germany ) . Using oligonucleotide primers specific for regions flanking SVV ORF63/70 at the 5’-end and RFP-specific sequences at the 3’-end , we amplified a 1748 bp DNA fragment containing RFP/kanamycin ( ORF63 mRFP Fw: TACCATCTGAATGTTACGTACATAAATAAAACGCTTCTCAATGGCCTCCTCCGAGGACG , ORF63 mRFP Rev: GACAGGGGTAACATGTTAGCGGCTCCCTATTGGGTAAGGGACTACAAGGCGCCGGTGGAG ) . The DNA fragment was used to transform E . coli GS1783 containing wild-type SVV BAC . We selected kanamycin-resistant colonies and extracted recombinant BAC DNA and confirmed recombination using Hind III digestion and agarose gel electrophoresis . We identified the recombinant BAC clones that contained RFP/kanamycin in place of SVV ORF63 and eliminated the kanamycin cassette . Complete replacement of SVV ORF 63 sequences by RFP was confirmed by sequence analysis . DHFR domains were introduced at the C-terminus of ORF63/70 using a plasmid containing the destabilization domain dihydrofolate reductase ( DHFR ) derived from E . coli ( kindly provided by Dr . Thomas Wandless , Stanford University , California ) . We introduced the kanamycin-cassette at a unique restriction site ( PmeI ) within the sequences encoding DHFR . We amplified DHFR/kanamycin with primers specific for SVV ORF63/70 ( ORF70 DHFR Fw: CCATCTGAATGTTACGTACATAAATAAAACGCTTCTCAATGATCAGTCTGATTGCGGCGTTAGCGGT , ORF70 DHFR Rev: CCATCTGAATGTTACGTACATAAATAAAACGCTTCTCAATGATCAGTCTGATTGCGGCGTTAGCGGT ) by PCR and transformed of E . coli GS1783 containing mutant SVV BAC in which SVV ORF63 was replaced with RFP . After elimination of the kanamycin cassette , mutant SVV BAC in which DHFR was fused at the amino terminus of SVV ORF70 was identified by HindIII digestion and gel electrophoresis . Proper fusion of DHFR to SVV ORF70 was confirmed by sequence analysis . The recombinant BAC was purified and used to transfect Vero cells . Infected cells were grown in the presence of 10 μM trimethoprim ( TMP ) to stabilize the ORF70-DHFR fusion protein . SVV plaques expressing eGFP and RFP were identified and isolated using a fluorescent microscope . Sequentially , mutant SVV was passaged four to five times in Vero-CRE cells . Passaging the virus allowed recombination of ORF70-DHFR to ORF63 location , which lead to the loss of RFP . In addition , BAC vector and eGFP sequences within the virus are flanked by loxP sites , thus passing the virus in Vero cells stably expressing cre recombinase resulted in the elimination of these non-viral sequences [39] . SVV plaques that were negative for both eGFP and RFP were purified and transferred from Vero-CRE cells to TRFs via serial passage . DNA extracted from Vero cells infected with SVV mutant was used for sequence analysis to confirm proper fusion of DHFR to the C-termini of both ORF63 and ORF70 . In vitro growth curves for wild type and mutant SVV were generated as described [41] . Briefly , a monolayer of uninfected Vero cells in 25 cm2 tissue culture flasks were infected with approximately 5X102 Vero cells previously infected with either wild type or ORF63-DHFR SVV . At 3 , 24 , 48 , 72 , 96 , 120 , 144 and 168 hours p . i . cells were trypsinized , diluted and seeded on triplicate dishes containing uninfected Vero cells . After approximately one week , infected cells were stained with crystal violet and infectious plaques were counted . SVV Delta , ORF63/70-DHFR SVV , SVV ORF63 gene , and SVV ORF32 gene: GenBank NC_002686 . VZV pOka and VZV ORF63 gene: GenBank AB097933 .
To determine if SVV interferes with type I IFN-mediated responses , we studied IFN-stimulated response element ( ISRE ) -dependent transcription in SVV-infected luciferase reporter cells . We used telomerized rhesus fibroblasts ( TRFs ) stably expressing firefly luciferase under the control of the ISRE as well as constitutively expressing renilla luciferase to control for differences in cell viability between the samples . TRF-ISRE cells were infected with SVV . eGFP at a ratio of 5:1 ( uninfected to SVV-infected cells ) and , after 42 hours , incubated with rhesus or human IFNα for 6 hours . Productive virus infection was confirmed by visualizing eGFP expression using immunofluorescence microscopy . The firefly and renilla signal was measured and the ratio of these values reflected ISRE activity . In mock-infected cells , incubation with increasing concentrations of rhesus IFNα corresponded with increased ISRE activity . However , only a minimal response to IFNα was observed in SVV-infected cells ( Fig 1A , left panel ) . The rhesus reporter cells were also activated by human IFNα and a comparable reduction in ISRE activity was observed in the SVV-infected cells ( Fig 1A , right panel ) . A dose-dependent increase of luciferase activity in SVV-infected cells was not due to the presence of uninfected cells since this was also observed when the infected cells were sorted for high GFP expression by flow cytometry before IFN-treatment ( Fig 1B ) , suggesting that high concentrations of IFN can partially overcome the inhibition by SVV . The ISRE element drives the expression of interferon stimulated genes ( ISG ) . To study if SVV inhibits IFNα-induced ISG-expression , we infected TRFs with SVV . eGFP for 40 hours and incubated with increasing concentrations of recombinant universal type I IFN ( uIFN ) for 8 hours . Productive SVV infection was confirmed by the detection of SVV ORF63 expression ( Fig 1C ) . Expression of ISG15 , ISG54 and Mx-1 was observed in all mock-infected IFNα-stimulated samples , but was absent in SVV-infected cells ( Fig 1C ) . These data show that SVV inhibits IFNα-mediated activation of ISRE-dependent reporter gene expression and ISG protein expression . The engagement of IFN with the IFN-receptor results in the activation of the JAK-STAT signal transduction pathway . The resulting phosphorylation of STAT1 and STAT2 allows their heterodimerization and association with IRF9 , forming the ISGF3 complex that subsequently shuttles to the nucleus to initiate ISRE-dependent transcription [42] . The nuclear translocation of the ISGF3 complex is thus essential for ISRE activation . We analyzed IFN-induced nuclear localization of STAT in SVV-infected cells . TRFs were infected at a 10:1 ratio with SVV . eGFP and incubated with uIFN for 40 minutes at 48 hours post infection ( p . i . ) . In uninfected cells , STAT2 was found predominantly in the cytosol in the absence of IFN and in the nucleus upon IFN-treatment ( Fig 2A ) . In contrast , STAT2 was not translocated to the nucleus in SVV-infected cells ( green/eGFP ) upon IFN-treatment ( Fig 2A ) . In addition , we isolated cytoplasmic and nuclear fractions of SVV . eGFP-infected Vero cells ( ratio 5:1 ) and determined the cellular localization of STAT2 by western blot . Separation of cytosol and nuclei was confirmed using GAPDH and the nuclear matrix protein p84 ( Fig 2B ) . In uninfected cells , STAT2 was found in the cytosolic fraction in the absence of IFN-treatment , whereas stimulation with uIFN led to the redistribution of STAT2 to both cytoplasmic and nuclear fractions . In contrast , STAT2 remained predominantly cytosolic in SVV-infected cells even upon IFN-treatment ( Fig 2B ) . The SVV ORF62 protein was found in both cytoplasmic and nuclear fractions . This distribution is consistent with reports for the homologous VZV protein that , while primarily nuclear during early times of infection , localizes to the cytoplasm at later times of infections as a results of phosphorylation by ORF66 [43 , 44] . Thus , in an asynchronous infection one would expect both nuclear and cytoplasmic expression of ORF62 . Taken together , these data suggest that SVV inhibits IFN-dependent ISG-induction by abrogating the IFN-associated translocation of STAT2 . Next we examined whether the inhibition of STAT2 nuclear translocation correlated with a SVV-mediated reduction in steady state levels of ISGF3 members or impaired STAT1/STAT2 phosphorylation . TRFs infected with SVV . eGFP for 48 hours were stimulated with IFN for 20 minutes . Steady state levels and IFN-induced phosphorylation of STAT1 were comparable between SVV- and mock-infected cells ( Fig 3A ) . In contrast , IFN-induced STAT2 phosphorylation was absent in SVV-infected cells and steady state levels of the protein also appeared to be reduced ( Fig 3A ) . Densitometric analysis of STAT2 protein using four independent experiments confirmed an approximately 25% decrease in STAT2 levels ( Fig 3B ) . However , this decrease was not statistically significant . In contrast , we observed a significant decrease of more than 50% in IRF9 levels by SVV ( Fig 3A and 3B ) . Interestingly , the reductions in STAT2 and IRF9 protein levels were observed regardless of IFN stimulation ( Fig 3B ) . Since IRF9 drives the nuclear translocation and retention of phosphorylated STAT1 and STAT2 [42] , we studied the localization of residual IRF9 in IFN-stimulated SVV-infected Vero cells by analyzing isolated cytoplasmic and nuclear fractions in western blots . In control cells , IFN-stimulation triggered the increased translocation of IRF9 from the cytosol to the nucleus . However , this increased nuclear translocation of IRF9 was not observed in SVV-infected cells ( Fig 3C ) . Furthermore , this experiment confirmed the SVV-mediated reduction in IRF9 expression levels . These data suggest that SVV abrogates JAK-STAT signaling by both preventing the phosphorylation of STAT2 and reducing STAT2 and IRF9 protein levels . Ambagala et al . showed that wild type VZV can replicate in cells preincubated with IFNα , but an ORF63 deletion mutant could not [17] . This observation suggested that VZV ORF63 might be involved in the ability of VZV to evade IFN responses . VZV and SVV ORF63 share 52% overall amino acid homology [4 , 45] . To determine if SVV ORF63 plays a role in the inhibition of IFN-stimulated responses observed in SVV-infected cells , we constructed a recombinant adenovirus expressing SVV ORF63 under the control of a tetracycline-responsive promotor ( AdORF63 ) . ORF63 expression is induced by co-infection with a recombinant adenovirus expressing the tetracycline-regulated transactivator ( AdTA ) [37] . These adenoviruses lack the E1-region [36] , and therefore unable to interfere with IFN-signaling [46] . TRFs were co-transduced with a multiplicity of infection ( MOI ) of 10 of AdTA and with increasing MOIs of AdORF63 . The expression of ORF63 was monitored at 48 hours p . i . ( S1A Fig ) . Increasing MOI correlated with increasing ORF63 expression levels as expected . However , higher ORF63 levels also resulted in decreased GAPDH expression , suggesting that high expression levels of ORF63 may be cytotoxic . The transactivator expressed by AdTA can be inactivated by the tetracycline derivative doxycycline ( Dox ) . To fine-tune ORF63 expression levels , we transduced TRFs with AdORF63 ( MOI 20 ) and AdTA ( MOI 10 ) in the presence of decreasing amounts of Dox . We detected robust ORF63 expression in cells that were incubated with 1 ng/ml Dox ( S1B Fig ) and these expression levels were comparable to ORF63 expression in cells infected with SVV ( S1C Fig ) . At 1 ng Dox , GAPDH levels were not affected whereas lower Dox concentrations resulted in decreased GAPDH levels reflecting reduced cell viability ( S1B Fig ) . To examine whether ORF63 inhibits IFN signaling we treated TRFs with IFN for up to 16 hours in the presence of 1ng Dox and studied ISG expression using qPCR ( Fig 4A ) . In addition we used 1000 ng/ml Dox to inhibit ORF63 expression and under these conditions we observed increased expression of Mx-1 and ISG54 mRNA , reaching peak expression at 8 and 4 hours of IFN stimulation , respectively . In ORF63 expressing cells , however , Mx-1 and ISG54 mRNA levels were severely reduced at all time points ( Fig 4A ) . ORF63 expression was confirmed by qPCR ( Fig 4A , lower right panel ) . We also examined ISG protein expression by western bloting in the absence or presence of ORF63: after 8 hours of stimulation with IFN , high levels of ISG15 , ISG54 and Mx-1 were detected in mock-transduced cells and in AdORF63-transduced cells treated with 1000 ng of Dox , whereas expression was absent or barely detectable in AdORF63-transduced cells treated with 1 ng Dox ( Fig 4B ) . Taken together , these data indicate that SVV ORF63 inhibits type I IFN-induced gene expression . The inhibition of IFN-induced ISG expression by ORF63 correlated with our observations in SVV-infected cells . Since reduced STAT2 phosphorylation as well as decreased amounts of STAT2 and IRF9 proteins were observed in SVV-infected cells ( Fig 3A and 3B ) , we examined whether expression of ORF63 leads to the inhibition of IFN-induced STAT2 phosphorylation and reduced steady state levels of STAT2 and IRF9 . Expression and phosphorylation status of members of the JAK-STAT pathway were examined in AdORF63/AdTA-transduced TRFs stimulated with uIFN for 20 minutes or 8 hours . Despite an inhibition of IFN-induced ISG expression in ORF63-expressing cells ( Fig 5A , lower panel ) the expression levels and phosphorylation status of STAT1 and STAT2 were unchanged ( Fig 5A , upper panel ) . However , we did observe a reduction in steady state levels of IRF9 when ORF63 was present ( Fig 5A ) . To confirm that ORF63 affects IRF9 expression , we transduced TRFs with AdORF63/AdTA with decreasing concentrations of Dox to obtain increasing ORF63 expression levels . Western blot analyses revealed that increasing levels of ORF63 inversely correlated with decreasing IRF9 levels ( Fig 5B and 5C ) . Interestingly , reduced IRF9 expression was observed in both unstimulated and IFN-stimulated samples ( Fig 5B ) , suggesting that ORF63 reduces IRF9 regardless of IFN signaling . To confirm that ORF63 does not affect STAT2 levels we transduced TRFs with AdORF63/AdTA in the presence of decreasing amounts of Dox . We observed a reduction in STAT2 levels in cells expressing high levels of ORF63 ( 0 . 1 and 0 ng/ul Dox ) ( Fig 5D ) . However , STAT1 and GAPDH expression were also affected in these samples , but when we normalized STAT1 and STAT2 expression to GAPDH expression the reduction in STAT1 or STAT2 levels was not significant when ORF63 was expressed ( Fig 5E ) . To determine whether ORF63 affects the transcription of IRF9 we studied IRF9 mRNA levels in TRFs transduced with AdORF63/AdTA in the presence of decreasing amounts of Dox . However , we did not observe a reduction of IRF9 mRNA levels upon increasing transcription of ORF63 suggesting that ORF63 does not affect IRF9 transcription ( Fig 5F ) . To analyze if ORF63 promotes IRF9 degradation via the proteasome we incubated ORF63-expressing cells with increasing concentrations of the proteasome inhibitor MG132 for 16 hours prior to lysing the cells . Treating ORF63-expressing cells ( + ) with increasing concentrations of MG132 reversed IRF9 degradation , but we also observed a slight increase in IRF9 levels in control cells ( - ) ( Fig 6A ) . To determine if the rescue of IRF9 expression in ORF63-expressing cells was due to reduced turnover of residual IRF9 or due to actively blocking ORF63-mediated IRF9 degradation , we averaged the ratio of IRF9 and GAPDH expression in MG132-treated ( 10 μM ) control or ORF63-expressing cells in four independent experiments ( Fig 6B ) . While MG132-treatment increased IRF9 expression in control cells , the difference was not statistically significant . In contrast , MG132-treatment of ORF63-expressing cells resulted in a fivefold increase in IRF9 expression ( Fig 5B , p = 0 . 007 ) . From these data we conclude that ORF63 promotes IRF9 degradation in a proteasome-dependent manner . In addition to IRF9 , related IRF proteins play an important role in the regulation of the expression of IFN and ISGs [47–49] . To determine whether SVV ORF63 affects the expression of IRFs other than IRF9 , we monitored the steady state expression of IRF1 and IRF3 in TRFs transduced with AdORF63/AdTA in the presence of decreasing amounts of Dox . Increasing levels of ORF63 reduced IRF9 expression levels , but not that of IRF1 or IRF3 ( Fig 6C ) . While we cannot formally rule out that SVV ORF63 might affect other IRFs it seems likely that SVV ORF63 specifically induces the degradation of IRF9 and thus preventing IFN-mediated ISG induction . To determine whether the reduction of IRF9 was sufficient to prevent ISG-induction , we attempted to recapitulate the ORF63-effect by reducing IRF9 levels using small hairpin RNA ( shRNA ) . Telomerized human fibroblasts ( THF ) stably expressing ISRE-luciferase ( THF-ISRE ) were transduced with lentivectors expressing IRF9-specific shRNA . Western blots of IRF9 showed that IRF9 expression levels were reduced in cells expressing shRNA-1 and completely absent in cells expressing shRNA-2 and -3 ( Fig 7A ) . To compare steady state levels of IRF9 upon translational inhibition by shRNA to that of post-translational degradation by ORF63 , we transduced THF-ISRE cells with AdORF63/AdTA in the presence of decreasing amounts of Dox . In these human cells , optimal ORF63 levels were observed at 0 . 1 ng/ml Dox or in the absence of Dox without reduction of GAPDH levels whereas only partial induction of ORF63 was observed at 1 ng Dox . A reduction of IRF9 levels consistent with ORF63 expression was observed . Reduced steady state levels of IRF9 were comparable to the partial reduction of IRF9 observed in shRNA-1 expressing cells while IRF9 was completely absent from shRNA-2 and 3 expressing cells ( Fig 7B ) . However , when the THF-ISRE cells expressing the three shRNAs were incubated with uIFN for 4 or 8 hours , ISG54 mRNA expression was only partially reduced in the presence of shRNA-1 , whereas shRNA-2 and shRNA-3 largely prevented ISG induction ( Fig 7C ) . Analysis of IFN-induced ISG54-expression by western blotting confirmed these results ( Fig 7D ) . These data demonstrate that removal of IRF9 is sufficient to inhibit ISG-expression and they are consistent with ORF63 affecting IRF9 protein turnover rather than IRF9 transcription or translation . To further determine whether IRF9 is the primary JAK/STAT-associated target of ORF63 , we took advantage of the fact that HEK 293T cells do not respond efficiently to type I IFN unless IRF9 is overexpressed [50] . Co-transfection of a plasmid encoding an ISRE-luciferase reporter with increasing amounts of rhesus IRF9-expressing plasmid resulted in an IRF9-dependent increase of luciferase expression upon treatment with IFN at 24 hours post transfection ( Fig 8A; black lined graph ) . Interestingly , optimal luciferase stimulation was observed with 25–100 ng of the IRF9 plasmid whereas higher IRF9 concentrations resulted in decreased luciferase activity . This decrease might be due to the fact that higher IRF9 levels activate ISRE activity even in the absence of IFN-treatment ( Fig 8B ) , and a prolonged stimulation might induce negative regulators of IFN signaling [51] . Co-transfection of 1 μg ORF63-expressing plasmid resulted in the inhibition of IRF9-induced luciferase expression both in the presence or absence of IFN ( Fig 8A and 8B; gray lined graphs ) suggesting that ORF63 inhibition cannot be overcome by increasing IRF9 levels . However , ORF63 needs to be in excess of IRF9 for complete inhibition since complete inhibition of IRF9-dependent ISG-induction was only observed when at least 250 ng of ORF63 plasmid was co-transfected with 50ng IRF9-expressing plasmid ( Fig 8C ) . Together with the finding that ORF63 inhibited IRF9-dependent ISRE-transcription even in the absence of IFN-stimulation ( Fig 8B ) these data further support the conclusion that degradation of IRF9 is the major mechanism by which ORF63 inhibits JAK/STAT signaling . To study the role of ORF63 in the context of virus-induced inhibition of JAK-STAT signaling we constructed a conditionally ORF63/ORF70-expressing mutant using a recombinant bacterial artificial chromosome containing the complete SVV genome ( SVV BAC ) [39] . Recently , using the SVV BAC an ORF63/ORF70 SVV mutant was constructed by introducing stop codons in both genes . These mutations severely affected replication of the virus in vitro consistent with ORF63 being essential for viral growth [29] . To generate a mutant virus in which expression levels of ORF63 could be conditionally regulated , we fused the destabilizing domain ( DD ) of dihydrofolate reductase ( DHFR ) to the C-termini of ORF63 and ORF70 using two-step red-mediated mutagenesis ( Fig 9A ) [40] . The addition of DD-DHFR to any protein results in rapid proteasomal degradation of the fusion protein unless DHFR is stabilized with trimethoprim ( TMP ) [52] . We were able to recover DD-DHFR-tagged SVV in the presence of TMP and growth curves confirmed that viral growth was reduced by about 50% during the first 96 hours of infection compared to that of unmodified BAC-derived SVV when grown in 10 μM TMP ( Fig 9B ) . A more severe reduction in viral growth was observed beyond that time point . To study the effect of TMP-removal on ORF63/70-DHFR SVV replication , we infected Vero cells in the presence of 10 μM TMP until viral plaques were detected ( Fig 9C , left panel ) . Removal of TMP and passaging ORF63/70-DHFR SVV-infected Vero cells resulted in very few plaques ( Fig 9C , middle panel ) . However , virus in these cultures could be rescued when 10 μM of TMP was added back to the culture ( Fig 9C , right panel ) . These observations confirmed that the virus expressing DD-DHFR-tagged ORF63/ORF70 was able to replicate in tissue culture in the presence of TMP and viral growth can be regulated by removal of TMP . To determine whether DHFR-fusion to ORF63/ORF70 affected viral inhibition of the JAK-STAT pathway , we infected TRFs with wild type or ORF63/70-DHFR SVV in the presence of 10 μM TMP and studied expression of STAT2 and IRF9 as well as phosphorylation of STAT2 after 20 minutes of stimulation with IFN ( Fig 9D ) . We observed a reduction in STAT2 and IRF9 expression levels as well as a complete inhibition of STAT2 phosphorylation in both wild type- and ORF63/70-DHFR-infected cells , indicating that the evasion of JAK-STAT signaling was not affected by the presence of the DHFR domains ( Fig 9D ) . Since the removal of TMP from the culture media triggers degradation of ORF63-DHFR and ORF70-DHFR , we infected TRF with ORF63/70-DHFR SVV and cultured the cells in varying concentrations of TMP . We observed a dose-dependent decrease in ORF63 expression and ORF63 was no longer detectable when the virus was grown in 0 . 02 μM or less TMP ( Fig 9E ) . This reduction in ORF63 levels led to increased levels of IRF9 , indicating that expression of these proteins was inversely correlated ( Fig 9E ) . However , the expression of ORF31 ( glycoprotein B ) was also affected by reducing TMP concentrations , similar to ORF63 expression ( Fig 9E , western blot and graph ) . This result revealed that ORF63/70 is required for ORF31 expression and most likely for the expression of other SVV genes as well [24] . Restoration of IFN-sensitivity could therefore not be unequivocally assigned to the absence of ORF63 since SVV ORF31 was absent as well . In the experiments described above , we established that SVV inhibits JAK-STAT signaling by interfering with IFN-induced phosphorylation of STAT2 and by modulating the degradation of STAT2 and IRF9 . Previous reports have shown that VZV interferes with IFN-mediated signaling [12 , 17] , however , the evasion mechanisms involved in this inhibition are largely unknown . To determine if VZV inhibits JAK-STAT signaling similar to SVV , we infected human fibroblasts , MRC-5 cells , with VZV . eGFP for 48 hours and activated JAK-STAT signaling by incubating the cells with uIFN for 20 minutes prior to harvesting the cells . Similar to SVV , expression levels of IRF9 and STAT2 were reduced in VZV-infected cells ( Fig 10A , upper panel ) . Moreover , complete inhibition of IFN-induced STAT2 phosphorylation was observed , indicating that VZV employs mechanisms of JAK-STAT evasion that are comparable to those of SVV ( Fig 10A , upper panel ) . However , unlike SVV ( Fig 5A ) , IFN-induced STAT1 phosphorylation is blocked by VZV ( Fig 10A , lower panel ) . To determine whether VZV ORF63 prevents ISG-induction , we co-transfected HEK 293T cells with the ISRE-luciferase reporter plasmid and plasmids encoding VZV ORF63 or , as a control , FLAG-tagged SVV ORF32 . The cells were stimulated with uIFN for 6 hours at 42 hours post transfection , after which ISRE-luciferase expression was measured . In contrast to untransfected controls or SVV ORF32 , expression of VZV ORF63 caused a reduction in IFN-induced luciferase expression ( Fig 10B ) . Expression of the viral proteins was confirmed by western blot ( Fig 10C ) . To assess if VZV ORF63 plays a role in the reduction of IRF9 expression observed in VZV-infected cells ( Fig 10A ) , we expressed the viral protein using the tetracycline-inducible adenovirus system described in Fig 4 . MRC5 cells were co-transduced with VZV AdORF63 and AdTA , incubated with the indicated concentrations of Dox and after 48 hours steady state levels of IRF9 were analyzed using western blot . We observed a prominent decrease in IRF9 expression levels in cells that expressed VZV ORF63 ( Fig 10D , left panel ) . To determine if the reduction in STAT2 expression levels and phosphorylation observed in VZV-infected cells was due to VZV ORF63 , we stimulated the transduced MRC5 with uIFN for 20 minutes to activate the pathway and studied STAT2 by western blot . STAT2 steady state levels and phosphorylation were unaffected by the presence of VZV ORF63 ( Fig 10D , left panel ) . The same experiment was performed in TRFs and we observed a VZV ORF63-induced reduction of IRF9 , but not of STAT2 , in those cells as well ( Fig 10D , right panel ) . Taken together these data show that SVV and VZV employ similar mechanisms to interfere with JAK-STAT signaling and that the ORF63 proteins of both viruses contribute to this inhibition by mediating the degradation of IRF9 . VZV ORF63 induced IRF9 degradation in both MRC5 and TRFs ( Fig 10D ) , indicating that the pathway’s inhibitory target is conserved between human and rhesus cells .
The data presented here show that both SVV and VZV inhibit IFN-mediated ISG induction and reduce the expression of IRF9 . In addition , we observed a marginal decrease in STAT2 levels and a complete inhibition of IFN-dependent phosphorylation of STAT2 in both SVV- and VZV-infected cells . Since IRF9 is essential for JAK-STAT signaling , the degradation of IRF9 by ORF63 is thus part of a multi-pronged inhibition of IFN-mediated antiviral gene induction . These observations are consistent with previous reports showing that VZV prevents induction of Mx-1 in brain fibroblasts and reduces STAT2 levels in infected brain fibroblasts [53] . We further demonstrate that SVV ORF63 promotes the degradation of IRF9 in a proteasome-dependent manner , but does not affect STAT2 protein levels or its phosphorylation . SVV ORF63 and VZV ORF63 share 52% amino acid identity and there is evidence that these proteins are expressed in latently infected ganglia [5 , 21 , 22 , 28 , 45 , 54–56] . We observed that inhibition of IFN-signaling by both proteins correlated with degradation of IRF9 whereas neither protein affected STAT2 expression or phosphorylation . Together with the previous report by Ambagala et al . demonstrating IFN-sensitivity of ORF63-deficient VZV [17] our data thus strongly suggest that ORF63 plays a central role in IFN-signaling inhibition by both SVV and VZV . In addition , our observations reveal that both viruses likely encode additional ORFs responsible for the inhibition of STAT2 phosphorylation and the reduction of STAT2 expression . The reduction in IRF9 levels observed in SVV/VZV-infected cells and ORF63-expressing cells did not result from reduced transcription ( Fig 5F and S2A Fig ) . Rather , ORF63-mediated the proteasomal degradation of IRF9 independently of IFN-signaling . Although this is the first description of IFN-evasion by IRF9 targeting for a herpesvirus , IRF9 ( p48 ) targeting has been demonstrated for several other viruses . The non-structural protein ( NSP ) 1 of the simian rotavirus strain SA11-4F was shown to induce proteasomal degradation of all IRF proteins that contain an IRF association domain ( IAD ) , which include IRF3 , IRF5 , IRF7 and IRF9 [57] . In contrast , our data suggest that ORF63 targets IRF9 , but not IRF3 which in VZV is degraded by ORF61 [15] . Adenovirus E1A protein blocks IFN-induced protein expression by reducing IRF9 expression levels , yet the mechanism of this immune evasion is unknown [46 , 58] . Incidentally , the presence of E1A in HEK293 cells [59 , 60] could be responsible for the requirement of exogenous IRF9 for IFN-dependent ISG-induction . In our co-transfection experiments ORF63 thus seemed to be able to eliminate IRF9 once the E1A-dependent IRF9-inhibition was breached . Additionally , the reovirus type 1 Lang μ2 protein causes nuclear accumulation of IRF9 , independent of IFN-stimulation , which results in severely impaired JAK-STAT signaling [61] . Finally , the human papillomavirus ( HPV ) 16 E7 oncoprotein was shown to interact with IRF9 thereby preventing ISGF3 formation [62] . We did not observe an interaction between IRF9 and ORF63 in immunoprecipitations studies performed in SVV-infected and ORF63-expressing TRFs . Therefore , the exact mechanism by which ORF63 elicits the degradation of IRF9 still needs to be elucidated . IRF9 is a key player in the JAK-STAT signaling pathway activated by type I IFN: unphosphorylated STAT2 is complexed with IRF9 and the pair continuously shuttles between the cytoplasm and the nucleus , which is driven by the nuclear localization signal of IRF9 and the nuclear export signal ( NES ) of STAT2 [63] . Upon activation of JAK1 and TYK2 , phosphorylated STAT1 and STAT2 dimerize , which results in the loss of the NES of STAT2 [63] . The requirement of IRF9 for anti-viral immune responses was demonstrated by Kimura et al . using IRF9 knock out murine cells . Replication of herpes simplex virus type 1 and vesicular stomatitis virus ( a rhabdovirus ) was greatly enhanced in IFNα-treated cultures of infected IRF9-/- cells , while the cytokine limited viral replication in wild type cells [64] . In addition , Maiwald et al . created a mathematical model based on experimental data that shows that IRF9 determines the peak time and intensity of type I IFN-induced responses [65] . Our data using IRF9-specific shRNA are consistent with these studies and demonstrate the requirement of IRF9 for efficient ISG induction in human cells . Whereas shRNA-expressing cells seemed to express lower levels of IRF9 than ORF63-expressing cells it needs to be considered that ORF63 needs protein expression to act on IRF9 whereas shRNAs prevent protein expression itself . Thus , we concluded that reduced IRF9 levels are responsible for inhibition of IFN-induced ISG expression in both shRNA and ORF63-expressing cells . We therefore propose that that ORF63 blocks the JAK-STAT pathway by reducing IRF9 levels . This conclusion is further supported by the ability of ORF63 to counteract IRF9-mediated ISG-induction in HEK 293T cells both in the presence and absence of IFN ( Fig 8 ) . Since IRF9-degradation was also observed in VZV-infected and VZV ORF63-transduced cells ( Fig 10 ) it seems highly likely that the restoration of IRF9-levels was responsible for the previously described hyper-sensitivity of ORF63-deficient VZV [17] . The VZV deletion mutant used in this study contained a truncation of ORF63 in which only the first 24 amino acids of ORF63 was expressed . This mutant did not replicate in the presence of IFNα , but was able to replicate , albeit at reduced levels , in some cell types such as the osteosarcoma cell line U2OS [17] . To determine whether IRF9-depletion would restore the ability of this VZV ORF63 deletion virus to replicate in fibroblasts we infected THF-ISRE and THF-ISRE shRNA-3 cells with the VZV-deletion mutant ( kindly provided by Jeff Cohen ) . However , we did no observe an increased growth of this virus upon IRF9-depletion . This indicates that additional functions of ORF63 , either directly affecting host pathways or indirectly via other viral proteins , contribute to the reduced growth of the deletion virus . Introducing stop codons in ORF63 and ORF70 severely impaired the growth of SVV in IFN-deficient Vero cells [29] . Similarly , when we fused a DHFR domain to the C-terminus of both ORF63 and ORF70 to create an inducible knock out for both proteins , removal of TMP resulted not only in the degradation of ORF63 and ORF70 but also prevented expression of other SVV genes that depend on ORF63 function . Because of the requirement of ORF63 for viral replication we were unable to directly address the biological significance of IRF9-degradation for evasion of JAK-STAT signaling by these varicelloviruses . Cohen et al . found that cells infected with the VZV ORF63 deletion virus were highly susceptible to IFN treatment [17] , suggesting that ORF63-induced degradation of IRF9 plays a prominent role in evasion of this pathway . However , since ORF63 is required for viral early gene expression and viral replication [23–25] , deletion of protein could also affect expression of the as yet unknown inhibitor of STAT2-phorphorylation as well . Thus , ORF63 likely impacts IFN-resistance both directly , by reducing IRF9 expression , and indirectly by regulating the expression of other IFN-inhibitory genes . Our data are thus consistent with a multipronged inhibition of JAK/STAT signaling by SVV and VZV to ensure efficient evasion of this innate immune pathway . It is not uncommon for a virus to target a signaling pathway at multiple levels . For example , VZV codes for at least three independent strategies devoted to inhibiting IRF3-driven expression of IFNs , which including ORF61 [15] , ORF47 [14] and IE62 [13] . In addition , it is conceivable that IRF9-inhibition by the immediate early gene ORF63 precedes STAT2 inhibition due to sequential expression of the respective inhibitory proteins during viral infection . The relative contribution of each inhibitory pathway during viral infection thus still needs to be elucidated . We observed that both SVV and VZV reduce levels and phosphorylation of STAT2 , whereas STAT1 levels were not affected . In contrast , reduced phosphorylation of STAT1 was only observed in VZV-infected cells . The diminished STAT2 levels did not result from reduced transcription ( S2B Fig ) . Previous reports demonstrated diminished levels of STAT1 in VZV-infected human fibroblasts treated with IFNγ [66] and reduced STAT1 phosphorylation in VZV-infected skin xenografts in the SCIDhu model [12] . A recent report confirmed VZV-mediated downregulation of STAT2 , but downregulation of STAT1 was inconsistent between the experiments and inhibition of STAT1 phosphorylation was not observed [53] . Since STAT1 is shared between type I and type II IFN signal transduction pathways , it represents an attractive target for viral innate immune evasion . However , observations concerning STAT1 expression and phosphorylation in VZV-infected cells are inconsistent ( our data , [12 , 53 , 66] ) , possibly due to the use of type II IFN and different cell lines . Therefore , we cannot rule out that SVV targets the phosphorylation of STAT1 in cell types other than fibroblasts . Since STAT1 phosphorylation was not reduced in SVV-infected TRFs it is unlikely that SVV interferes upstream of STAT1/STAT2 since the binding of IFN to its receptor triggers the activation of the tyrosine kinases JAK1 and TYK2 which in turn phosphorylate STAT1 and STAT2 resulting in the formation of the ISGF3 complex [67 , 68] . If SVV would target either JAK1 or TYK2 one would expect a reduction in STAT1 phosphorylation . For VZV however , this possibility cannot be ruled out since STAT1 phosphorylation is inhibited . Although SVV and VZV-infected cells displayed lower expression levels of STAT2 the remaining STAT2 was not phosphorylated . Therefore , SVV and VZV most likely affect STAT2 phosphorylation directly . Primary VZV infection starts with respiratory mucosal inoculation [69] . Recently , type III IFNs ( IFNλ1–3 ) have been implicated in playing an important role in limiting ( herpes ) viral replication in mucosal tissues [70–72] . These cytokines bind to the IL28Rα/IL10Rβ receptor complex [73 , 74] , which is predominantly expressed on epithelial cells [75] . Engagement of the receptor results in the activation of the JAK-STAT pathway and induction ISG expression [74] . The Kaposi’s sarcoma-associated herpesvirus protein vIRF2 was shown to inhibit both IFNα and IFNλ-mediated ISG expression , by reducing the levels of STAT1 and IRF9 [76 , 77] . Similarly , the inhibition of JAK-STAT signaling by VZV is likely to block IFNλ-mediated signaling in epithelial cells , thereby immediately limiting the host anti-viral responses to allow further spreading . In addition to inhibiting IFN-dependent signal transduction , VZV inhibits activation of the IFN-gene itself and IFN-independent ISG-induction . The VZV proteins IE62 , ORF47 , and ORF61 all target IRF3-mediated induction of IFNβ and ISG genes , such as ISG15 , ISG54 and IS56 [14 , 15 , 22] . VZV ORF61 also blocks the TNFα-mediated activation of NFκB by inhibiting IκBα [16] . Taken together with our observations , it appears that multiple VZV and SVV ORFs are devoted to interfering at sequential steps along the induction of IFN and anti-viral ISG . Despite efficiently counteracting IFN activation and IFN-dependent signaling , several reports have indicated the importance of type I IFNs in limiting VZV replication and spread in vivo . Children that were treated for leukemia and were suffering from varicella showed significantly reduced dissemination of the virus in response to the administration of intra-muscular IFNα [78] . In addition , experiments in severe combined immunodeficiency mice engrafted with VZV-infected human skin ( SCIDhu mice ) showed that preventing IFN signaling with antibodies specific for the interferon receptor resulted in larger cutaneous lesions compared to mice that were untreated [12] . These in vivo responses are most likely explained by the fact that the establishment of an anti-viral state prior to infection is more difficult to overcome than IFN-responses in infected cells . IFNα-treatment of melanoma cells prior to VZV infection led to a reduction in plaque formation [17] . Thus , IFN likely prevents viral spread during the acute phase of infection by inducing an antiviral state in target cells . However , further sensitizing VZV to IFN by therapeutically blocking viral IFN-evasion mechanisms could improve the ability of IFN to prevent spread and/or reactivation of VZV in vivo . This concept could be experimentally tested in non-human primates using the SVV model . Since ORF63 expression have been demonstrated in latently-infected ganglia of SVV-infected rhesus macaques [5] , it is also conceivable that IFN evasion is essential for maintaining viral latency . Dissecting the role of ORF63 in limiting IFN signaling could thus lead to a better understanding of the role of IFN in viral latency and reactivation .
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In this manuscript we demonstrate that the immediate early protein ORF63 encoded by varicella zoster virus ( VZV ) and simian varicella virus ( SVV ) interferes with interferon type I-mediated activation of JAK-STAT signaling and thereby inhibits the expression of interferon stimulated genes . ORF63 blocks this pathway by degrading IRF9 , which plays a central role in JAK-STAT signaling . In addition , both viruses code for immune evasion mechanisms affecting the JAK-STAT pathway upstream of IRF9 , which results in the inhibition of STAT2 phosphorylation . By fusing a degradation domain derived from dihydrofolate reductase ( DHFR ) to ORF63 we further demonstrate that this protein is essential for SVV growth and gene expression , indicating that ORF63 also affects IFN-signaling indirectly by regulating the expression of other immune evasion genes .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
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Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms
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The RNA synthesis machinery of vesicular stomatitis virus ( VSV ) comprises the genomic RNA encapsidated by the viral nucleocapsid protein ( N ) and associated with the RNA dependent RNA polymerase , the viral components of which are a large protein ( L ) and an accessory phosphoprotein ( P ) . The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs , including capping , cap methylation and polyadenylation . Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation . The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs . Here , we report the length at which viral transcripts first gain access to the capping machinery during synthesis . By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP , we found that transcripts that were 30-nucleotides in length were uncapped , whereas those that were 31-nucleotides in length contained a cap structure . The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions . This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA . We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization .
The RNA synthesis machinery of the non-segmented negative-strand ( NNS ) RNA viruses contains at its core a large polymerase protein ( L ) that possesses all the enzymatic activities for genome transcription and replication . During transcription , L catalyzes nucleotide polymerization [1]–[3] as well as each step of mRNA cap addition [4]–[9] and polyadenylation [10] . Those activities are intimately coordinated such that failure to cap the mRNA results in the premature termination of RNA synthesis [7] , [11]–[13] , and failure to methylate the mRNA can result in the hyper polyadenylation of the mRNA [13]–[15] . In this study , we sought to examine how the different L activities are coordinated to ensure the correct synthesis of a capped and methylated mRNA by precise determination of the point at which those 5′ mRNA processing reactions occur during transcription . Our understanding of the activities of L protein has been largely shaped by studies of a prototype of the NNS RNA viruses , vesicular stomatitis virus ( VSV ) . The NNS RNA virus L proteins are homologous and share six regions of sequence conservation ( CRI-VI ) [16] that were thought to contain the conserved functions . The RNA dependent RNA polymerase ( RdRP ) was readily identified by the presence of a set of motifs in CRIII [1] . Consistent with this assignment , substitution of an aspartic acid residue predicted to coordinate a catalytically essential magnesium ion ablates nucleotide polymerization in vitro . Although L protein was known to possess the enzymatic activities for mRNA cap addition , their identity proved difficult to pin down unambiguously . This is because the enzymatic activities themselves are unusual , in that the cap is added by the action of a polyribonucleotidyltransferase ( PRNTase ) that transfers pRNA onto a GDP acceptor through a covalent L-pRNA intermediate [7] , [9] , [17] This contrasts with all other known capping reactions which involve an RNA guanylyltransferase that transfers GMP onto a diphosphate RNA acceptor [18] . Substitutions to residues in CRV of VSV L that are conserved throughout all NNS RNA viruses led to the ablation of capping activity in vitro and defined a motif GxxT[N]HR that was essential for capping , implicating CRV as the PRNTase [7] . Subsequently , the conserved histidine was shown to be essential to form the covalent L-pRNA intermediate , further substantiating this assignment [17] . Polymerases that were defective in mRNA cap addition terminated transcription prematurely , further underscoring the link between correct 5′ mRNA processing and nucleotide polymerization [7] . Following cap formation , the cap itself is methylated at guanine-N-7 and ribose-2′-O positions [5] , [19]–[25] . Those reactions also differ for VSV since they are catalyzed by a single methyltransferase domain [8] . Moreover , in contrast to the order of cap methylation events in other viruses and organisms , the ribose 2′-O methylation precedes and facilitates the subsequent guanine-N-7 methylation [26] . Sequence alignments identified a methylase like domain in CRVI of L [27] , [28] , and substitutions within this region block both 2′-O and G-N-7 methylation [6] , [8] . Like capping , the act of methylation can also influence the properties of the RdRP , since in some circumstances a failure to methylate the mRNA cap is accompanied by the production of a large polyadenylate tail by excessive stuttering of the polymerase on a U7 tract [13]–[15] . This intimate co-ordination of the 5′ mRNA processing reactions with nucleotide polymerization may help ensure production of correctly modified transcripts , which in turn could minimize triggering of cellular pathways that recognize either uncapped or unmethylated RNA [29] . Recently , we obtained a first view of the molecular architecture of the VSV L polymerase protein [30] . Single particle electron microscopy revealed that the capping machinery of L resides within 3 globular domains that are appended to a core ring-like RdRP domain . Moreover the architecture of L rearranges significantly following complex formation with the essential viral polymerase cofactor P , and this rearrangement likely positions the domains in the correct orientation to ensure the modification of the nascent mRNA chain [30] . In this study , we sought to determine at what stage a VSV mRNA acquires a cap structure . Since the PRNTase and the RdRP are localized within different regions of the L protein , a minimal length of RNA may serve as an important check point regulating the distinct activities . For decades RNA synthesis reactions have been carried out in vitro with VSV using either virus in which the membrane is disrupted with detergent [31] , or purified polymerase ( L and P ) and the N-RNA template [32] , [33] . From those reactions , the shortest transcripts that were identified as being capped were 37-nucleotides [34] . It was not clear , however , whether those transcripts were capped during synthesis or had at some level gained access to the PRNTase following release from the polymerase . Recent experiments have shown that short 5-nt transcripts corresponding to the beginning of a VSV mRNA can be capped in trans by L , but such experiments cannot address at what stage during transcription the RNA chain is modified . In the present study , we established a system to provide a “freeze-frame” view of VSV transcription using templates that lacked sites for UTP incorporation . By stalling transcription reactions at precisely defined chain lengths we identify a minimal length at which the transcript becomes capped . This work reveals the spatial arrangements of the capping activities of the VSV L protein in relation to the RNA dependent RNA polymerase domain during active transcription .
The promoter for VSV mRNA synthesis includes the 3′ leader region and the first 10-nt of the conserved gene-start element [35]–[38] . The sequences of those elements specify the incorporation of each of the four NTP's into the nascent RNA chain ( Figure 1A ) , creating a challenge in selecting a nucleotide that can control polymerase stalling . We elected to engineer the template such that it lacked sites for UTP incorporation . We chose UTP because of the known requirement for a high ATP concentration for initiation of RNA synthesis , and because previous work had revealed the essential nature of sites for CTP and GTP incorporation in the cis-acting signals in the mRNA start sequence [11] , [39] . To do this , we engineered the leader region and the gene-start element of a non-essential 60-nt gene that was inserted at the leader-N gene junction of an infectious cDNA clone of VSV ( Figure 1A ) . Specifically , we modified the leader sequence such that it lacked adenosine nucleotides except for those at position 48–50 which are typically not transcribed by the polymerase . We generated recombinant viruses in which those mutations were engineered within the leader region , purified the virus and confirmed that the mutations were present in the viral genome ( data not shown ) . To examine the effect of the leader mutations on viral transcription , we performed transcription reactions in vitro . Briefly , 10 µg of purified virus was incubated with detergent to disrupt the viral membrane and NaCl to liberate the M protein from the RNP core and permit transcription . The detergent-disrupted particles were incubated in transcription buffer containing ATP , CTP , UTP and [32P]-GTP and the products of transcription purified and analyzed by electrophoresis on acid-agarose gels ( Figure 1B ) . Although overall levels of transcription were reduced ( compare rVSV ( 60 ) and rVSV ( A- ) 60 ) , indicating that the U- leader promoter was less efficient than the wild type sequence , each of the viral mRNAs were synthesized . This result demonstrates that the sequence of the viral leader region could be engineered to eliminate sites of UTP incorporation which forms the basis of the templates to stall transcription within the downstream 60-nt transcriptional unit . The 60-nt gene sequence was next modified such that the first place the polymerase would encounter sites for UTP incorporation were at positions +11–13 , 21–23 , 31–33 , 41–43 , or 51–53 with respect to the first gene-start ( Figure 2A ) . Infectious recombinant VSV was recovered from each of those clones and while all the mutants grew less well than wild type VSV , there we no obvious differences in the plaque morphologies of the mutants ( Figure 2B ) . Sequencing of the genomic RNA confirmed that the first sites for UTP incorporation were at the desired location ( Figure 2C and data not shown ) . To determine whether transcription could be stalled at the inserted adenylates , we used purified recombinant viruses and performed RNA synthesis in the absence of UTP . Briefly , 10 µg of purified virus was disrupted with detergent , incubated in transcription buffer containing ATP , CTP and [32P]-GTP and the products of transcription purified and analyzed by electrophoresis on polyacrylamide gels . The VSV recombinants that were designed to stall transcription at positions +30 , 40 and 50 , generated RNA of the anticipated length , thus demonstrating that transcription can be specifically stalled by omission of UTP from the reactions ( Figure 2D ) . We did not observe significant quantities of a read-through transcript where polymerase incorporated another nucleotide in place of UTP to generate a full-length 60-nt transcript ( Figure 2D ) . This indicates that the polymerase error rate is insufficient to bypass three sites of UTP incorporation through mis-incorporation of an alternate nucleotide . Treatment of the RNA products with the cap cleaving enzyme tobacco acid pyrophosphatase ( TAP ) revealed that the stalled 30-nt transcript was insensitive to cleavage as evidenced by its unaltered mobility , whereas the 40-nt and 50-nt transcripts shifted in mobility by a single nucleotide following cleavage ( Figure 2D ) . This indicates that the 30-nt transcripts were uncapped , whereas the 40 and 50-nt long transcripts were capped . Using a 5′-3′ exonuclease ( +Exo ) that cleaves uncapped RNA , we further confirmed that the 40-nt and 50-nt transcripts were capped , since those RNAs were resistant to digestion , whereas the 30-nt transcript was sensitive ( Figure 2D ) . As expected , each of the recombinant viruses also produced a leader RNA as evidenced by the collection of transcripts around 47-nt ( Figure 2D ) . Leader RNA synthesized in vitro is known to contain at least 4 distinct 3′ termini which likely accounts for the multiple bands observed for the altered leader sequence [40] . Those leader transcripts lack an mRNA cap structure as shown by their insensitivity to cleavage by TAP , and their hydrolysis by the 5′-to 3′ exonuclease . Collectively , this analysis indicates that a VSV mRNA gains access to the mRNA capping machinery at an RNA chain length of >30-nt , but <40-nt . This experiment , however , could not determine whether the RNA was capped within a range of 30–40 nt or whether capping required a specific chain length . To more precisely define the point at which the RNA chain gains access to the capping machinery , we constructed a set of recombinant viruses designed to stall transcription at intermediate points between 30 and 40-nt ( Figure 3A ) . Specifically , we recovered viruses designed to stall transcription at 31–37 nts ( Figure 3B ) and examined the products made by those viruses following transcription reactions performed as above . As expected , each of those viruses generated the characteristic profile of leader RNAs around 47-nt , as well as specifically stalled transcripts ( Figure 4A ) . The mobility of the majority of the transcripts matched the anticipated position of stalling as shown by those RNAs that were 30 , 31 , 35 , 36 and 37-nt long ( Figure 4A ) . The mobility difference between the transcripts produced by the viruses designed to stall transcription at 30 vs . 31-nt appears to be 2-nt , indicating that the 31-nt transcript was fully capped . By contrast , stalling appeared relatively inefficient and somewhat heterogeneous for the viruses designed to stall transcription at positions +33 and +34 extending from the anticipated length ( indicated by the * ) to 1–2 nt larger . Stalling of transcription at +32 appeared inefficient with only low levels of the stalled transcript visible on the gel ( Figure 4A ) . An enhanced contrast view of the gel is provided to illustrate the low levels of stalled transcripts obtained with the virus designed to stall transcription at +32 ( Figure 4A , lower ) . Although we do not know the reason for this altered stalling efficiency it correlates precisely with the point immediately following mRNA cap addition . The ratio of leader to stalled transcript appears to vary for several of the recombinant viruses . We do not know the basis for this variation , but with the exception of the virus designed to stall transcription at +32 , each of the viruses generated sufficient stalled transcripts for us to determine the cap status . To definitively determine whether those transcripts contained an mRNA cap structure , we compared the mobility of the stalled transcripts before and after TAP digestion . As expected , the leader RNA , and the +30 RNA were insensitive to TAP cleavage indicating that they lack an mRNA cap structure ( Figure 4B ) . By contrast the transcripts stalled at +31 , +35 , +36 and +37 showed a clear single nucleotide shift in their mobility following TAP cleavage ( Figure 4B ) . Moreover , the collection of transcripts synthesized following stalling at positions +33 and +34 shifted in mobility indicating that they were capped ( Figure 4B ) . Collectively , these data indicate that a VSV mRNA gains access to the capping machinery at a chain length of 31-nt . The above experiments show that the transcript must be 31-nt long to be capped . To provide further support for this , we took advantage of our previously characterized cap defective polymerase ( L-H1227A ) [7] . As expected , the transcripts synthesized following stalling of transcription at +30 were uncapped ( Figure 5 ) , and identical products were generated by L-H1227A . Using wild type L , stalling of transcription at position +31 and above demonstrated that the transcripts were capped . Consistent with this , the cap defective polymerases generated stalled transcripts that were 1-nt shorter . This experiment confirms the identity of the uncapped stalled transcripts and further supports that a VSV mRNA gains access to the mRNA capping machinery at an RNA chain length of 31-nt . We also noted that the cap defective polymerase appears to stall more readily on the rVSV ( A- ) 32 template . This raises the possibility that the inability to efficiently stall RNA synthesis reflects a transition of the polymerase related to cap addition . Electron microscopic structural analyses of VSV L indicate that the capping and cap methylation activities reside within distinct globular domains that are connected to a ring-like domain containing the RdRP . The capping enzyme resides in a globular domain that is proximal to the RdRP , whereas the MTase located to a more distal globular domain . The position of the MTase domain showed a degree of variability suggesting a high degree of flexibility within L . Significant structural rearrangements occur in L , following complex formation with the essential cofactor P rendering a straightforward identification of the capping and MTase domains within the active polymerase complex difficult . We therefore sought to determine whether there was an additional length requirement for mRNA cap methylation . To do this , we performed transcription reactions on the engineered templates in the absence of UTP and the presence of [3H]-SAM . The resulting transcripts were purified , analyzed by electrophoresis on a 20% polyacrylamide gel and detected by fluorography ( Figure 6A ) . The 30-nt uncapped transcripts were not labeled by [3H]-SAM , demonstrating that lacked methyl groups at both ribose 2′-O and G-N-7 positions of the cap structure . By contrast the 31-nt and larger transcripts were labeled by [3H]-SAM . To evaluate whether a methyl group was present at both G-N-7 and 2′- O positions , the transcripts were exposed to TAP prior to gel electrophoresis . Following TAP cleavage , approximately 50% of the label remained associated with the transcript ( Figure 6B ) , consistent with the removal of the 7mGp structure . Because this reduction in intensity of labeling was apparent starting at the +31 transcript , the results suggest that there is no distinct length requirement for 2′-O and guanine-N-7 methylation . To quantify this effect further , we measured the amount of label associated with purified RNA before and after TAP cleavage by scintillation counting . To eliminate the released 7mGp we purified RNA that was longer than 20-nt prior to scintillation counting . This analysis demonstrates that 50% of the [3H]-SAM signal is lost upon TAP cleavage , and indicates that the RNA cap structures are guanine-N-7 and ribose 2′-O methylated . Collectively , these data indicate that the RNA chain gains access to the MTase domain at the same length as the PRNTase domain .
The evidence presented here shows that a VSV mRNA must be 31-nt long to gain access to the mRNA capping machinery . This conclusion comes from stalling transcription at precisely that point and evaluating whether the RNA contains an mRNA cap structure . In these experiments , however , the nascent RNA chain remains stalled at 31-nt and the relative time at which the cap is added is not certain . Thus while our experiments demonstrate that the RNA chain can gain access to the PRNTase once it reaches 31-nt , they cannot determine over what range during transcription cap addition actually occurs . This will be impacted , for example , by the rate at which the PRNTase catalyzes transfer of the RNA onto GDP , and indeed the formation of the GDP acceptor . Consequently , it seems likely that cap addition occurs over a range of nucleotide positions and that the earliest possible point of capping is position +31 . We and others have reported the existence of abortive uncapped transcripts that range in size up to several 100 nucleotides [7] , [11] , [12] . We anticipate , however , that since failure to cap leads to premature termination of mRNA synthesis , it seems likely that capping typically occurs within a short window of +31 . Consistent with this latter idea it is possible to generate a short capped and polyadenylated transcript from an artificial transcription unit of 60-nt inserted between the leader and N genes of VSV [41] . The presence of uncapped transcripts that range in size up to several 100 nucleotides would then simply reflect the variable termination of transcripts that failed to gain access to the capping apparatus within the optimal window for cap addition . Our study defines the lower limit of that optimal window as 31-nt during transcription . We do not precisely know the upper limit of the window for capping nascent RNA , but since we do not observe large quantities of an uncapped 60-nt mRNA transcript synthesized in vitro [41] it seems likely that the window is quite narrow . The finding that the transcript gains access to the methyltransferase active site at the same length , +31-nt , demonstrates that there is no further elongation requirement for cap methylation . An earlier study that examined the kinetics of mRNA cap methylation catalyzed by the VSV New Jersey polymerase provided evidence that cap methylation occurs over a nascent RNA chain length window that spanned several 100 nucleotides [42] . It seems likely , therefore , that during transcription the nascent RNA chain continues to grow while the RNA is being capped and methylated and that the precise point at which the chain gets methylated is not critical . Such a scenario therefore implies that the influence of methylation on mRNA polyadenylation , where failure to methylate can result in the production of large polyadenylate tails , is unlikely to be a coupling of the two reactions directly . Rather , the act of methylation itself in some way influences the extent of polymerase stuttering on the U7 tract present at the viral gene-end sequence and not by influencing the cessation of stuttering during the reiterative transcription process . The effects of several mutations in L that ablate mRNA cap methylation were previously examined for their ability to promote hyper polyadenylation . The majority of the methylation defective mutants synthesized mRNA with normal levels of polyadenylate [15] . The synthesis of large polyadenylate may instead be related to the affinity of the various L mutants for the inhibitor SAH , such that the hyperpolyadenylation phenotype may simply reflect increases in the Km for SAM binding rendering the polymerase mutants hypersensitive to levels of SAH . Although the present study defines 31-nt as a minimal length of mRNA cap addition , it remains possible that there are additional gene-position or sequence dependent effects that may alter the precise position of cap addition for specific genes . For example , the sequence of the transcript may influence the length at which the RNA gets capped simply by influencing the elongation properties of the polymerase , or possibly by favoring specific nascent RNA structures that alter access of the nascent transcript to the capping site . If such gene specific effects occur , they may serve as a means to regulate the amount of full-length transcripts made from specific genes , since failure to cap would lead to premature termination , down-regulating specific gene expression . We have not yet succeeded in extending the freeze-frame methodology to study sequential transcription from the viral genome , consequently , further experiments will be required to investigate this possibility . Previous experiments have shown that short exogenous synthetic RNA oligonucleotides ( 5–10 nts ) are of sufficient length to be capped when added to VSV L in trans . In the system used in the current study , where capping is evaluated co-transcriptionally , mRNAs shorter than 31 nts fail to be capped by the polymerase . These results indicate that while an exogenously added RNA is able to freely diffuse into the capping active site , the access of an endogenous nascent RNA to that site is regulated . Such a regulation is likely achieved through anchorage of the mRNA 3′ end by the RdRP domain preventing the 5′ end from accessing a physically distant capping active site . In combination with the recent electron microscopic structures , the present findings provide new insights into the structural and functional organization of the VSV polymerase . EM images of L alone showed the organization of L into a ring domain ( 90–100 Å ) containing the RNA polymerase and an appendage of three globular domains ( approximately 45 Å each ) containing the cap-forming activities [30] . The capping enzyme maps to a globular domain that is juxtaposed to the ring and the cap methyltransferase maps to one of two more distal and flexibly connected globules . Notably , the position of the globules relative to one another appears rather flexible such that the methylase domain can be positioned adjacent to the RdRP . This arrangement of the capping and methyltransferase domains is consistent with the results of this study: the 5′ end of the mRNA originating from the RdRP domain requires a minimal length to reach the capping apparatus . The length of 100 Å is approximately the distance spanned by 31 nts , and is consistent with the distance between the center of the ring and the proximal globular domain in the appendage . The fact that the same length is sufficient to gain access to the MTase is consistent with the high degree of flexibility exhibited by the distal globular domains . However , it should be emphasized that our EM studies showed that complex formation with P induces a significant conformational rearrangement of L including the loss of the globular features of the appendage ( Figure 7 ) . It is this L-P complex that is the active form of the polymerase for RNA synthesis . The L-P complex , however , retains some features of the L protein alone , including a ring-like domain that presumably includes the RdRP activity and an altered appendage that presumably includes the capping machinery ( Figure 7 ) . The work presented here supports the idea that in the rearranged appendage , the capping active site is approximately 100Å from the RdRP active site , and the MTase domain is sufficiently flexible to not require further elongation of the mRNA . Our findings also have implications for the path the RNA chain takes between the RdRP domain and the capping apparatus . Although , the length of the RNA would permit the transcript to traverse from the core ring domain to the PRNTase appended to the ring itself , as well as the flexible cap methylation apparatus of the same L molecule , we cannot exclude the possibility that the cap formation is catalyzed by an adjacent L molecule . Indeed , our electron microscopic examination of the L-P complex provided evidence for polymerase dimers , and genetic experiments with Sendai virus are consistent with the presence of at least a dimeric polymerase complex . It remains unknown , however , whether cap defective VSV polymerase molecules can complement RdRP defective VSV polymerase molecules . If indeed the RNA chain is capped by an adjacent polymerase molecule , such complementation should be achievable . Additionally , by incorporation of thio substituted nucleotides within the nascent RNA chain , it should now be possible to decipher the path that the RNA chain traverses in its transit from the RdRP domain to the PRNTase domain . During transcription , the capping activities of L are known from biochemical experiments to influence the polymerization activity . Specifically , failure to cap the mRNA leads to premature termination of transcription [7] , [11]–[13] , and inhibition of methylation of the mRNA cap can lead to hyperpolyadenylation [13]–[15] . We previously investigated the influence of sequence on premature termination by polymerases that are defective in cap addition [13] . Analogous to authentic termination , premature termination was favored at AU rich elements in the template and was significantly suppressed during copying of CG rich templates . This implies that the strength of a hybrid between the nascent strand and template can influence polymerase processivity . Although speculative , a relatively simple mechanism by which capping could influence termination is by regulating the stability of the hybrid between the nascent strand and template in the RdRP domain . Since the capping domain is juxtaposed to the ring , capping itself may induce a tightening of the grip of the polymerase stabilizing the hybrid between the template and nascent mRNA strand thus rendering the polymerase fully processive . The physical sequestration of the 5′ end of the nascent RNA strand within the PRNTase domain and then the cap methylation domain may itself also favor the RNA polymerization reaction becoming fully processive , so that the nascent RNA chain has elongated to the point at which the polymerase can only terminate in response to a highly specialized transcription stop sequence . The length requirement for mRNA 5′ modification is similar to that previously reported for vaccinia virus [43] , RNA polymerase II and the apparent length at which a reovirus transcript gains access to the capping enzyme [44] . In those other cases the capping enzyme is encoded by a separate polypeptide that appears associated with the RNA polymerase either physically in the case of the reovirus polymerase as a structural component of the virus capsid , or by recruitment to the phosphorylated C-terminal domain of RNA polymerase II [45] . For RNA polymerase II it has been shown that the nascent RNA chain is capped over a range of nucleotide positions , in this case the polymerase pauses prior to the act of mRNA cap addition , and the act of cap addition is linked to full polymerase elongation . We do not know whether a similar pausing event occurs during VSV transcription . The properties of the polymerase , however , do appear distinct immediately upon cap addition . This is illustrated by the fact that while we were able to efficiently stall the polymerase at positions +30 , +31 , +35 , +36 , +37 , +40 , +50 during transcription , stalling at positions +32 , +33 and +34 result in a ladder of products that range up to 1–2 nucleotides longer than anticipated . Since the cap was just added at position +31 , capping itself may render the polymerase somewhat resistant to stalling and result in the production of slightly longer transcripts . A second possible explanation for the production of stalled transcripts that vary in their precise point of termination relates to the intrinsic ability of the polymerase to stutter on homopolymeric tracts . The best characterized slippage sequence for VSV is that of the gene-end AUACUUUUUUUG in which the AUAC element regulates the extent of stuttering by L on the U7 tract [46] . We considered that the template sequence CCGUUUGUCUUU present at positions 25–36 of the unperturbed 60-nt gene may itself favor some slippage that is enhanced by the insertion of three adenylates at positions 32 , 33 , and 34 . Although the presence of G or C within a U tract blocks slippage during transit of polymerase across a gene-junction [46] , the general A/U rich nature of the present sequence may be sufficient to destabilize the elongating polymerase complex in vitro . Definitive assessment of the precise point of termination of the stalled transcripts will require sequencing of these small RNA products . The ability to stall transcription at specific places on the template , through the use of engineered recombinant templates now provides us an ability to study more precisely the steps of transcription . This methodology should be readily adaptable to probe the length of the nascent RNA chain that is protected by the polymerase during transcription and to determine whether the RNA chain becomes accessible prior to mRNA cap addition . Moreover , combined with our ability to image the L-P complex by electron microscopy , this approach may permit us to examine the polymerase bound to the RNA template at various stages of transcription . Our work also has implications for understanding transcription in other nonsegmented negative-strand RNA viruses in that they likely require a minimal length of transcription prior to cap addition . We anticipate that those lengths will vary for each virus , and as outlined above may perhaps show gene-specific variation . Since the production of triphoshphate RNA itself may serve as an activator of specific cytosolic sensors of the innate immune system , what appears to be a specific and efficient position of mRNA cap addition may also serve to decrease the production of pppRNA transcripts in infected cells . Moreover , since the act of capping serves as a positive regulator of transcriptional elongation this minimal length represents a key check-point in the transcription cycle .
Plasmid pVSV1 ( + ) 60 , containing an infectious cDNA clone of the VSV genome with a 60-nt long non-essential gene inserted at the leader-N gene junction , was generated as described previously [41] . The adenylates at positions 19 , 20 , 22 , 23 , 25 , 28 , 29 and 37-nt in the VSV genome were replaced using site-directed mutagenesis to generate an ( A- ) leader region ( Figure 1A ) . In order to introduce three adenylates at positions 11 , 21 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 41 and 51 in the 60-nt long non-essential gene site-directed mutagenesis was performed . The presence of the mutation was confirmed by sequence analysis . Recombinant VSV was rescued from cDNA by transfection of BHK-21 cells infected with a recombinant vaccinia virus ( vTF7-3 ) that expressed T7 RNA polymerase as described previously [47] , [48] . The generated viruses were designated rVSV ( A- ) -10 , -20 , -30 , -31 , -32 , -33 , -34 , -35 , -36 , -37 , -40 and -50 , respectively . Cell culture supernatants were collected at 48 to 96 h post transfection , and virus was amplified once in BHK-21 cells . Individual plaques were isolated on Vero cells , and large stocks were generated in BHK-21 cells and purified as described previously [12] . Viral titer was determined by plaque assay on Vero cells , and protein content was measured with the Bradford reagent ( Sigma Chemical Co . , St Louis , MO ) . The ( A- ) leader and 60-nt gene of the purified viruses were sequenced again , and these stocks were used for in vitro transcription reactions . Viral RNA was synthesized in vitro as described previously [31] , [38] . 10 µg of the purified recombinant virus was activated by incubation with detergent for 5 min at room temperature . RNA synthesis reactions were performed in the presence of nucleotide triphosphates ( 1 mM ATP and 0 . 5 mM each of CTP and GTP ) . The reaction mixtures were supplemented with 20 µµCi of [α-32P]-GTP ( 3 , 000 Ci mmol-1 ) ( Perkin-Elmer , Wellesley , MA ) . Total RNA was extracted , purified , and used for secondary manipulations as follows . Where indicated , the RNA cap structure was removed by tobacco acid pyrophosphatase ( TAP; Epicenter ) as previously described [6] . For exonuclease digestion , total RNA was dephosphorylated using Antarctic phosphatase ( New England Biolabs [NEB] ) , and a single phosphate was added using T4 polynucleotide kinase ( NEB ) according to the manufacturer's instructions . The resulting RNAs were subsequently treated with Terminator exonuclease ( Epicenter ) according to the manufacturer's instructions [12] . The products of RNA synthesis were analyzed on a 6% polyacrylamide gel and visualized by a phosphorimager ( GE Healthcare; Typhoon ) . The N-RNA template was purified from rVSV ( A- ) -30 , -31 , -32 and-33 as described previously [6] . Briefly , 4 mg purified virus was disrupted on ice for 1 h in 20 mM Tris-HCl ( pH 8 . 0 ) , 0 . 1% Triton X-100 , 5% glycerol , 5 mM EDTA , 3 . 5 mM dithioerythritol , 20% dimethyl sulfoxide , and 1 . 0 M LiCl . The template was recovered by centrifugation ( 190 , 000× g , 3 . 5 h ) through a step gradient of 0 . 25 ml each of 40 , 45 , and 50% glycerol in TED buffer ( 20 mM Tris-Cl [pH 8 . 0] , 1 mM EDTA , 2 mM dithioerythritol ) supplemented with 0 . 1 M NaCl . The pellet was resuspended in 0 . 3 ml of TED buffer plus 10% glycerol and disrupted on ice , except that the Triton X-100 and EDTA concentrations were reduced to 0 . 05% and 1 mM , respectively . The N RNA was isolated by banding in a 3 . 6-ml 20 to 40% ( wt/wt ) CsCl gradient ( 150 , 000× g , 2 . 5 h ) , recovered by side puncture and diluted fourfold with 10 mM Tris-Cl ( pH 8 . 0 ) , 0 . 1 mM EDTA . The N-RNA was recovered following centrifugation ( 150 , 000× g , 1 . 5 h ) through a 0 . 5-ml cushion ( 50% glycerol , TED buffer , 0 . 1 M NaCl ) . Recombinant L was expressed from recombinant baculoviruses in Spodoptera frugiperda 21 cells , and P was expressed in BL21 ( DE3 ) as described previously [6] . At 72 h postinfection , the cells were collected , washed twice with ice-cold phosphate-buffered saline , and recovered by centrifugation . The cells were suspended in lysis buffer ( 50 mM NaH2PO4 , 10% glycerol , 0 . 2% NP-40 , 300 mM NaCl , 10 mM imidazole [pH 8 . 0] ) supplemented with EDTA-free protease inhibitor cocktail ( Roche ) and 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication . The L and P proteins were purified by Ni-nitrilotriacetic acid–agarose ( Qiagen ) , followed by ion-exchange chromatography as described previously [6] . Reactions were carried out in the absence of rabbit reticulocyte lysate using 5 µg of N-RNA template , 4 µg of purified L , 2 µg of purified P and nucleoside triphosphates ( 1 mM ATP and 0 . 5 mM each of CTP and GTP ) as described previously [6] . The reaction mixtures were supplemented with 20 µCi of [α-32P]GTP ( 3 , 000 Ci mmol−1 ) ( Perkin-Elmer , Wellesley , MA ) . After 5 h of incubation at 30°C , the RNA was purified by phenol and chloroform extraction and analyzed on 6% polyacrylamide gel and visualized by a phosphorimager ( GE Healthcare; Typhoon ) . For methylation of the transcripts , the same reaction condition was used as described above with the exception that 20 µM 3H-labeled S-adenosyl-L-methionine ( 3H-SAM ) ( 76 Ci/mmol , Perkin-Elmer , Wellesley , MA ) was used for labeling the RNA instead of [α-32P]GTP . After 5 h of incubation at 30°C , the RNA was purified by phenol and chloroform extraction and analyzed on 20% polyacrylamide gel followed by autoradiography .
|
Using a prototype of the nonsegmented negative strand RNA viruses , vesicular stomatitis virus , we probed the spatial relationship between the RNA dependent RNA polymerase and 5′ mRNA capping and methylation activities of the large polymerase protein . Because the 5′ mRNA processing reactions dramatically impact the nucleotide polymerization activity of the protein , they may function as a quality control step in viral transcription . We developed a means to stall transcription at precisely defined locations following initiation and analyzed the cap status of the stalled transcripts . We show that 30-nt transcripts are uncapped whereas those that are 31-nt long gain are capped and methylated at both guanine-N-7 and ribose-2′-O positions . Combined with our recent work that determined the molecular architecture of the VSV polymerase , this work reveals the spatial relationship within a functional polymerase complex of the polymerase domain and the 5′ mRNA processing domains of the L protein .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"molecular",
"cell",
"biology",
"rna",
"processing",
"virology",
"gene",
"expression",
"viral",
"enzymes",
"biology",
"microbiology",
"viral",
"replication",
"viral",
"replication",
"complex",
"molecular",
"biology"
] |
2011
|
A Freeze Frame View of Vesicular Stomatitis Virus Transcription Defines a Minimal Length of RNA for 5′ Processing
|
The shoot apical meristem ( SAM ) maintains a pool of indeterminate cells within the SAM proper , while lateral organs are initiated from the SAM periphery . Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development . Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf ( P0/P1 ) or in the SAM proper verified the precision of the microdissections . Genes involved in cell division/growth , cell wall biosynthesis , chromatin remodeling , RNA binding , and translation are especially upregulated in initiating leaves , whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper . In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1 . P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation . Reverse genetic analyses of asceapen1 ( asc1 ) , a maize D4-cyclin gene upregulated in the P0/P1 , revealed novel leaf phenotypes , less genetic redundancy , and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis . These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize .
The maize shoot apical meristem ( SAM ) is a complex signaling network of distinct structural and functional domains that performs two essential developmental functions during plant shoot development: ( 1 ) self-maintenance and ( 2 ) organogenesis . Responsible for the development of all above ground organs in the plant , the SAM must maintain a precise equilibrium during which cells lost to newly-initiated leaves are replenished to maintain the SAM proper . Comprised of two tissue layers , the single-celled tunica ( L1 ) and a multilayered corpus ( L2 ) , the maize SAM displays histological zonation that is correlated with its functions ( Figure 1A ) . Determinate lateral organs arise from the peripheral zone ( PZ ) whereas the central zone ( CZ ) is comprised of more slowly dividing meristem initial cells that replenish the SAM . Although Caspar Wolff first recognized the SAM as the organogenic center of the plant shoot almost 250 years ago [1] , detailed mechanisms of SAM function remain a fundamental question in plant biology . Molecular genetic analyses have identified a growing number of genes contributing to the complexity of SAM function in maize . The homeobox gene knotted1 ( kn1 ) is required for meristem indeterminacy; null kn1 mutants fail to maintain the SAM [2] , [3] . Down-regulation of KN1 accumulation in the PZ precedes lateral organ initiation , and is correlated with auxin transport and expression of the knotted1-homeobox ( KNOX ) regulator rough sheath2 ( rs2 ) in the PZ [4]–[7] . SAM size is also controlled by the cytokinin-inducible RESPONSE REGULATOR ABPHYL1 , in which mutations increase SAM size and lead to disrupted phyllotaxy in the maize shoot [8] , [9] . Maize leaves are formed via recruitment of ∼200 leaf founder cells from the PZ of the SAM [10] , followed by differentiation along three developmental axes ( proximo-distal/medio-lateral/adaxial-abaxial ) . Genetic analyses have identified several maize genes involved in these SAM functions , including those required for leaf initiation and phyllotaxy ( terminal ear1 and aberrant phyllotaxy1 ) , proximodistal patterning ( rs2 and semaphore1 ) , mediolateral development ( narrow sheath1&2 , ragged seedling2 , wavy auricle in blade1 ) , and adaxial-abaxial patterning ( rolled1 , miR166 , leafbladeless1 , milkweed pod1 ) [11] , [8] , [12]–[19] . Elucidation of the regulatory networks that coordinate these intersecting developmental functions will be bolstered by the use of genomic approaches to generate testable models for the SAM interactome , followed by comprehensive genetic and biochemical analyses to test and extend these hypotheses . The complementary expression domains of the molecular markers rs2 and kn1 clearly illustrate that indeterminate cells of the SAM proper are immediately juxtaposed to leaf founder cells within the maize shoot apex ( Figure 1B ) . The close proximity of these distinct functional domains presents technical barriers to comparative analyses of these discrete SAM functions . However recent technical advances have enabled a genomics approach toward the molecular dissection of SAM function . The relatively large size of the maize SAM , 50–250 founder-cells are recruited into the incipient leaf versus 25–30 in Arabidopsis [10] , [20] , renders the maize plant especially tractable to laser-microdissection technologies . Laser-microdissection permits the precise isolation of specific tissues , organs , or cells from fixed and sectioned plant tissues adhered to microscope slides [21] . Nanogram quantities of RNA extracted from less than 1 mm2 of microdissected tissue ( comprising five to ten whole SAMs ) can be linearly amplified using T7 RNA polymerase to generate microgram quantities of RNA sufficient for transcriptional profiling using microarray technology [22]–[26] . Owing to its unique ability to sample discrete microdomains in plant tissues , laser-microdissection eliminates the transcriptional noise contributed by adjacent or contaminating unrelated tissues and thereby enables transcriptional profiling that is focused on the cells and tissues of interest . Laser microdissection-microarray technology was utilized in comparative transcriptional analyses of functional domains in the maize SAM . Gene expression within SAM microdomains encompassing the initiating maize leaf ( P0/P1 ) and the stem cells of the SAM-proper was analyzed; 962 maize genes were differentially expressed in this comparison . Control genes of known expression domain confirmed the accuracy of the laser microdissections and validate the dataset . Genes predicted to function during cell division/growth , chromatin remodeling , RNA-binding , cell wall biosynthesis and translation are especially upregulated in initiating leaves , whereas genes involved in protein fate and DNA repair are prevalently expressed within the SAM-proper . In situ hybridization analyses , and qRT-PCR analyses of apices that are arrested in leaf initiation identified twelve maize genes predicted to function during leaf initiation . Reverse genetic analyses of the maize D-cyclin gene asceapen1 ( asc1 ) confirmed its predicted function during maize leaf and shoot development; novel mutant phenotypes revealed differing levels of genetic redundancy and divergent patterns of subfunctionalization among cyclin paralogues in maize and Arabidopsis . Our data provide a unique database that provides insight into SAM function and a useful platform for reverse genetic and biochemical analyses of maize shoot development .
Maize seedlings were grown under controlled conditions and processed for laser microdissection of SAM domains ( see Materials and Methods ) . Although KNOX immunohistolocalization analyses clearly delineate the leaf/non-leaf boundary in the maize shoot apex ( Figure 1B ) , these treatments require crosslinking fixatives that preclude the extraction of RNA from microdissected tissues . Therefore , in lieu of molecular makers , two distinct SAM domains were captured using morphological/anatomical cues ( Figure 1 C–D ) . The “SAM-proper” comprised the apical crown and central stem of the shoot apex , and is estimated to include the CZ . Tissue extracted from the “P0/P1” domain included the PZ and the newest-initiated lateral organ that formed a protruding buttress on the SAM flank . Care was taken to avoid the SAM peripheral zone during captures of the SAM-proper domain; likewise the P0/P1 samples were harvested from a depth of no more than three cell-layers in order to avoid tissues that typically accumulate KNOX proteins ( Figure 1B ) , markers of meristematic identity [2] . Tissues derived from ten total SAMs were pooled into domain-specific samples comprising a single biological replicate . Following RNA extraction and amplification ( see Materials and Methods ) , six such biological replicates were utilized in microarray hybridizations to 29 , 600 total elements ( including approximately 23 , 000 unique maize genes ) contained on the customized maize cDNA microarrays SAM1 . 1 and SAM3 . 0 [described in 25] , [26] . Replete with genes identified from meristematic tissues , SAM 1 . 1 contains over 7 , 500 cDNAs derived from maize inflorescences and SAM 3 . 0 contains over 10 , 500 cDNAs derived from vegetative apices ( i . e . SAM plus four leaf primordia ) . For each array platform , three of the six cDNA pairs were labeled with Cy3 from the SAM-proper and Cy5 for the P0/P1 . Dye assignments were reversed for the other three replications . Normalized Cy5 and Cy3 signals were used to test for evidence of differential expression among the SAM domains using a linear model analysis for each gene ( see Materials and Methods ) . A total of 1 , 312 array elements were differentially expressed in these SAM domains utilizing cut-off parameters of P-value<2 . 93 E-4 and fold change>2 . 0 . Alignment of these 1 , 312 cDNA sequences to predicted genes within the sequenced maize genome ( see Materials and Methods ) identified 962 maize gene contigs ( MGC ) that were differentially expressed in the SAM-proper and P0/P1 leaf primordia ( Table S1 ) . These included 542 genes upregulated in the P0/P1 leaf and 420 genes upregulated in the SAM-proper ( Figure 2 ) . Notably , 48 ( i . e . 3 . 6% ) of the 1 , 312 differentially expressed array elements did not align to any sequenced MGC; the EST accession numbers of these unaligned genes are thus listed among the 962 genes contained in Table S1 . None of these unaligned EST sequences are predicted to comprise repetitive retrotransposons , but presumably correspond to a portion of the maize gene space that is as yet unrepresented in the sequenced portion of maize genomic DNA . Approximately 31 . 8% of the cDNA array dataset aligned with equal affinity to multiple MGCs , and are likely to comprise gene family members for which the available cDNA sequence does not distinguish between close paralogs ( Table S1 ) . The estimated false discovery rates were 1 . 1% for SAM1 . 1 and 0 . 5% for SAM3 . 0 . Bioinformatic predictions of function were performed for all differentially expressed genes as described [27] , and are presented at the SAM-The Maize Shoot Apical Meristem Project database created during this project ( http://sam . truman . edu/geneva/geneva . cgi ) . A total of eighteen different Gene Ontology ( GO ) functional categories are identified as detailed below ( Figure 2 ) , including: DNA repair; photosynthesis related; RNAi; transposable element; other; cytoskeletal; extracellular matrix/cell wall; signal transduction; cell division/growth; protein fate; RNA binding; stress related/defense; vesicle trafficking/transport; transcription; chromatin; metabolism; translation; and unknown . Control genes whose expression in either the SAM-proper or the P0/P1 is described previously attested to the precision and accuracy of the SAM domain microdissections ( Figure 1D; Table 1 and references therein ) . For example , the meristem maintenance gene knotted11 ( kn1; [2] , see Dataset S1 for a list of the MGC accession numbers corresponding to superscripted numerals in this text ) , the phyllotaxy regulator terminal ear12 ( te1; [11] ) , the trans-acting siRNA ( tasiRNA ) biogenesis gene leafbladeless13 ( lbl1; [28] ) and the maize homolog of the sterol biosynthetic gene fackel4 [29] were all identified in our microarrays as up-regulated in the SAM-proper , in agreement with published expression analyses . Likewise , the knox-regulatory gene rough sheath25 ( rs2; [6] , [7] ) , a maize homolog of growth-regulating factor16 ( grf1; [30] ) , a maize auxin response factor5/monoteros17 ( arf5/monopteros; [31] ) , and several members of the yabby gene family of transcription factors ( yab158 , yab109; Zm-drooping leaf-like10; [16] , [26] ) were all up-regulated in the P0/P1 domain , as predicted from previous studies . Control genes also up-regulated in the initiating leaf included maize orthologues of the auxin transporters pinformed111 ( pin1 ) and auxin insensitive112 ( aux1 ) , as well as the cell wall-loosening gene beta expansin813 ( expb8 ) , all of which are known to be expressed during leaf initiation in maize and/or Arabidopsis [32]–[35] . Microarray analyses of the SAM-proper and P0/P1 apical domains reveal discrete GO functional categories of preferentially expressed genes ( Figure 2; Table S1 ) . For example , significantly more ( P<0 . 036 ) genes involved in protein fate/ubiquination were found to be upregulated in the SAM-proper ( 42 ) as compared to the P0/P1 ( 24 ) , including multiple paralogues encoding a predicted E2 UBIQUITIN CONJUGATING ENZYME-LIKE14 . Also , three-fold more DNA-repair genes were upregulated in the SAM-proper than in the P0/P1 , including maize orthologues of rad2315 , radA16 , mus117 and the SNF2 domain/helicase protein18 . Genes comprising five predicted functional categories were significantly upregulated in the P0/P1 , including those functioning in the extracellular matrix/cell wall ( P<6 . 10E-05 ) , cell division/growth ( P<0 . 005 ) , RNA binding ( P<0 . 009 ) , chromatin ( P<2 . 64E-04 ) , and translation ( P<4 . 76E-07 ) . Fifteen genes involved in cell wall biology were upregulated in the P0/P1 , whereas none were upregulated in the SAM-proper . These included genes encoding cell wall GLYCOPROTEINs19 and GLYCOSYLASES20 , an ALPHA-EXPANSIN21 , a BETA-EXPANSIN13 , and a CELL WALL-ANCHORED PROTEIN22 . Differentially expressed genes encoding proteins involved in cell division and growth in the P0/P1 outnumbered those identified in the SAM-proper twenty-three to seven ( Figure 2 ) . Reflecting the increased mitotic activity found in the peripheral zone and initiating leaf as compared to the SAM central zone , these included genes encoding various CYCLINs23–27 and a putative maize homolog of mammalian growth regulating factor16 . Also identified are at least four maize paralogs of the TRANSLATIONALLY-CONTROLLED TUMOR PROTEIN ( TCTP28–31 ) , guanine exchange factors that control organ size in Drosophila and mammals by regulating a specific dRheb-GTPase within the target of rapamycin ( TOR ) signaling pathway [36] . Recent analyses of a TCTP gene in Arabidopsis revealed increased expression in rapidly growing tissues; reverse genetic mutant plants exhibited a range of developmental defects including reduced cell size and leaf expansion , and decreased sensitivity to auxin [37] . Three distinct argonaute1-like maize paralogues32–34 were identified in our microarray data , all of which were upregulated in the P0/P1 ( Table S1 ) . Arabidopsis contains two close paralogues , argonaute1 ( ago1 ) and pinhead1/zwille1 ( pnh1/zll1 ) , which encode components of the multi-subunit RNA-induced silencing complex ( RISC; [38] ) . In keeping with their partially overlapping roles in the miRNA-regulated control of leaf polarity and of SAM maintenance , ago1 is evenly expressed throughout the Arabidopsis SAM and young leaf primordia [39]–[42] , whereas pnh1/zll1 transcripts accumulate preferentially in leaf primordia and in the vasculature [43]–[45] . Owing to the nearly identical amino acid sequences of AGO1 and PNH1 , it is not possible to predict which of maize ago-like genes are ago1 orthologues and which are pnh1/zll1 orthologues . However , in situ hybridization analysis of a maize ago1-like gene that was upregulated more than seven-fold in the P0/P1 confirmed our microarray data , and revealed a pnh1/zll-like expression pattern ( Figure 3A ) . Although transcripts are indeed detected in the SAM crown and center , more abundant transcript accumulation is observed in the leaf founder cells , the SAM periphery , and in young leaf primordia ( Figure 3A ) . In contrast to the miRNA regulatory ago1 genes identified in the P0/P1 , the tasi-RNA gene lbl13 [28] and the siRNA effector protein gene argonaute435 ( ago4; [46] , [47] ) were upregulated in the SAM-proper . Moreover , significantly more RNA-binding genes were preferentially expressed in initiating maize leaves compared to the SAM-proper , including four genes predicted to encode RIBONUCLEOPROTEINs36–39 , numerous GLYCINE-RICH RNA-BINDING PROTEIN40–44 paralogs , and a maize homolog of the Arabidopsis flowering-time regulator flowering locus K45 gene ( flk; [48]; Table S1 ) . A preponderance of gene elements predicted to function during chromatin structure and remodeling were upregulated in the P0/P1 versus the SAM-proper ( 45 versus 16 , respectively; Figure 2 ) . For example , a cytosine-5-methyltransferases46 and three methyl-CpG DNA binding domain47–49 genes are specifically upregulated in the P0/P1 . Likewise , whereas hdt3–like50 and sir2-like51 histone deacetylase gene are upregulated in the leaf initials , a hdt2-like histone deacetylase52 is highly expressed in the SAM-proper and three swib-domain56–58 gene paralogs are detected only in the SAM-proper . Although the number of putative transcription factors ( TFs ) preferentially expressed in either the SAM-proper or the P0/P1 is exactly equal at thirty-three each ( Figure 2 ) , each SAM domain exhibited upregulation of various distinct TF genes not identified in the other . For example , the founding member of the knotted-like homeobox ( knox ) gene family kn11 is differentially expressed in the SAM-proper , while the related knox gene gnarley159 ( gn1 ) is upregulated in the P0/P1 . Although a previous report detected gn1 expression in the shoot apex [49] , the SAM domain specificity of gn1 expression was not described previously . Likewise , a maize homologue of the Arabidopsis leunig co-repressor60 [50] gene is identified in the SAM-proper , as were three paralogs encoding B3-domain61–63 TFs . Developmental regulators of embryo and meristem development , many B3-domain TFs are shown to function via interaction with auxin or ABA signaling pathways [51] , [52 , and references therein] . In contrast , rs25 , auxin response factor264 ( arf2 ) , and multiple members of the yabby8–10 gene family were identified in the P0/P1 . RS2 represses knox gene expression in developing leaves [6] , [7] , whereas arf2 accumulates in Arabidopsis lateral organs [53] and maize yabby genes are transcribed in the P0 and leaf primordia ( Figure 3B; [16] , [17] , [26] . Lastly , the largest single gene category identified in our microarray analyses comprised genes of unknown predicted function , which contained 142 genes upregulated in the SAM-proper and 133 genes in the P0/P1 ( Figure 2 ) . Distinct gene paralogs of the histone-methylating SET DOMAIN-encoding gene53–55 family are upregulated in the immediately adjacent apical domains that comprise the P0/P1 and SAM-proper . In addition , paralogs of six other maize gene families including auxin response factor165 , 66 ( arf1 ) , histone367 , 68 , histone469 , 70 , ubiquitin-conjugating enzyme E271 , 72 , ADP-ribosylation factor/Secretion-associated and Ras-related73 , 74 protein ( ARF/SAR ) , and the ubiquitin-ligase subunit gene S-phase kinase-associated protein175 , 76 ( skp1 ) exhibit preferential expression within the SAM-proper and the P0/P1 , and thus provide intriguing evidence for subfunctionalization of these gene families within discrete functional zones of the maize SAM . Focusing on previously uncharacterized maize genes implicated during leaf development , six genes upregulated in the P0/P1were subjected to in situ hybridization analyses in order to verify the domain-specific transcript accumulation predicted from our microarray data and identify novel patterns of gene expression . In addition to the maize ago1-like gene described in Figure 3A , five genes whose functions are yet to be demonstrated in maize were analyzed . These included a putative oligopeptide transporter77 , a yabby gene drooping leaf110 , a predicted growth-regulating factor6 , a lipid-transfer protein78 , and a D4-class cyclin25 . In all cases the pattern of transcript accumulation observed in the in situ hybridizations correlated with the microarray data . Stronger signals were observed in the SAM periphery , P0 , and small leaf primordia as compared to the SAM crown and center ( Figure 3 ) , which verified the P0/P1 upregulated expression observed in our microarray analyses . Auxin transport is the earliest-demonstrated prerequisite to KNOX downregulation and leaf initiation from the SAM flank; disruption of auxin transport by the chemical inhibitor N-1-naphthylphthalamic acid ( NPA ) leads to the arrest of lateral organogenesis in plant shoots [54] , [4] , [5] . Therefore , NPA-induced inhibition of leaf initiation provides a compelling experimental system with which to monitor SAM gene expression during very early events in leaf development . Toward this end , 14-day-after-germination seedling shoot apices were dissected to remove all organs except the SAM and the six youngest leaf primordia and placed in tissue culture with or without 30 mM NPA as described [4] . As shown in Figure 4A–B , NPA-cultured SAMs became greatly elongated but failed to initiate any new leaves , whereas equivalent sibling apices generated 6–7 new leaf primordia in NPA-free culture . After 14 days in culture , samples were processed for SAM laser-microdissection mediated qRT-PCR analyses as described [55] . Genes found to be upregulated during leaf initiation but down-regulated during NPA-induced arrest of organogenesis are especially implicated to function during early stages of maize leaf development . Transcript accumulation analyses were performed for nine genes that were significantly upregulated in the P0/P1 in our microarray analyses ( Table S1 ) . As shown in Figure 4D , qRT-PCR analyses revealed that transcripts of six of these nine genes were also down-regulated in leaf-arrested apices , including a second maize ago1-like32 gene , a putative brassinosteroid response factor79 gene , an E2 ubiquitin-conjugating-like71 paralog , the aux112 auxin transporter gene , a yabby 158 gene , and the growth-regulating factor16 . One gene ( a putative seven-in-absentia-like ubiquitin ligase80 ) was weakly down-regulated in NPA-treated apices , whereas two genes ( a tctp-like31 gene paralog and a maize AMP-dependent synthetase81 ) were unchanged in NPA-treated versus untreated shoots . Thus , six genes identified as upregulated in the P0/P1 are downregulated in shoot apices that are arrested in leaf initiation . We speculate that the three genes whose expression levels were unchanged following NPA treatment may mark a domain within the PZ that functions upstream or independent of auxin transport during leaf initiation , since accumulation of some PZ markers has been shown to persist in Arabidopsis pin1 mutants and in tomato apices treated with NPA [54] . Alternatively , these NPA-unaffected genes may not be preferentially expressed during early leaf initiation . The differential gene expression data presented in this study identify genes implicated in SAM domain-specific functions during maize shoot development . Validation of these predicted functions , however , requires biochemical or genetic analyses . A reverse genetic strategy was implemented ( see Materials and Methods ) to investigate the function of a D4-class cyclin25 gene that was identified as upregulated in the leaf primordia , which we have named asceapen1 ( asc1 ) . In situ hybridization of seedling shoot apices verified the P0/P1 upregulated asc1 expression observed in the microarray analyses ( Figure 3E ) . The asc1 gene contains six exons ( Figure 5A ) and is located at position 39 , 743–41 , 597 of contig 45 on maize chromosome 7 . The 1068 bp open reading frame is predicted to encode a protein of 355 amino acids , which contains the canonical LxCxEx RETINOBLASTOMA-interaction domain that is characteristic of D-CYCLINS ( Figure 5B;Wang et al . , 2004 ) . In addition , ASC1 contains the conserved amino cyclin box and a CYCLIN recognition motif . Limited expression profiles are described for four related maize D-cyclins ( including a D2-cyclin , a D4-cyclin , and two D5-cyclins [56] , although expression within the vegetative SAM was not examined . No genetic analyses of D-CYCLIN function have been performed previously in maize . D-CYCLINS perform an evolutionarily conserved growth-regulatory function to regulate progression through the G1 phase of the cell cycle [57] . Genetic analyses in Arabidopsis suggest that D-CYCLINS function as important regulators of asymmetric cell division , a process that is critical to developmental differentiation and has played a pivotal role in the evolution of multicellularity [reviewed in 58] , [59] . Arabidopsis has 10 CYCD genes comprised of six subgroups ( CYCD1 , CYCD2 , CYCD4 ( 2 genes ) , CYCD3 ( 3 genes ) , CYCD5 , CYCD6 , and CYCD7 [60] . Overexpression analyses suggest that as a group , D-CYCLINS may regulate the developmental progression from cell proliferation to differentiation [61] , [62] , [63] . Genetic analyses reveal redundant functions for the three CYCD3 genes in Arabidopsis [64] . Single CYCD3 mutations yield non-mutant phenotypes; triple mutations condition small yet fertile plants with narrow leaves , a small SAM , and decreased cytokinin response . CYCD4;1 is expressed in both shoot and root apices , although CYCD4;2 is not detected in the SAM . Single mutations in CYCD4;1 and CYCD4;2 render no macrophenotype , although reduced numbers of anatomically normal stomata develop in mutant hypocotyls [65] . A phylogenetic analysis was performed on the maize ASC1 protein and thirteen additional plant D-CYCLINS for which transcriptional analyses and/or genetic analyses are documented [66] , [56] , [67] , including four additional maize D-CYCLINS , ten Arabidopsis D-CYCLINs , and three D-CYCLINs from Antirrhinum majus . Utilizing the D1-CYCLIN from the moss Physcomitrella patens as an outgroup . ASC1 was placed on a well-supported clade together with the D4-CYCLINS and D2-CYCLINS from Arabidopsis ( Figure 5C ) . All the other D-CYCLIN proteins were placed on separate clades; the D3-CYCLINS from Arabidopsis and Antirrhinum comprise a well-supported separate clade from ASC1 . Reverse genetic analyses of asc1 were instigated in order to investigate the function of this D4-CYCLIN in maize . F2 seedlings were obtained from self-pollination of over 3 , 000 maize plants with Mutator ( Mu ) transposon activity , a maize transposon with an unusually high forward mutation rate [68] . A PCR-based reverse genetic strategy similar to previously published protocols ( [69] , [70]; see Materials and Methods ) identified two independently-segregating Mu-insertion alleles of the asc1 gene , designated asc1-M1 and asc1-M2 ( Figure 5 ) . The asc1-M1 allele harbors a Mu4 insertion in position 47 of the 129 bp intron 1 , whereas the predicted null asc1-M2 allele harbors a Mu1 at position 31 of the 87 bp second exon ( Figure 5A ) . RNA gel-blot hybridization analyses reveal that asc1 transcript accumulation is greatly diminished in asc1-M1 homozygotes and is virtually absent in asc1-M2 homozygous plants , relative to non-mutant siblings ( Figure S1 ) . F2 progeny of plants heterozygous for asc1-M1 or asc1-M2 each segregate for short , infertile plants with very narrow leaves ( Figure 6A–B ) . These mutant phenotypes co-segregate with homozygosity for asc1 mutations , and interallelic crosses of plants heterozygous for asc1-M1 and asc1-M2 fail to complement ( Figure 6N ) . No female inflorescences ( ears ) are observed in homozygous asc1 mutant plants , and male inflorescences form only rudimentary tassels with sterile branches and no floral morphogenesis ( Figure 6B–D ) . Histological examinations of asc1 mutant seedlings reveal extremely narrow leaves and small vascular bundles with reduced numbers of xylem and phloem vessels , as well as reduced SAM size ( Figure 6E–J ) . Both mutant alleles conditioned equivalent phenotypes , although the range of phenotypes is more severe in plants homozygous for the exon-insertion allele asc1-M2 . Especially striking are the effects of asc1 mutations on stomatal patterning and anatomy . Comprised of two subsidiary cells that surround and appress two smaller guard cells , interspaced stomatal complexes are formed via a series of ordered , asymmetric cell divisions in the leaf epidermis ( Figure 6K; reviewed in [71] ) . Analyses of the asc1 mutant leaf epidermis reveal irregular stomatal patterning ( Figure 6L–N ) . Two mutant stomatal complexes often form immediately adjacent to one another , a pattern not observed in non-mutant leaves . Other abnormalities include enlarged , distorted , and supernumery subsidiary cells and guard cells , which often develop immediately adjacent to completely normal stomatal complexes .
Novel transcriptomic comparison of the functionally distinct microdomains within the maize SAM are presented , an analysis that was enabled by the relatively large size ( ∼120 µm ) of the maize SAM as compared to Arabidopsis . The differential expression of 275 unknown genes within a particular SAM domain thereby provides a first suggestion of their potential function . Moreover , the documentation of seven instances wherein gene paralogues exhibited subfunctionalized preferential expression within distinct SAM microdomains provides insight into the evolution of specific gene families in maize . The entirety of this unique expression database is publicly available ( http://sam . truman . edu/geneva/geneva . cgi ) and represents a starting point for subsequent reverse genetic analyses of SAM function in maize . In addition , these genomic analyses are likely to uncover genes whose functions that are not amenable to traditional genetic analyses , owing to the embryo/seedling lethality that may result from mutations in genes required for early events in SAM ontogeny and/or leaf initiation . Genes whose SAM domain-specific expression are previously described in maize or Arabidopsis ( Table 1 ) served as experimental controls for the analyses presented here , and attest to the power and precision of laser-microdissection for analysis of transcript accumulation within plant microdomains . Three distinct paralogs of ago132–34 are identified , each of which was upregulated in the P0/P1 . Arabidopsis contains two ago1-like genes , one of which ( pnh1/zll1 ) is preferentially expressed in leaf primordia whereas the other ( ago1 ) is evenly expressed throughout the SAM and young leaves [39]–[45] . Although the extreme amino acid conservation observed in PNH1 and AGO1 precludes the identification of specific maize orthologues from homology alone , in situ hybridization analysis verified the leaf-preferential expression of one maize ago gene ( Figure 3A ) whereas a separate ago1 paralog was downregulated in NPA-treated apices that are arrested in leaf initiation ( Figure 4D ) . Our results are analogous to the reported leaf-upregulated expression of the rice pinhead/zwille orthologue OsPNH1 [72] . We speculate that maize co-orthologs whose expression domains mirror that of the Arabidopsis ago1 gene would not be identified in our microarray analyses , owing to the relatively equivalent transcript accumulation in the SAM-proper and P0/P1domains . In support of this hypothesis , the SAM1 . 1 and SAM3 . 0 gene chips contain additional ago1 co-orthologs that were not detected as differentially expressed in this analysis . It appears likely that as in Arabidopsis , the maize ago1 gene family has expanded and paralogs became subfunctionalized to perform specialized tasks during leaf development and/or shoot meristem maintenance . Plant miRNAs are described that function in the ARGONAUTE1-directed regulation of leaf initiation and polarity , including miR166 , and miR156 ( reviewed in [73] , [74] ) . Although these regulatory RNAs and/or their mRNA precursors are detected in both the SAM-proper and the initiating leaves of maize , mature microRNAs preferentially accumulate in the P0 and leaf primordia [75] , which may be functionally correlated with the differential expression of one or more of the ago1-co-orthologs identified herein . We speculate that the SAM-upregulated expression of a maize ago4-like35 gene , predicted to function during regulation of siRNA-induced gene silencing and maintenance of DNA methylation [46] , [47] , may be elicited in response to the pronounced upregulation of retrotransposon transcription that is observed in the maize SAM [25] . Moreover , six genes predicted to function during DNA repair are upregulated in the SAM-proper versus just two in the P0/P1 ( Figure 2 ) , which may reflect selective pressures to maintain a mutation-free DNA template in the indeterminate , stem cell population of the meristem . Ultimately , the SAM is the source of all the somatic cells comprising the plant shoot , as well as the germinal cells within floral organs . While DNA repair is certainly occurring in the P0/P1 , spontaneous mutations in the DNA of sterile , determinate leaf primordia may be subjected to weaker selective pressure as compared to the SAM . Of the 66 protein fate genes upregulated in our microarray analyses , 42 were identified in the SAM-proper ( Figure 2 ) . Although ubiquitination and additional mechanisms of proteolysis are widespread throughout plant tissues , these data suggest the particular importance of these proteolytic pathways during SAM function . Previous studies in Arabidopsis and rice revealed that 26S proteaosome-dependent proteolysis is required for shoot meristem maintenance and identity [76]–[79] . Our data suggest that 26S proteaosome-dependent proteolysis is also important during the function of the maize SAM , and likewise implicates ubiquitin-related proteases , serine carboxy peptidases , OTU-like cysteine proteases , CLP proteases , aspartic proteases and various SUMO proteins during SAM function ( Table S1 ) . Multiple categories of gene function are identified as upregulated during leaf initiation ( Figure 2 ) . Genes involved in cell wall biosynthesis and cell division/growth are logically co-regulated , and both gene categories are significantly upregulated in the P0/P1 . Although it is true that cell division is absolutely required in the CZ of the SAM in order to replace cells lost during organogenesis and to maintain the meristematic stem cell population , live imaging in Arabidopsis has shown that mitotic activity in the PZ during leaf initiation is more expansive and proceeds at a faster rate than in the SAM proper [80] . Therefore , our array data are in agreement with both classical ( reviewed in [81] ) and recent descriptions of differential cell division rates within SAM functional zones . Although the maize yabby-like8–10 genes upregulated in the P0/P1were placed in the separate GO category of transcription , the YABBYs are likewise presumed to function during expansive organ growth [17] , [26] , [82] . A maize homolog ( Zm-grf16 ) of a family of transcription factors that regulate cell expansion in Arabidopsis leaves and cotyledons [30] was also identified in the P0/P1 dataset ( Table 1 ) . Bioinformatic analyses reveal that grf1 homologs in Arabidopsis and rice have complementary target sites for miR396 , a relatively rare small RNA that is either expressed at very low levels or in a limited number of cells/tissues [83] . Zm-grf1 is expressed in the SAM periphery ( Figure 3D ) and leaf primordia and is downregulated in leaf-arrested SAMs ( Figure 4 ) , implicating a function very early in maize leaf development . Zm-grf1 also harbors the conserved miR396 recognition motif , and thus represents an intriguing candidate gene for reverse genetic analyses of microRNA-regulated leaf development . The grf genes function redundantly in Arabidopsis [30] and at least eight grf1sequence paralogues are present in maize , suggesting that reverse genetic analyses utilizing RNAi approaches or miR396-resistant transgenes may be more informative than characterization of Zm-grf1 knockout alleles . Nearly three times as many gene elements involved in chromatin structure and remodeling were upregulated in the P0/P1 as in the SAM-proper ( 45 versus 16 ) . These data may reflect the fundamental and widespread changes in chromatin that are predicted to accompany the switch from meristematic to leaf developmental programs . Alterations in chromatin structure are inherent when changing from the propagation of an extant developmental state ( i . e . the SAM ) to the installation of a new developmental program ( i . e . leaf initiation ) , and may be further enhanced during the transition from an indeterminate to a determinate developmental field . Previously uncharacterized in maize , the asc1 gene was selected for reverse genetic analysis because our microarray and in situ hybridization analyses revealed significantly upregulated expression in leaf primordia ( Table S1; Figure 3F ) . Moreover , the related genes Zm-cycD4 and Zm-cycD2 are also contained on the SAM 3 . 0 gene chip used in these assays , although neither gene was identified as differentially-expressed . This failure to detect redundant , differential expression of D4-CYCLIN paralogs in the maize SAM suggested that potential mutant phenotypes conferred by asc1 mutations may not be masked by paralogous gene functions . As shown in Figure 6 , single mutations in asc1 condition infertile plants with extreme reductions in leaf width and plant height . Interestingly , these mutant phenotypes are more widespread and severe than those observed in Arabidopsis triple mutant plants homoyzygous for mutations in each of three D3-cyclin paralogs [64] , which are phylogenetically distinct from the CD4/CD2 cyclins ( Figure 5C ) . In addition , whereas mutations in each of the paralogous D4-cyclin Arabidopsis genes condition mild reductions in hypocotyl stomatal number [65] , solo asc1 mutants exhibit profound abnormalities in leaf stomatal patterning ( Figure 6 ) . These asc1 mutant phenotypes suggest that ASC1 is required for normal maize leaf development , and that subfunctionalization of D-cyclin gene function has proceeded quite differently within the maize and Arabidopsis lineages . These data further demonstrate that laser microdissection-microarray analysis is a tractable approach toward the identification of important gene functions within adjacent yet distinct microdomains during maize shoot development .
Seedlings of the maize inbred B73 were raised in a growth chamber on a 15 hr light cycle . Samples were incubated at 25°C during the light cycle and 20°C during the dark cycle . Seedlings were harvested for dissection and fixation at 14 days after germination . For use in shoot-apex culture , maize shoot apices were hand-dissected from 14-day-old seedlings to remove all except the four youngest leaf primordia as described [4] . Dissected apices were cultured on maize culture medium ( MCM; described in [4] ) containing 30 µmol N-1-naphthylphthalamic acid ( NPA ) dissolved in DMF , or in maize tissue culture media containing equal amounts of DMF but no NPA . Apices were incubated for 14 days on a 14 hour light cycle at 28°C ( light period ) or 24°C ( dark period ) . EST clone AW067338 was used to identify maize core gene AC196112 . 3_FG024 located at location 39 , 743–41 , 597 of contig 45 on chromosome 7 ( Maize Genome Browser; http://www . maizesequence . org/index . html ) . As the second D4 cyclin gene characterized in maize , this locus was named leaf cyclinD42 ( asc1 ) . For reverse genetic analyses of asc1 , DNA samples were prepared from pooled F2 seedling progeny obtained via self-pollination of 3 , 456 F1 plants containing active Mutator ( Mu ) transposon systems and subjected to PCR-based screens using nested asc1 gene-specific primers ( lcd1-CTTGCATCCTCCACTTGAGC and lcd2-AGCAGCTGTGTCATCCAAGC ) and a Mu specific primer ( MuTIR-AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC ) . To rule out false-positive results derived from multiple Mu insertions , control reactions were performed with the Mu primer only . PCR reactions with specific products only from the nested PCR amplifications were sequenced to verify the Mutator transposon insertion . Sibling seed from PCR-positive families were planted in a corn nursery in Aurora , NY , screened for developmental phenotypes and outcrossed for two generations to inbred B73 . Interallelic crosses of plants heterozygous for independent Mu-insertion alleles of asc1 failed to complement , indicating the mutant phenotype observed in F2 progeny of self-pollinated plants harboring asc1-Mu insertion alleles are due to mutations in asc1 . Alignments were performed on protein sequences translated from ten Arabidopsis proteins AtCYCD1;1 ( NM105689 ) , AtCYCD2;1 ( NM127815 ) , AtCYCD3;1 ( NM119579 ) ; AtCYCD3;2 ( NM126126 ) , AtCYCD3;3 ( NM114867 ) , AtCYCD4;1 ( NM125940 ) , AtCYCD4;2 ( NM121082 ) , AtCYCD5;1 ( NM119926 ) , AtCYCD6;1 ( NM116565 ) , AtCYCD7;1 ( NM120289 ) , a D1-CYCLIN from Physcomitrella patens ( CAD32542 ) , three CYCLINS from Antirrhinum majus including AmCYCD1 ( AJ250396 ) , AmCYCD3a ( AJ250397 ) and AmCYCD3b ( AJ250398 ) , ASC1 and the maize CYCLINS ZmCYC2;1 ( AF351189 ) , ZmCYC4;1 ( AF351191 ) , ZmCYCD5;1 ( AF351190 ) and ZmCYCD5;2 ( AY954514 ) . Sequences were aligned using CLUSTALX 2 . 0 . 4 , and cladograms were generated with PAUP 4 . 0 using the Maximum Parsimony method and after treating gaps in the alignment as missing data . Equivalent cladograms were generated using the Neighbor Joining method and without removing gaps; bootstrapping values were calculated for 1 , 000 replicates . Maize seedlings harvested at 14 days after germination were fixed in FAA , paraffin-embedded , sectioned at 10 µm , and stained in either Toluidine Blue O or Safranin-Fast Green using Johanssen's method as described [84] . Immunohistochemical analyses of KNOX protein accumulation were performed as described [13] . Epidermal images were obtained using cyanoacrylate glue surface impressions as described [85] . All micrographs were imaged on a Zeiss Z1-Apotome microscope ( Thornwood , NY ) . Seedling shoots were fixed by incubating in acetone , paraffin-embedded as described and sectioned at 10 µm as described [25] , [26] . All laser-microdissections were performed using a P . A . L . M . Laser Microbeam ( P . A . L . M . Microlaser Technologies , Bernried , Germany ) . SAM tissue domains were captured from 5–10 sections per sample , comprised of 0 . 3 mm2–2 mm2 of tissue . Six biological replicate samples were obtained . RNA was isolated from laser-microdissected tissue as described [25] , [26]; RNA amplifications were performed using the RiboAmp™ HS kit ( Arcturus , Mountainview , CA ) according to the manufacturers protocol . The SAM cDNA-enriched SAM1 . 1 and SAM3 microarrays used in these experiments were as described [26] . The MIAME guidelines utilized , hybridization protocols , and array scanning procedure were as described [25] , [26] . All microarray data are available at Gene Expression Omnibus ( GEO; http://www . ncbi . nlm . nih . gov/geo ) . Six biological replicate array hybridizations were performed . One of the six SAM1 . 1 slides was excluded from analysis due to poor hybridization quality and areas of very high background . Data from the other 11 slides were normalized within slides using loess normalization and across slides within each platform using scale normalization [86] . The limmaGUI R package [87] was used to conduct a linear model analysis for each gene following described methods [88] . The method of Benjamini and Hochberg [89] was used to estimate the false discovery rate associated with the identified sets of differentially expressed genes . Annotations of the predicted GO functions for all differentially expressed genes were performed as described [27]; annotated data is presented at SAM-The Maize Shoot Apical Meristem Project ( http://sam . truman . edu/geneva/geneva . cgi ) . EST sequences of the 1 , 312 microarray elements that were found to be differentially-expressed in the P0/P1 and SAM datasets were sorted by BLAST homology analyses to the contigs sequenced maize genomic DNA ( i . e . maize contigs ) in order to convert array elements ( ESTs ) into maize gene contig ( MGC ) groups . For multiple ESTs that hit a single MGC , the EST list was collapsed under the single MGC identity , and mean and standard error of mean ( sem ) were calculated for P-values and fold change of those ESTs . Sample size ( n ) value for mean and sem calculations is represented by the number of ESTs for a single MGC group . Multiple ESTs linked to a single MGC group were searched in both P0/P1 and SAM datasets to detect potential dual hits; such MGC groups containing P0/P1- and SAM-specific ESTs were removed from the dataset . To this end , 10 MGC groups representing 20 P0/P1- and 9 SAM-specific ESTs were removed . In some cases , a MGC could not be assigned for an EST , as indicated by the identifier ‘contig:NONE’ . In other cases , single or multiple ESTs hit multiple MGCs ( Table S1 ) . A test based on the binomial distribution was used to identify functional categories for which the discrepancy between the number of genes upregulated in SAM and the number upregulated in P0/P1 was significant . Conditioning on n = total number of genes identified in a given category , the null hypothesis that the proportion of genes upregulated in SAM was equal to 1/2 was tested against the alternative that the proportion was not 1/2 . A p-value was obtained by comparing the observed number of genes upregulated in SAM to a binomial distribution with n trials and success probability 1/2 . For example , given that 34 genes predicted to function in RNA binding were identified as differentially expressed , the probability that only 9 of these 34 would be up in SAM relative to P0/P1 is approximately 0 . 0045 according to a binomial distribution with 34 trials and success probability 1/2 . This yields a two-sided p-value of 2*0 . 0045 = 0 . 009 and suggests that discrepancy ( 9 up in SAM vs . 25 up in P0/P1 ) cannot easily be explained by a simple chance mechanism . qRT-PCR analyses of NPA-treated and untreated shoot apices ( described in Plant Materials , above ) were performed on cDNA prepared from tissue-cultured/laser-microdissected SAMs as described [55] . Analyses utilized three technical replications performed on pooled cDNA prepared from ten microdissected SAMs . Gene-specific primer pairs used in these analyses were as follows: AI855049 ( 5′-CAGAATCATCACCTACACCT-3 and 5′-GAGTAGTAGAAGATTGCTGTGAG-3′ ) ; DN220821 ( 5′-GCTAATGAGCATAGTATGCC-3′ and 5′-CTGCTCATTACCATGTCCTG-3′ ) ; CD527823 ( 5′-TCCGTCTTGTACATGTGAG-3′ and 5′-TCTCGACATTCTTAAGGAGC-3′ ) ; CD670256 ( 5′-GGTCTCTAAAGTCACTGAAACC-3′ and 5′-GAGCTGATCCCTTAGTTAAGTC-3′ ) ; BG840831 ( 5′-GATCAAATCATAGACCTAGAGTCC-3′ and 5′-ATTGGTGTAGTTTCCTAGCTG-3′ ) ; AY313902 ( 5′ CCTCAAGAAGACCTTCAAGAC-3 and 5′-TTATTAGAATGGAGTGATGCCC-3′ ) ; CB380920 ( 5′-TCACCGTCAGAATTTACGTC-3′ and 5′-GCATAAACAACCACTGAACC-3′ ) ; CA998660 ( 5′- TTGAACTCATCCGCTTTCTC-3′ and 5′-TTGACACATTCCGTCTACAG-3′ ) ; BM073971 ( 5′-CCTCAAGGCATTCAGATCTC-3′ and 5′-AGATGATGTCTTCCTGTCGT-3′ ) . Fourteen-day-after-germination maize B73 seedlings were processed for in situ hybridization as described [90] with modifications [91] . Gene-specific probes were synthesized from the cDNA clones DN229322 , BM073398 , CB381076 . BG840831 , AW067338 . For each gene-specific probe analyzed , at least six replicate samples were hybridized . For use in RNA gel-blot hybridizations , total RNA was extracted from 14-day seedlings using the Trizol lysis method and prepared for Northern transfer as described [92] . The gene-specific asc1 probe was prepared from genomic DNA using the primer pair 5′-CGGTTTCCTGGAGTCTGAGG-3′ and 5′- CTTGCATCCTCCACTTGAGC-3′ , which amplifies a 776 bp fragment spanning exons 1–4 ( Figure 5A ) .
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All the organs of plant shoots are derived from the shoot apical meristem ( SAM ) , a pool of plant stem cells that are both organogenic and self-sustaining . These dual SAM functions take place in distinct yet adjacent meristematic domains; leaves are derived from the peripheral zone ( PZ ) of the SAM whereas cells lost during organogenesis are replenished from the central zone ( CZ ) . Deciphering the global patterns of differential gene expression within these discrete SAM functional domains is integral toward understanding the molecular-signaling networks that regulate plant development . We utilized laser-microdissection technology to isolate tissues from the SAM crown and center ( SAM-proper ) and from initiating leaves ( P0/P1 ) at the SAM periphery for use in microarray comparisons of gene expression within these SAM functional domains . Nine hundred and sixty-two maize genes were differentially expressed , confirming that the distinct functions of these meristematic domains involve widespread differences in gene expression . Genes involved in cell division , cell wall biosynthesis , chromatin structure , and RNA binding are especially upregulated in initiating leaves , whereas genes regulating protein stability and DNA repair are upregulated in the SAM proper . Mutations in a D-cyclin gene that was upregulated in the P0/P1 render narrow-leafed mutant plants with defective stomatal patterning , providing functional genetic data for a previously uncharacterized maize gene .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"plant",
"biology/plant",
"growth",
"and",
"development",
"plant",
"biology/plant",
"genetics",
"and",
"gene",
"expression",
"plant",
"biology"
] |
2009
|
Microdissection of Shoot Meristem Functional Domains
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Human toxocariasis is a zoonotic infection caused by the larval stages of Toxocara canis ( T . canis ) and less frequently Toxocara cati ( T . cati ) . A relationship between toxocariasis and epilepsy has been hypothesized . We conducted a systematic review and a meta-analysis of available data to evaluate the strength of association between epilepsy and Toxocara spp . seropositivity and to propose some guidelines for future surveys . Electronic databases , the database from the Institute of Neuroepidemiology and Tropical Neurology of the University of Limoges ( http://www-ient . unilim . fr/ ) and the reference lists of all relevant papers and books were screened up to October 2011 . We performed a systematic review of literature on toxocariasis ( the exposure ) and epilepsy ( the outcome ) . Two authors independently assessed eligibility and study quality and extracted data . A common odds ratio ( OR ) was estimated using a random-effects meta-analysis model of aggregated published data . Seven case-control studies met the inclusion criteria , for a total of 1867 participants ( 850 cases and 1017 controls ) . The percentage of seropositivity ( presence of anti-Toxocara spp . antibodies ) was higher among people with epilepsy ( PWE ) in all the included studies even if the association between epilepsy and Toxocara spp . seropositivity was statistically significant in only 4 studies , with crude ORs ranging 2 . 04–2 . 85 . Another study bordered statistical significance , while in 2 of the included studies no significant association was found . A significant ( p<0 . 001 ) common OR of 1 . 92 [95% confidence interval ( CI ) 1 . 50–2 . 44] was estimated . Similar results were found when meta-analysis was restricted to the studies considering an exclusively juvenile population and to surveys using Western Blot as confirmatory or diagnostic serological assay . Our results support the existence of a positive association between Toxocara spp . seropositivity and epilepsy . Further studies , possibly including incident cases , should be performed to better investigate the relationship between toxocariasis and epilepsy .
Human toxocariasis is a parasitic zoonosis caused by the larval stages of the ascarids Toxocara canis ( T . canis ) , the common roundworm of dogs , and by the roundworm of cats , Toxocara cati ( T . cati ) [1] . The reported prevalence of soil contamination with Toxocara spp . eggs is variable between studies , going from a percentage of 6 . 6 to 87 . 1% [2]–[9] . Therefore toxocariasis is one of the most prevalent zoonotic helminth infections , occurring whenever the man–soil–dog relationship is particularly close . High seroprevalence rates of Toxocara spp . ( presence of sera anti-Toxocara spp . antibodies ) have been found in tropical countries , where the humid climate favours the survival of parasite eggs in the soil , and in rural settings , where the poor hygiene and the rare administration of anthelmintic treatments to dogs increases the probability of human infection [10]–[12] . Nevertheless , the reported seroprevalence in apparently healthy adults from urban areas of Western countries is of 2–5% [13] , whit a wider range ( 2 . 4%–31 . 0% [14] , [15] ) when considering all the studies carried out in Europe , independently from age of participants and type of setting . Despite being the most prevalent human helminthic infection in some industrialized countries [16] , toxocariasis remains relatively unknown to the public [17] and the true magnitude of the global burden of Toxocara spp . -associated human disease has still to be evaluated [18] . Humans are infected by the accidental ingestion of embryonated Toxocara spp . eggs present in contaminated soil or food , or by the ingestion of encapsulated larvae contained in the raw tissues of paratenic hosts , such as cows , sheep or chickens [1] , [19] . The clinical manifestations of human toxocariasis vary from asymptomatic infection to severe organ injury , depending on the parasite load , the sites of larval migration and the host's inflammatory response [20] . Two severe clinical syndromes are classically recognised: visceral larva migrans ( VLM ) , systemic disease caused by larval migration through major organs , and ocular larva migrans ( OLM ) , in which the disease is limited to the eyes and the optic nerves . Two less severe syndromes have also been described: ‘covert toxocariasis’ , seen mainly in children and characterized by fever , headache , behavioural and sleep disturbances , cough , anorexia , abdominal pain , hepatomegaly , nausea and vomiting , and ‘common toxocariasis’ , seen predominantly in adults with weakness , pruritus , rash , difficult breathing and abdominal pain [20] . Clinical involvement of the central nervous system ( CNS ) in visceral larva migrans is thought to be rare , although in experimental animals the larvae frequently migrate to the brain [21]–[23] . The CNS migration may lead to a variety of neurological disorders such as meningo-encephalitis , myelitis , cerebral vasculitis , optic neuritis [23] , [24] and probably cognitive [25] and behavioural [26] disorders . Concerning epilepsy , early reports have suggested a high exposure rate to Toxocara spp . among people with epilepsy ( PWE ) [27] , [28] . In particular , in 1966 Woodruff et al . [27] found that 7 . 5% of PWE had a positive skin reaction to an antigen prepared from adult T . canis , in contrast to 2 . 1% of apparently healthy persons . In addition , they noted a statistically significant association between contact with dogs and positive skin test to toxocaral antigen in PWE . Following these preliminary observations and prompted by the development of serodiagnostic tests with improved sensitivity and specificity , further studies have been carried out in different populations to investigate the possible association between Toxocara spp . seropositivity and epilepsy , suggesting that toxocariasis could play a role in the incidence of epilepsy in endemic areas [29]–[31] . Considering that toxocariasis is one of the most common helminthiasis worldwide and that it is a potentially preventable disease , a correct estimate of the association between toxocariasis and epilepsy is necessary . We carried out a systematic literature revision and a meta-analysis to evaluate the possible association between human toxocariasis and epilepsy and to highlight some methodological points to be taken into account for the elaboration of future surveys .
A systematic search without past time or language restriction was conducted to identify published and unpublished articles dealing with the association between toxocariasis and epilepsy . The following online databases were independently examined by two researchers ( GQ and BM ) : MEDLINE , IngentaConnect , ScienceDirect ( Elsevier ) , Refdoc ( ex ArticleScience ) , Scopus , Highwire . In addition , the database from the Institute of Neuroepidemiology and Tropical Neurology of the University of Limoges ( IENT ) : Virtual Library on African Neurology , BVNA ( http://www-ient . unilim . fr/ ) , which contains more than 9000 references of medical dissertations , theses and articles dealing with tropical neurology and parasitology , was examined . In MEDLINE combined text words and Medical Subject Headings ( MeSH ) terminology were used . The following search key words and Boolean operators were entered: “toxocariasis” AND “epilepsy” AND “epidemiology” . The term “toxocarosis” as an alternative to “toxocariasis” was also considered . The literature search was adapted for the other databases . Titles and available abstracts were scanned for relevance , identifying papers requiring further consideration . Reference lists of all available reviews , primary studies and books found were screened manually . When necessary , corresponding authors of relevant studies were contacted . Experts in the field were also contacted to find out other eventual non-published studies . The systematic search was realized up to October 2011 . Considering epilepsy as the outcome and toxocariasis as the exposure , all the studies meeting the following eligibility criteria were included: Studies including only acute symptomatic seizures or specific seizure patterns or epileptic syndromes were excluded . Full copies of all reports identified by the electronic or hand searching were obtained and two reviewers ( GQ and BM ) independently assessed their eligibility and extracted data . The following data were independently recorded in an ad hoc created collecting form: author , country , study design , study population ( number , age group , gender , setting ) and recruitment methods . For toxocariasis , specific information was recorded on methods used for diagnosis . Considering epilepsy , details on definition and assessment were extracted . Discrepancies between reviewers were rechecked and consensus was achieved by discussion . For each survey , the crude odds ratio ( OR ) on the association between toxocariasis and epilepsy and the relative 95% confidence interval ( CI ) were recalculated . Furthermore , statistical power was calculated as a priori and a posteriori . A priori statistical powers were calculated following the hypothesis that the objective of the survey was to identify a minimum OR of 2 ( i . e . , Toxocara spp . exposure leads to twice more epilepsy ) with one control per case , based on the number of PWE and the percentage of Toxocara spp . seropositivity in PWOE . The a posteriori statistical powers were calculated upon the results of the surveys . In both cases a 5% alpha risk was considered . Powers were calculated using Epi-Info 6 . 04 [32] . To estimate the association between toxocariasis and epilepsy we performed a meta-analysis applying a random effects model , assuming that the true effect size of exposure varies from one study to the other , and that the studies in our analysis represent a random sample of effects that could have been observed [33] . A common risk was estimated as a common OR from all the studies . The homogeneity was tested by the Cochran Q test of heterogeneity . In order to account for the different age groups considered , the analysis was then separately applied to the studies including an exclusively juvenile population [30] , [31] . Furthermore , considering that Western Blot ( WB ) is as sensitive but more specific than enzyme-linked immunosorbent assay ( ELISA ) [34] , we also conducted an analysis restricted to the studies using WB as diagnostic or confirmatory test [35]–[38] . The meta-analysis was performed using EasyMA , 2001 version [39] . The PRISMA ( Preferred Reporting Items for Systematic reviews and Meta-Analyses ) statement [40] was used as a guide in the reporting of this study .
A flowchart summary of the literature search is shown in Figure 1 . A PRISMA flowchart is also shown ( Figure S1 ) . Electronic search produced 131 publications , among which 25 dealt with epilepsy and toxocariasis . The removal of duplicate citations and the screening of abstracts permitted to isolate 8 documents [27] , [31] , [35]–[38] , [41] , [42] . Two additional publications [28] , [30] were found by hand searching ( reference lists check ) . Full text review of the 10 documents permitted to exclude 3 of them for not fulfilling the inclusion criteria: one [27] was excluded because methods to assess epilepsy were not reported and toxocariasis infection was detected through a skin test; furthermore the included cases consisted of a highly selected group of severe patients with epilepsy . Another study [28] was excluded because toxocariasis infection was exclusively assessed in a sample of PWE without control group . The last study [42] was excluded because of the lack of reporting of aggregated data for each group . Considering the 7 articles meeting the inclusion criteria , the materials and methods of the study reported by Nicoletti et al . ( 2007 ) [36] had been previously detailed in Nsengiyumva et al . [43] , while the study population of Winkler et al . ( 2008 ) [38] has been better described in Winkler et al . ( 2009 ) [44] . The methodological aspects of these articles have been therefore assessed considering both the publications . Seven case-control studies [30] , [31] , [35]–[38] , [41] were included , providing a total subjects number of 1867 ( 850 PWE and 1017 PWOE ) . Two of them [30] , [31] considered a population aged 1–17 years while one excluded children aged 10 years or younger [38] . The studies were carried out in 6 different countries ( USA , Italy , Bolivia , Turkey , Burundi and Tanzania ) , both in rural [35] , [36] , [38] and urban [30] , [31] , [37] , [41] settings . In the study by Akyol et al . [41] 10% of participants were from rural areas , but no significant relationship was found between residency and seropositivity rate . The general characteristics of the included studies are shown in table 1 . Three surveys had a matched case-control design [35]–[37] among them age was the only common matching criteria . Only one study was a population-based survey [35] . The epilepsy definition proposed by the International League Against Epilepsy ( ILAE ) in 1993 [45] was applied in 3 studies [35]–[37] while Glickman et al . [30] considered the definition proposed in 1972 by Alter [46] , and Winkler et al . [38] defined epilepsy according to the World Health Organization ( WHO ) Neurosciences Research Protocol proposal [47] . In the work by Arpino et al . [31] a general definition of “positive seizure history” was considered as cases entry criteria . Considering seizures , 4 studies [35]–[37] , [41] applied the classification of epilepsies and epileptic syndromes proposed by the ILAE in 1981 [48] , while one [38] used an adjusted classification for rural African hospitals suggested in 2007 [49] . All PWE were prevalent cases and none of the studies clearly specified if active or lifetime epilepsy was considered , the second being more probable . Controls were out- or in-patients attending the same hospital of cases [30] or people going to hospital for vaccination or haematological check [36] , [37] or volunteers [41] . A negative history for seizures [31] , [36]–[38] , [41] and for both seizures and other neurological diseases [31] , [36] , [37] was considered for controls definition . In the population-based survey controls were selected from the same community , but different households , of cases [35] , whereas another study selected controls from the same province of PWE excluding blood relationship [36] . In an attempt to determine the accuracy of the seizures classification EEG recordings were examined in some studies [31] , [35]–[37] . A neurologist confirmed both cases and controls through anamnesis and complete neurological examination in 4 studies [35]–[38] . In order to obtain demographic data and information concerning factors possibly associated with Toxocara spp . exposure a questionnaire was administered to cases and control subjects in 5 studies [30] , [31] , [36] , [37] , [41] . Data were usually obtained by the patient's mother when the study population was infantile [30] , [31] . The questionnaire version used was specified only in one study [36] and interviewers qualifications were stated only in 2 surveys [36] , [37] . Presence of anti-Toxocara spp . antibodies in sera was assessed using antibodies-ELISA ( Ab-ELISA , commercial or in-house kits ) [30] , [31] , [41] , or immunoblot [36] , [37] or Ab-ELISA followed by WB confirmation [35] , [38] . Laboratories performing the analysis were blind to the case-control status of sera samples in 3 studies [35]–[37] . The results of the included studies are shown in Table 2 . Toxocara spp . seropositivity ranged from 6 . 5% to 50 . 8% in the control group and from 12 . 0% to 59 . 7% in PWE . Seroprevalence rate was higher among PWE than control subjects in all the 7 included surveys , even if the association between Toxocara spp . seropositivity and epilepsy was statistically significant in 4 of them [30] , [31] , [35] , [37] . In one study the crude OR bordered on statistical significance , anyway , after adjustments on other variables according to a multivariate model using the conditional logistic regression , a stronger and significant association was found [36] . Significant crude ORs ranged between 2 . 04 and 2 . 85 . A priori statistical power ranged 32 . 8–90 . 9% and a posteriori statistical power 8 . 0–89 . 6% . A meta-analysis was at first performed on all the 7 studies included . Results are presented in figure 2 . A significant ( p<0 . 001 ) common OR of 1 . 92 ( 95%CI 1 . 50–2 . 44 ) was estimated . The test of heterogeneity was not significant ( p = 0 . 545 ) , indicating homogeneity of the studies included . When analysis was restricted to the 2 studies considering only a juvenile population [30] , [31] , as shown in figure 3 , a common OR of 2 . 23 ( 95% CI 1 . 35–3 . 69; p = 0 . 002 ) was found . The test for heterogeneity was also not significant ( p = 0 . 655 ) . The meta-analysis was at last restricted to the 4 studies using WB test [35]–[38] , as shown in figure 4 , leading to an OR of 1 . 91 ( 95% CI 1 . 33–2 . 75 , p<0 . 001 ) and a non significant test for heterogeneity ( p = 0 . 430 ) .
We performed a systematic literature revision and a meta-analysis of available data to evaluate the association between epilepsy and toxocariasis . To our knowledge this is the first meta-analysis on this argument . Based on our literature search , we analyzed data from 7 case-control studies carried on in rural or urban settings and in various countries worldwide . We are confident that our literature search is exhaustive as conducted on several electronic databases and also on a specific database containing literature on tropical neurology and parasitology including theses and memos unpublished in international or electronic databases . Seroprevalence rate of anti-Toxocara spp . antibodies was higher among PWE than control subjects in all the 7 studies analysed [30] , [31] , [35]–[38] , [41] even if only 4 showed a significant positive association between Toxocara spp . seropositivity and epilepsy [30] , [31] , [35] , [37] and a fifth reached statistical significance only after adjustment for other variables [36] . In our meta-analysis we found evidence of positive association , with a common OR of 1 . 92 ( 95%CI 1 . 50–2 . 44 ) and a lack of heterogeneity between the studies . Our result is noteworthy for coming from studies across different populations in disparate geographic locations and socioeconomic climates . This consistency of observations among different populations is in favor for a causal relationship between toxocariasis and epilepsy [50] . Anyway , various important points should be taken into account when interpreting our data . First of all , the studies evaluated were retrospective case-controls ascertaining both Toxocara spp . seropositivity and epilepsy in a cross-sectional manner; thus , the inclusion of “prevalent” rather than “incident” cases does not permit to demonstrate a temporal relationship between the exposure ( Toxocara spp . ) and the outcome ( epilepsy ) and doesn't allow to exclude a possible “reverse causality” . It has been in fact hypothesized that the abnormal behavior patterns ( e . g . pica and hyperactivity ) and the elevated number of falls to the ground of PWE ( especially children or mentally retarded ) could predispose them to Toxocara spp . exposure rather than the contrary [30] . In particular , evidence of association has been reported between Toxocara spp . seropositivity and mental retardation [51] , [52] . We underline anyway that the study by Nicoletti et al . ( 2002 ) [35] found no statistical difference in seroprevalence between PWE with or without mental retardation . On the other hand , a significant difference in the frequency of mental retardation between seropositive and seronegative subjects was found by Nicoletti et al . ( 2008 ) [37] , but it lead to only a slight reduction of association after restriction of the analysis to the PWE without mental retardation . Selection of cases and controls represents one of the most important pitfalls in case-control studies . In the studies evaluated , with the exception of the only population based survey [35] , PWE and PWOE were generally enrolled from a hospital setting , and their source population was often not clearly defined . This constitutes a possible recruitment bias , especially in rural settings , where people receiving care are not representative of the general population . In particular , concerning controls , hospital controls might resemble cases more than population controls , biasing OR towards the null [53] . Furthermore , a volunteer bias could have affected the study by Akyol et al . [41] , where the control group was composed by volunteers coming from an undefined source . However , the population based survey showed a positive association , similar to the results found by the positive hospital based studies , suggesting that the selection bias effect could be limited . Considering cases and controls characteristics , PWE and PWOE should be comparable at least for age , because the prevalence of both epilepsy and anti-Toxocara spp . antibodies vary with age , and for geographical provenience and education , which are likely related to the level of exposure to Toxocara spp . On this point , the studies examined are often lacking of detailed descriptive data . We report as an example , the comment by Quet et al . [54] on the study by Akyol et al . [41] , which noticed how the greater number of students observed in the control group could suggest a higher education in controls than cases . The educational level was in fact expressed as a binary variable ( less or more than primary school ) in this study , which could give an unreliable estimation of education; in such cases the number of school years might be a more precise measure . In order to account for the different age groups included , and considering that young age seems to contribute to Toxocara spp . exposure [1] , we restricted our meta-analysis to the studies with an exclusively juvenile population and we obtained also in this case a significant positive association ( OR 2 . 23 , 95% CI 1 . 35–3 . 69 , p = 0 . 002 ) . In particular , in the study by Arpino et al . [31] the relationship was more remarkable in children under 5 years old . Based on these findings , it has been suggested that the parasite may act as a cofactor in determining the occurrence of seizures in children with a predisposing background [31] . Only a prospective cohort follow-up study could avoid these biases . However such a design , leaving subjects exposed to toxocariasis and without intervention , is ethically not acceptable . A potential weakness of our study is the use of different and not always clears epilepsy definitions in the included articles . On this point , considering that the lag time between Toxocara spp . infection and epilepsy occurrence is not yet defined , we underline the importance of including lifetime and not only active epilepsy , as likely properly done in the studies examined . On the other hand , the lack of exhaustive descriptive data on the age of onset , on the probable etiology and on seizures classification didn't permit us to differentiate our analysis for these factors . The significant positive association found in some studies between Toxocara spp . seropositivity and partial epilepsy could in fact be biologically plausible , given the higher prevalence of idiopathic epilepsy among the generalized forms [35] , [37]; while the lack of association between partial epilepsy and toxocariasis found by Nicoletti et al . ( 2007 ) [36] has been related by the authors to a lack of power . In the study by Akyol et al . [41] , besides the lack of a precise definition of epilepsy , the authors reported a higher frequency of generalized epilepsies , as expected , because of the inclusion of only cryptogenic ( in the abstract ) or idiopathic ( in the methods ) epilepsies [54] . This could have affected the results , showing no statistically significant association between toxocariasis and epilepsy . A correct classification of seizures , possibly with the help of EEG recordings , is therefore an important element that should be taken into account in future studies to permit a correct interpretation of the results . Regarding the diagnosis of toxocariasis , the major limitation in confronting different studies consists in the heterogeneity of techniques ( Ab-ELISA or WB or both ) used to detect sera anti-Toxocara spp . antibodies , mostly due to different cost and availability . Also when considering ELISA , different kits ( commercial or in house ) with different sera dilutions were utilized and a serum pre-adsorption with larval Ascaris extracts was carried on only in some studies . It would have been interesting evaluating and reporting the sensitivity and specificity of the used assays , which has never been done in the studies examined . Considering that the WB confirmation of positive results from the ELISA ( especially where pre-absorption is not carried out ) has been recommended [55] , and given the higher specificity of WB [34] , we restricted our meta-analysis to the studies applying WB , obtaining results similar to the global analysis ( OR 1 . 91 , 95%CI 1 . 33–2 . 75 , p<0 . 001 ) . When interpreting these data , we are of course aware that other factors , such as Toxocara spp . excretory-secretory ( TES ) antigen preparations , parasite strains , and WB technical procedures , could have influenced the results obtained by different investigators . It should also be kept in mind that a single seropositivity has limited pathological significance and could probably represent past rather than recent infection . Furthermore , the presence of sera antibodies against Toxocara spp . does not provide evidence of either an active systemic infection or a CNS involvement . Diagnosis of neurotoxocariasis is in fact based on the history; blood tests , including differential blood cell count and determination of serum total IgE; CSF investigation , including the detection of anti-Toxocara spp . antibodies and neuroimaging [13] . The absence of significant results was associated with a lower power ( type II error ) , making not really surprising the lack of statistical confirmation of the difference found . The statistical power of a study can be improved performing surveys in areas with high level of exposure assessed with the most sensitive assay or , when the number of cases is small , increasing the ratio of controls to cases up to 4/1 [53] . The low a posteriori power of the studies by Winkler et al . [38] ( 8 . 0% ) and Akyol et al . [41] ( 11 . 3% ) could be mostly accounted to the small sample size and in particular the lower number of controls than cases , highlighting one more time the central role of the elaboration of the control group . In our paper we referred to toxocariasis etiological agent as Toxocara spp . and not only T . canis . TES in fact are not species-specific and the differentiation between T . canis and T . cati remains challenging . Considering the prominence historically given to T . canis , the role of T . cati in human toxocariasis could have been underestimated . Further work should be encouraged to differentiate the two parasites and to better address future prevention strategies [56] . The most frequent infectious agent involved in the differential diagnosis of subjects with late-onset epilepsy or inflammatory brain nodules is the larval stage of Taenia solium ( T . solium ) , aetiological agent of neurocysticercosis ( NCC ) . Concomitant T . solium and Toxocara spp . seropositivity is a possible event in areas endemic for both helminthes . Anyway , albeit there is yet no evidence on the mechanisms underlying toxocariasis-induced epilepsy , according to us toxocariasis should not be ruled out as an accidental association . First of all , the presence of anti-T . solium antibodies , as in the case of toxocariasis , could represent only a previous exposure without established infection . Furthermore , considering the studies included in our analysis , in the study by Nicoletti et al . ( 2002 ) [35] only 7 PWE over a total of 113 were positive to both T . solium and Toxocara spp . and in the study by Nicoletti et al . ( 2007 ) [36] , finding a positive association between Toxocara spp . seropositivity and epilepsy , seropositivity for cysticercosis was considered as a variable in the multivarate analysis . Of course , the interpretation of serological results should always take into account the background seroprevalence of both Toxocara spp . and T . solium in the studied population and cysticercosis seropositivity should always be evaluated as a possible confounder when carrying on surveys on infectious agents and epilepsy . In conclusion , a positive association between Toxocara spp . -seropositivity and epilepsy could be hypothesized; nevertheless , even the modestly strong association demonstrated in our meta-analysis does not necessarily prove causality ( i . e . , Toxocara spp . infestation caused the epilepsy ) . Further studies , considering incident rather than prevalent cases and with a population-based design , should be performed . An internationally accepted epilepsy definition and seizures classification should be applied and cases and controls should be comparable at least for age , sex , geographic provenience , education and socio-economic background . Pica , pet owning , mental retardation and other possible toxocariasis risk factors should be assessed trough a validated questionnaire administered by trained investigators and assessors and laboratory personnel should be blind to the status of participants . The improvement of techniques permitting to distinguish recent from past infections , such as antigen-ELISA ( Ag-ELISA ) , should be encouraged in order to better investigate the time relationship between Toxocara spp . infection and epilepsy occurrence . Assessing the link between toxocariasis and epilepsy is of interest as toxocariasis is a potentially preventable disease . Nowadays , despite the implementation of regular and intensive de-worming programs in western countries , the parasite still prevails , indicating that prevention is not easy in practice . Good hygiene practices should be encouraged and further strategies to prevent Toxocara spp . transmission should be identified and applied , permitting to experimentally investigate the causation hypothesis [50] . The existence of a causal relationship and the estimation of the impact of toxocariasis on the global burden of epilepsy may strongly contribute in encouraging further programs on toxocariasis prevention worldwide , in order to control both the Toxocara spp . transmission and the related epilepsy burden .
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Human toxocariasis is an infection caused by the larval stage of the worms Toxocara canis and less frequently Toxocara cati , common parasites of domestic and peridomestic dogs and cats . It is a cosmopolitan infection , occurring whenever the man-soil-dog relationship is particularly close , especially in tropical countries , where the humid climate favours the survival of parasite eggs in the soil , and in rural settings , where the poor hygiene increases the probability of human infection . Epilepsy affects nowadays at least 65 million of people worldwide and is particularly common in tropical areas , probably because of the presence of cases caused by infectious diseases largely absent in industrialized countries . For several decades , researchers have investigated the possible association between toxocariasis and epilepsy . In this study we conducted a statistical analysis of all the data available on the relationship between these two conditions . The combined results of the 7 studies included indicate an association between the two diseases . Further studies are necessary to demonstrate a causal relationship ( i . e . toxocariasis causes epilepsy ) . Considering that toxocariasis is a preventable and common disease , a better understanding of the relationship between toxocariasis and epilepsy may contribute to improving prevention of epilepsy worldwide .
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2012
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Toxocariasis and Epilepsy: Systematic Review and Meta-Analysis
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The ability to discriminate tones of different frequencies is fundamentally important for everyday hearing . While neurons in the primary auditory cortex ( AC ) respond differentially to tones of different frequencies , whether and how AC regulates auditory behaviors that rely on frequency discrimination remains poorly understood . Here , we find that the level of activity of inhibitory neurons in AC controls frequency specificity in innate and learned auditory behaviors that rely on frequency discrimination . Photoactivation of parvalbumin-positive interneurons ( PVs ) improved the ability of the mouse to detect a shift in tone frequency , whereas photosuppression of PVs impaired the performance . Furthermore , photosuppression of PVs during discriminative auditory fear conditioning increased generalization of conditioned response across tone frequencies , whereas PV photoactivation preserved normal specificity of learning . The observed changes in behavioral performance were correlated with bidirectional changes in the magnitude of tone-evoked responses , consistent with predictions of a model of a coupled excitatory-inhibitory cortical network . Direct photoactivation of excitatory neurons , which did not change tone-evoked response magnitude , did not affect behavioral performance in either task . Our results identify a new function for inhibition in the auditory cortex , demonstrating that it can improve or impair acuity of innate and learned auditory behaviors that rely on frequency discrimination .
Frequency discrimination is a fundamental task in everyday hearing and can be vitally important , as spectral differences can be used to distinguish dangerous and safe sounds [1–3] . However , our knowledge of the neuronal mechanisms that support frequency discrimination remains incomplete . The auditory cortex ( AC ) is involved in many auditory behaviors [4–13] , with some studies suggesting that it controls frequency discrimination [14–16] ( but see [5 , 17] ) . It remains poorly understood which aspects of neuronal circuits in AC contribute to behavioral frequency discrimination performance . Neurons in AC exhibit frequency selectivity in their responses to tones [18–24] , and modify their tuning properties with auditory learning [25–27] , providing support for the involvement of AC in frequency discrimination . Many aspects of neuronal responses to tones , including magnitude of neuronal responses and width of tuning , can in principle affect behavioral performance [28] . Furthermore , learning and experience can lead to changes in tone-evoked response patterns in AC [25 , 27 , 29–31] , affecting neuronal frequency tuning and selectivity . At present , a detailed understanding of the relation between tone response properties of AC neurons and frequency discrimination behavior remains missing . Neurons in AC form mutually coupled excitatory–inhibitory networks , which shape the responses of neurons to sounds [32 , 33] . Electrophysiological recordings and pharmacological studies demonstrate that GABA-ergic inhibition controls tone-evoked response amplitude , spontaneous firing rate and frequency selectivity [25 , 27] , among other aspects of excitatory neuronal responses . The most common type of interneurons , parvalbumin-positive interneurons ( PVs ) [11 , 34–38] , which target the pyramidal cell bodies , gate feed-forward thalamocortical auditory inputs [38] . We postulated that optogenetically modulating PV activity would affect tone-evoked responses in the AC , thereby enabling us to examine the effect of changing tone response properties of AC neurons on auditory behavior and learning . We focused on two behaviors , frequency discrimination—driven prepulse inhibition ( PPI ) of the acoustic startle response ( ASR ) , and differential auditory fear conditioning ( DAFC ) . Frequency discrimination-driven PPI relies on an innate behavior—startle response to loud noise , and is controlled by subcortical circuits [39] . Because PPI decreases if the startle noise is preceded by a change in an acoustic stimulus , it can be used to measure frequency discrimination acuity [3 , 40] . By contrast , DAFC requires both learning and memory and is controlled by interactions between the cortex and a complex circuit including the amygdala and the hippocampus [41–44] . While these two behaviors rely on different brain circuits , they can affect each other [45] , with the AC facilitating this interaction [3] . We found that cortical inhibition controls frequency discrimination acuity and frequency specificity of auditory fear conditioning . These behavioral changes were correlated with changes in the magnitude of tone-evoked neuronal activity .
To manipulate the level of activity of a specific type of inhibitory interneuron , PVs , in AC , we drove them to express Channelrhodopsin ( ChR2 ) or Archaerhodopsin ( Arch ) , using targeted viral delivery to AC ( Fig 1A and 1F ) [34 , 38 , 46] . Arch is a light-driven proton pump that hyperpolarizes the neuron when activated with green light [35] . Conversely , ChR2 is a light-gated cation channel that depolarizes the neuron when activated with blue light [47] . We injected a modified adeno-associated virus ( AAV ) , which carried the antisense code for either opsin under the FLEX cassette in AC of PV-Cre mice . Following an incubation period , PVs in AC expressed ChR2 or Arch efficiently and with high specificity ( Fig 1B and 1C and Fig 1G and 1H ) . Analysis of light-evoked responses of putative PVs showed that PVs have a distinct waveform with relatively deep troughs ( S1 Fig ) . We used spike waveform shape as a criterion for exclusion of putative PVs from the pool of analyzed neurons . Throughout the study , we compared the effects of interneuron modulation with that of direct increase in the activity of excitatory neurons by photostimulation . This control allowed us to test whether a simple elevation of the activity level of excitatory neurons can account for the observed results . In order to activate excitatory neurons directly , we drove them to express ChR2 in AC using targeted viral delivery in mice that express Cre recombinase in neurons under CamKIIα promoter . This resulted in efficient and specific expression of ChR2 in putative excitatory neurons in AC ( Fig 1K–1M ) . To verify the effectiveness of optogenetic modulation , we measured the effect of the laser on the spontaneous firing rate of AC neurons . Spiking activity of neurons in AC of awake , head-fixed mice was recorded during acoustic presentation of a random tone sequence , a stimulus designed to measure the frequency tuning curve of neurons . Locally shining either blue ( 473 nm ) or green light ( 532 nm ) suppressed or activated the activity of putative excitatory neurons confined to AC , respectively ( S2 Fig ) . Activation of PVs ( 473 nm , 0 . 2 mW/mm2 intensity at the fiber tip ) significantly reduced the spontaneous firing rate ( FRbase , computed during the baseline period , 0–50 ms prior to tone onset ) in a large fraction of recorded neurons ( Fig 1D and 1E ) , resulting in a reduced mean spontaneous firing rate over the recorded neuronal population . The effect scaled with increasing light intensity: the index of change in FRbase increased with increased activation of ChR2 ( S3A and S3B Fig ) . Conversely , suppression of PVs ( 532 nm , 10 mW/mm2 ) increased mean FRbase ( Fig 1I and 1J ) . These changes in spontaneous firing rate demonstrate that the optogenetic manipulation of PV activity efficiently altered neuronal activity in the AC . Direct optogenetic manipulation of excitatory neurons was similarly effective: photoactivation of CamKIIα neurons by blue light increased the spontaneous activity of neurons ( Fig 1N and 1O ) . The effect of light in the CAMKIIα-ChR2 group was either the same or larger than in the PV-Arch group ( S2 Fig ) , allowing for comparison of effects of suppression of PVs in PV-Arch group to direct activation of excitatory neurons in CAMKIIα-ChR2 group . We next tested the function of PVs in behavioral frequency discrimination acuity . To determine whether PV activity affects behavioral frequency discrimination acuity , we measured the change in frequency discrimination threshold ( Th ) , while modulating PV activity . Th was determined by measuring the percent inhibition of the ASR due to a shift in frequency between a background and a pre-pulse tone for varying frequencies of the pre-pulse [3 , 40] ( Fig 2A ) . Strong PPI of the ASR indicates that the mouse detected the shift in frequency between the background and prepulse tones ( Fig 2B , 2D and 2F ) . As previously reported [3] , PPI increased with larger frequency shifts between the background and prepulse tones . This method thus provides psychometric response curves for frequency discrimination over the course of a single session that lasts less than 1 hr and does not require training the subject . Th was computed as the percent difference in frequency between the background and the prepulse tone that elicited 50% of the maximum PPI ( S4 Fig ) . To test the effect of PV activity on Th , the laser was turned on during half of the behavioral trials , overlapping with the startle and prepulse stimuli ( light-On trial ) . On the remaining ( light-Off ) trials , the laser was turned on at a quasirandom time during intertrial interval . In an additional test session , the light was not used throughout ( no-light ) . Optogenetic modulation of PV activity significantly affected behavioral frequency discrimination acuity . Activating PVs improved frequency discrimination acuity , as evidenced by a reduction in Th for light-On trials as compared to light-Off trials and no-light session in PV-ChR2 mice ( Fig 2C ) . Suppressing PV activity reduced frequency discrimination acuity , leading to a significant increase in Th in PV-Arch mice ( Fig 2E ) . Combined , these results demonstrate that the level of PV activity bidirectionally controls behavioral frequency discrimination acuity . We performed several controls to ensure that the effects of photomodulation of PV activity were specific to the shift in frequency and could not be explained by a change in the ability of the mouse to respond to and to hear the stimuli . First , we tested whether light alone affected Th . In a control group of PV-Cre mice , in which PVs were driven to express only the fluorescent marker , but not the opsin , light did not affect Th ( S5 Fig ) . This indicates that the observed change in Th required the expression of opsins in PVs . In mice expressing ChR2 or Arch , neither activation nor suppression of PVs affected the magnitude of ASR elicited by startle stimulus alone ( S6A Fig ) . Therefore , the observed change in Th was not simply due to a change in the magnitude of ASR . Furthermore , activating or suppressing PVs did not lead to a change in the maximum PPI elicited by the pre-pulse tone ( S6B Fig ) . These tests indicate that photomodulation of PV activity did not affect the ability of the mouse to detect large shifts in frequencies . To further test that photomodulation of PVs did not impair the mouse's ability to hear test tones , we measured PPI due to the prepulse tones alone , without the background , as an estimate of how strongly the mouse could detect the prepulse tone . PPI elicited by the pre-pulse tones was not significantly different on light-On and light-Off trials ( S7 Fig ) . Furthermore , there was no difference in PPI elicited by the prepulse tone at all six frequencies tested , indicating that mice detected the different tones similarly well on both light-On and light-Off trials . Taken together , these controls demonstrate that the observed change in Th cannot be explained by changes in more basic aspects of mouse hearing or a non-specific effect of photostimulation . We repeated the experiments , activating the excitatory neurons directly in the CamKIIα-ChR2 group . In striking contrast to the effect of PV inactivation in PV-Arch mice , direct activation of principal neurons did not affect Th ( Fig 2G ) . This result demonstrates that the change in Th due to photosuppresson of PV activity is specific to the effect of inhibitory interneurons , and is not simply due to an increase in the mean firing rate of excitatory neurons during PV suppression . Can the changes in neuronal activity in AC evoked by the different types of optogenetic manipulation explain the behavioral results ? To answer this question , we measured how strongly photostimulation of PVs affected the responses of neurons during tone presentation in a frequency band of one octave centered at the best frequency ( BF ) . To estimate the relative strength of population neuronal responses to tones , we computed the tone-evoked response magnitude measured as a difference between mean firing rate during tone presentation ( FRtone ) and FRbase ( Fig 3 ) . Photomodulation of PVs resulted in a significant change in the magnitude of normalized tone-evoked response over the population of putative excitatory neurons . Photoactivation of PVs increased the tone-evoked response magnitude ( Fig 3A and 3B ) . This effect was due to a relatively weaker decrease in FRtone as compared to the decrease in FRbase evoked by PV photoactivation ( Fig 3A and S3 Fig ) . By contrast , photosuppression of PVs led to a decrease in tone-evoked response magnitude ( Fig 3C and 3D ) . This effect was due to a relatively weaker increase in FRtone as compared to FRbase ( Fig 3C ) . These results were consistent with the mean behavioral results for changes in Th: PV photoactivation , which improved behavioral frequency discrimination acuity , also increased mean tone-evoked responses; whereas PV photosuppression , which impaired behavioral frequency discrimination acuity , also suppressed mean tone-evoked responses in AC . The effects of PV inactivation differed between subjects . Therefore , we computed a correlation between neuronal responses and behavioral performance over subjects by comparing the mean tone-evoked response magnitude over all neurons and behavioral Th for each mouse . Changes in neuronal responses caused by photomodulation of PV activity were significantly inversely correlated with changes in Th measured behaviorally ( Fig 3G ) . This correlation suggests that the measured change in magnitude of tone-evoked responses in AC is a good predictor for the change in behaviorally measured frequency discrimination acuity . By contrast , direct photoactivation of excitatory neurons in the CamKIIα-ChR2 group did not affect the tone-evoked response magnitude ( Fig 3E and 3F ) . This result is due to the strong increase in both the spontaneous and tone-evoked activity of recorded neurons by direct photoactivation of excitatory neurons ( S8A and S8B Fig ) . These results are consistent with the lack of change in behavioral frequency discrimination acuity due to photoactivation of excitatory neurons . Combined , our findings support the interpretation that both the bidirectional modulation of Th due to PV stimulation , and the lack of modulation due to excitatory neuronal stimulation , are due to changes in the magnitude of tone-evoked responses relative to the baseline firing rate of AC neurons . Frequency discrimination may be controlled not only by the firing rate of neurons but also by their frequency tuning properties [48] . Therefore , we next quantified the effect of PV photo-modulation on the frequency tuning properties of putative excitatory neurons . The mean firing rate of neuronal responses to tones was used to construct a tuning curve for the frequency and intensity level of the tones , computed on light-Off and light-On trials , separately ( Fig 4A–4C ) . It has previously been suggested that excitatory and inhibitory inputs to the same neurons exhibit similar frequency tuning properties in AC [49] . We therefore expected that the BF ( the frequency of the tone eliciting the highest firing rate ) would not be affected by PV photostimulation . Indeed , the BF of recorded units was not affected by PV photoactivation and photosuppression ( Fig 4D and 4E , respectively ) . However , PVs exhibit tuning that is similar [50] or lower [51] in selectivity to excitatory neurons . Therefore , manipulation of PV activity would likely affect the frequency selectivity of putative excitatory neurons to tones . Indeed , photostimulation modulated frequency selectivity of neuronal responses . We quantified frequency selectivity by two measures: the width of frequency tuning and the sparseness of the frequency response function . Tuning width was computed as twice the standard deviation of the Gaussian fit to the frequency response function . Tuning width decreased during activation of PVs and increased during suppression of PVs ( Fig 4G and 4H , respectively ) . We used sparseness as an additional measure for frequency tuning selectivity , because it is less sensitive to the magnitude of the firing rate as well as spontaneous firing rate than tuning width . In addition , sparseness does not assume a specific shape of the frequency response function . A sparseness value of 1 indicates that the neuron responds to tone at only one frequency , whereas a sparseness value of 0 indicates that the neuron responds equally strongly to tones at all frequencies . Activating PVs significantly increased sparseness over the population of putative excitatory neurons ( Fig 4J ) . The strength of the effect of photoactivation on neuronal sparseness increased with light intensity ( S9A and S9B Fig ) and was significantly correlated with the change in the baseline firing rate ( S9C Fig ) . Conversely , suppressing the activity of PV interneurons significantly reduced the sparseness of neuronal responses to tones ( Fig 4K ) . As expected , the effects of photomodulation on sparseness and tuning width were significantly correlated ( Fig 4M and 4N ) . Combined , we found that up- or down-regulating activity of PVs did not affect the BF of neurons , but modulated the tuning selectivity of principal AC neurons , such that activating PVs increased neuronal frequency selectivity , whereas suppressing PVs reduced neuronal frequency selectivity . On average , the mean changes in frequency selectivity were consistent with behavioral results: activation of PVs , which improved frequency discrimination acuity , increased frequency selectivity in AC neurons , whereas suppression of PVs , which impaired frequency discrimination acuity , decreased frequency selectivity in AC . However , when examined on an animal-by-animal level , there was no significant correlation between frequency sparseness and the change in behavioral threshold when tested using either parametric or nonparametric tests ( Fig 4P ) . This result suggests that mean frequency selectivity may not be as important for behavioral frequency discrimination acuity as the response magnitude for tones of preferred frequencies . We next tested whether photoactivation of excitatory neurons affected mean neuronal frequency tuning . Over the population of recorded neurons , the BF was not affected ( Fig 4F ) . However , the tuning width increased significantly ( Fig 4I ) , whereas sparseness of frequency responses decreased ( Fig 4L ) . As in PV-Cre mice , the tuning width and sparseness significantly correlated with each other ( Fig 4O ) . These measurements contrast with the behavioral findings that photoactivation of excitatory neurons does not affect frequency discrimination acuity , further supporting the interpretation that frequency selectivity may not be as important for behavioral frequency as changes in tone-evoked response magnitude . Thus far in the behavioral test , we examined frequency discrimination acuity using a modified procedure that relied on measuring inhibition of the startle response by a tone preceding the startle noise—an innate behavioral response measured as PPI . We then tested whether inhibition in AC also modulated auditory associative learning [52] . In DAFC , the mouse is presented with two tones of different frequencies , one of which ( CS+ ) is associated with an aversive stimulus ( mild electric foot shock ) and one that is not ( CS− ) ( Fig 5A and S10 Fig ) . 24 h later , the mice typically exhibit an increase in conditioned response ( freezing ) during presentation of CS+ and a smaller increase in freezing during presentation of CS− . For different subjects , the freezing response may be specific to CS+ or generalize to tones at frequencies beyond CS− [3] . We hypothesized that specificity of freezing after conditioning may be controlled by PVs in AC . To test this hypothesis , we measured whether up- or down-regulating the activity of PVs in AC during conditioning affects the specificity of the freezing response . We subjected four groups of mice to DAFC , overlapping light and tone presentation . In the PV-Arch group , suppression of PVs during conditioning led to activation of putative excitatory neurons . In the PV-ChR2 group , photoactivation of PVs during conditioning led to suppression of putative excitatory neurons . In the control group of mice , which were injected with control vector that encoded only fluorescent protein , PVs were not affected by the laser . In CamKIIα-ChR2 group , the activity of excitatory neurons was enhanced during conditioning . 24 h following DAFC , we tested the level of freezing to CS+ , CS− and two additional tones during the LS test , designed to measure how specific freezing response was to conditioned tones ( Fig 5A ) . We then assessed the level of specificity of conditioned response by measuring the relative difference in freezing response to the CS+ tone and mean freezing response to test tones ( LS index , Methods ) . In all groups , mice exhibited an increase in the freezing response to CS+ ( Fig 5B and 5C ) . However , mice in which PVs were suppressed during conditioning did not exhibit differential freezing response to CS+ and CS− . By contrast , mice in both the PV-ChR2 and the control groups exhibited a significant reduction in freezing to CS− as compared to CS+ . Furthermore , the specificity of learned freezing as measured by LS was significantly lower than for mice in PV-Arch group than for mice in control group ( Fig 5D ) . Interestingly , direct activation of excitatory neurons in CamKIIα-ChR2 group did not result in significant change of LS ( Fig 5C and 5D ) . Thus , we find that suppressing PV activity during conditioning led to a decrease in specificity of auditory fear conditioning , whereas either increasing PV activity or increasing the general level of activity of excitatory neurons did not have a significant effect on the specificity . As expected , between subjects , the level of specificity of conditioned fear varied . If inhibition in AC controls both the frequency discrimination acuity and specificity of the conditioned response via a similar mechanism , we expected the behavioral measures for acuity and specificity to be correlated . Indeed , change in behavioral frequency discrimination acuity due to photostimulation was significantly correlated with the change in specificity of auditory fear conditioning ( Fig 5E ) . Furthermore , there was a significant correlation between LS and the effect of photomodulation of PVs activity on neuronal tone-evoked response magnitude but not sparseness ( Fig 5F ) . Combined , these findings demonstrate that neuronal response magnitude in AC regulates not only behavioral frequency discrimination acuity measured through a test of innate behavior , but also specificity of associative learning . We investigated a model of excitatory–inhibitory circuit interactions to better understand why manipulation of activity of PVs , but not excitatory neurons , affects the magnitude of tone-evoked responses . We constructed a firing-rate model as an extended Wilson-Cowan model of mutually connected excitatory and-inhibitory neuronal populations [53–55] . In this model , the inhibitory neuronal population integrates depolarizing currents from tone-evoked inputs and inputs from the excitatory neurons , whereas the excitatory neurons integrate tone-evoked inputs and hyperpolarizing currents from inhibitory neurons ( Fig 6A , S14A Fig and Methods ) . Optogenetic modulation was modeled as an additional input current delivered to either excitatory or inhibitory neuronal populations . This simple simulation provided for a biological implementation of the circuit that is consistent with our experimental findings . Inputs from PVs to excitatory neurons have been shown to exhibit synaptic depression [56 , 57] . We incorporated synaptic depression at the PV to excitatory synapse in the model ( Fig 6 ) . The model here did not assume a specific form ( e . g . , pre- or postsynaptic ) of depression . Rather , we modeled synaptic transfer function as a nonlinearity , using a closed form solution for the relation between the output of the inhibitory neuronal firing rate and the input current for the excitatory neuronal population assuming depressing synaptic dynamics ( see Methods ) . A simulation of excitatory neuronal responses exhibited the differential effects of inhibitory and excitatory stimulation of interneurons as well as lack of effect of stimulating the excitatory neurons directly on tone-evoked responses: Activating PVs increased the tone-evoked responses , whereas suppressing PVs decreased the tone-evoked responses of the excitatory population ( Fig 6B and 6C ) . By contrast , activating excitatory population directly did not change the tone-evoked response magnitude ( Fig 6B and 6C ) . This simulation thus provides for one plausible biological implementation of the circuit that is consistent with our experimental findings . To develop a more basic understanding of the circuit , we implemented an instantaneous sigmoidal input–output nonlinearity with varying coefficients after synaptic integration for either excitatory or inhibitory neurons ( S14 Fig ) . We used three different scenarios ( S14A Fig ) : under scenario 1 , the nonlinearity operates in a linear regime for both the excitatory and the inhibitory populations; under scenario 2 , the nonlinearity is saturating for the excitatory , and linear for the inhibitory , neuronal population; under scenario 3 , the nonlinearity operates in a saturating regime for the inhibitory population , and a linear regime for the excitatory population . Only scenario 3 ( S14B–S14E Fig right ) supported our experimental findings that a ) suppressing PV activity increased the magnitude of tone-evoked responses ( Fig 3A ) ; b ) increasing PV activity decreased the magnitude of tone-evoked responses ( Fig 3C ) ; and c ) activating excitatory neurons directly did not affect tone-evoked response magnitude ( but increased both the spontaneous and the tone-evoked firing rate by the same amount ) ( Fig 3E ) . Under scenario 1 , activation of excitatory neurons did not affect tone-evoked response amplitude , but neither did activation or suppression of inhibitory neurons ( S14B–S14E Fig , left ) . Under scenario 2 , activation or suppression of inhibitory neurons decreased or increased tone-evoked response magnitude , respectively ( S14B–S14E Fig center ) ; however , activation of excitatory neurons decreased tone-evoked response magnitude . Therefore , the scenario 3 , under which the excitatory neurons integrate their inputs close to linear , but the inhibitory inputs are passed through a saturating nonlinearity , is consistent with our data ( S14B–S14E Fig right ) . The synaptic depression model ( Fig 6 ) can be viewed as a special case of scenario 3 , in which the transfer function between inhibitory and excitatory neuronal population saturates . Indeed , a number of other circuits , for example activation of an additional class of interneurons , such as somatostatin-positive interneurons [51] , could potentially provide for a saturating transfer function .
Our results demonstrate that auditory cortical neurons regulate auditory behaviors that rely on frequency discrimination , and that this regulation can be facilitated by the overall activity level of a specific type of inhibitory , but not excitatory neurons . Optogenetic modulation of the level of activity of PV-positive interneurons drove changes in frequency discrimination acuity and specificity of auditory conditioning ( Fig 2 and Fig 5 ) . At the neuronal level , we find that modulating the level of PV activity differentially affects the spontaneous and the tone-evoked responses of putative excitatory neurons ( Fig 1 and Fig 3 ) . The changes in tone-evoked responses magnitude were correlated with behavioral performance ( Fig 3G and Fig 5F ) . Whereas activating PVs during fear conditioning preserved specificity of conditioned fear , consistent with a previous pharmacological study [58] , suppressing PVs increased generalization of fear responses . These effects of PVs extend beyond controlling the overall firing rate of excitatory neurons as changing the gain of excitatory neuronal responses directly did not lead to similar changes in behavioral performance ( Fig 2G and Fig 5C ) . This difference can be attributed to a nonlinear relationship between inhibitory input from PVs and output firing rate of excitatory neurons , consistent with a mechanism of synaptic depression that has been identified at the synapse from PVs to excitatory neurons ( Fig 6 ) [57] . Combined , our results support the view that PVs regulate signal-to-noise ratio of responses of principal neurons , extending beyond the effect of a global gain control , and that this dual effect on the spontaneous and tone-evoked activity affects behavioral frequency discrimination . Our electrophysiological results demonstrating that activating PVs leads to narrower frequency tuning of putative excitatory neurons whereas suppressing PVs leads to broader frequency tuning ( Figs 3 and 4 ) are consistent with previous pharmacological and electrophysiological investigations of inhibitory neuronal responses [59–63] . Behaviorally , while PVs have been implicated in two separate auditory behaviors: detection of temporal gap in sound [11] and in disinhibition of responses to tones during aversive stimulus presentation in AFC [43] , our results provide for the initial demonstration of the role of PVs in auditory tasks relying on frequency discrimination . Our findings are thus consistent with those in the visual system , where PVs have been found to modulate responses of principal cells to visual stimuli and affect visual discriminative behavior [64–67] . Optogenetic approaches act on different timescales than lesion studies , or pharmacological methods for neuronal activity suppression . Lesioning or pharmacologically inactivating AC previously provided mixed effects on frequency discrimination , with some studies resulting in small , if any impairments in frequency discrimination performance [3 , 5 , 17 , 68] , whereas other studies exhibited stronger effects [69] . These results are not inconsistent with the present findings: lesioning and pharmacological studies are performed on much longer time scales ( hours to days [59] ) , as compared to the millisecond timescale of optogenetic perturbation . Therefore , lesioning or pharmacologically suppressing AC potentially allows for other neuronal circuits to take over frequency discrimination function , similarly to brain reorganization in response to injury [70] or simply abnormal lack of activity . Our results therefore support a modulatory , but not necessary , role for AC in frequency discrimination: when AC is “online” , excitatory–inhibitory circuits control frequency discrimination behavior , and their perturbation modulates frequency discrimination behaviors . By contrast , lesioning or suppressing AC pharmacologically for extended periods of time potentially allows for other brain areas to take over control of frequency discrimination . Behavioral frequency discrimination acuity was tested through a task that is based on an innate , rather than learned response [3 , 40 , 71] . Implementing the PPI-based behavioral task has the advantage that the animal does not need to be trained on the task , and therefore allows for dissociation of perceptual report and learning . A recent study has found that corticocollicular feedback affects learning-induced changes in auditory spatial learning [72] . Here , similarly , AC may affect PPI through corticocollicular feedback , as PPI is controlled by the inferior-colliculus to pedunculopontine nucleus connection [39 , 73] . Future studies , including a test of the effect of inactivation of corticocollicular feedback , are needed to determine which of the possible circuits downstream of AC drive the observed behavioral changes . Regulation of auditory frequency discrimination by the AC is not restricted to the PPI circuit , as we find that AC also regulates how specific conditioning is to a particular frequency of the tone . A number of studies have demonstrated that the AC plays an important role in fear conditioning [30] . Our results identify that the AC shapes frequency specificity of DAFC: suppressing the activity of interneurons decreased the specificity of DAFC , as the subjects generalized the conditioned response to the full range of tones on which they were tested ( Fig 5 ) . Several circuits may underlie this effect: AC projects to the amygdala , a crucial brain area in auditory fear conditioning , via the secondary AC or via feedback through the thalamus [41 , 74] . Applying selective manipulation to elements in these circuits in future studies will be necessary to learn how AC controls associative learning . Interestingly , activating PVs did not increase the specificity of auditory associative learning , measured by LS , as would have been expected from frequency discrimination results . This suggests that the limits to specificity of auditory associative learning may not only be set by the AC , but may also rely on other brain regions , which would have a lower frequency resolution than the AC . Furthermore , it points to an asymmetry between the effects of activation or suppression of circuit elements: taking out a crucial element of a circuit led to a qualitatively different effect than increasing the activity of an already present element . Our results point to remarkable robustness of frequency discrimination to the overall level of activity in the AC . Whereas direct photoactivation of excitatory neurons dramatically increased the overall firing rate in the cortex , at the behavioral level , we did not observe a change in either behavioral frequency discrimination , as measured by Th , or in specificity of DAFC ( Fig 2F and 2G , Fig 5C ) . This robustness to the mean firing rate level may underlie important perceptual effects , such as the ability to preserve acoustic discrimination or speech comprehension in different acoustic environments . Our results provide for a mechanism by which the AC may modulate learning-driven changes in frequency discrimination following emotional learning [3] . Previously , we found that frequency discrimination acuity and specificity of learning were correlated across subjects , pointing to a common mechanism that controls the two behaviors . We identified AC as a candidate brain area for controlling frequency discrimination acuity and DAFC , as pharmacological inactivation of AC abolished DAFC-induced change in frequency discrimination acuity [3] . Inhibitory neurons differentially process auditory information and are affected by auditory learning and experience [13 , 75 , 76] . Our present results are consistent with the possibility that the learning-driven changes in frequency discrimination may be due to inhibitory interneuron activity or plasticity in inhibitory–excitatory connections . Combined , we find that modulating frequency response properties of neurons in AC via activity of PVs modulates frequency discrimination acuity and specificity of auditory associative learning , confirming an important role for inhibitory circuits in AC in auditory behavior . While PVs are the most common type of interneurons in AC , other interneuron types , such as somatostatin-positive and vasoactive intestinal peptide-expressing inhibitory interneurons , may play additional complementary roles in shaping frequency discrimination , through more complex circuits . It will be important to tease apart the function of different cortical circuits in the processing of spectral information .
All experiments were performed in adult male mice ( supplier: Jackson Laboratories; age , 12–15 wk; weight , 22–32 g; PV-Cre mice , strain: B6; 129P2-Pvalbtm1 ( cre ) Arbr/J; CamKIIα-Cre: B6 . Cg-Tg ( CamKIIα-Cre ) T29-1Stl/J; wild-type control: C57BL/6J ) housed at 28°C on a 12 h light–dark cycle with water and food provided ad libitum , less than five animals per cage . In PV-Cre mice Cre recombinase ( Cre ) was expressed in PPI , and in CamKIIα-Cre , Cre was expressed in excitatory neurons [77] . All animal work was conducted according to the guidelines of University of Pennsylvanian IACUC and the AALAC Guide on Animal Research . Anesthesia by isofluorane and euthanasia by carbon dioxide were used . All means were taken to minimize the pain or discomfort of the animals during and following the experiments . All behavioral experiments were performed during the animals' dark cycle . At least 10 d prior to the start of experiments , mice were anesthetized with isoflurane to a surgical plane . The head was secured in a stereotactic holder . The mouse was subjected to a small craniotomy ( 2 x 2 mm ) over AC under aseptic conditions . Viral construct was injected using syringe pump ( Pump 11 Elite , Harvard Apparatus ) targeted to AC ( coordinates relative to bregma: −2 . 6 mm anterior , ±4 . 2 mm lateral , +1 mm ventral ) . Fiber-optic cannulas ( Thorlabs , Ø200 μm Core , 0 . 22 NA ) were implanted bilaterally over the injection site at depth of 0 . 5 mm from the scull surface . Craniotomies were covered with a removable silicon plug . A small headpost was secured to the skull with dental cement ( C&B Metabond ) and acrylic ( Lang Dental ) . For postoperative analgesia , Buprenex ( 0 . 1 mg/kg ) was injected intraperitonially and lidocaine was applied topically to the surgical site . An antibiotic ( 0 . 3% Gentamicin sulfate ) was applied daily ( for 4 d ) to the surgical site during recovery . Virus spread was confirmed postmortem by visualization of the fluorescent protein expression in fixed brain tissue , and its colocalization with PV or excitatory neurons , following immuno-histochemical processing with the appropriate antibody . Modified AAV vectors were obtained from Penn VectorCore . Vector encoding light-gated proton pump Archaerhodopsin ( Arch ) under FLEX promoter was used for selective suppression of PVs ( Addgene plasmid 22222 , AAV-FLEX-Arch-GFP [35] ) . Modified AAV encoding ChR2 under FLEX promoter ( Addgene plasmid 18917 AAV-FLEX-ChR2- tdTomato , ChR2 [78] ) was used for activation of either PVs iin PV-Cre mice and or excitatory neurons in CamKIIα-Cre mice . Modified AAV vectors encoding only GFP or tdTomato under FLEX promoter were used as a control for the specific action of Arch and ChR2 on the neuronal populations . Brains were extracted following perfusion in 0 . 01 M phosphate buffer pH 7 . 4 ( PBS ) and 4% paraformaldehyde ( PFA ) , postfixed in PFA overnight and cryoprotected in 30% sucrose . Free- floating coronal sections ( 40 μm ) were cut using a cryostat ( Leica CM1860 ) . Sections were washed in PBS containing 0 . 1% Triton X-100 ( PBST; 3 washes , 5 min ) , incubated at room temperature in blocking solution ( 10% normal goat serum and 5% bovine serum albumin in PBST; 3h ) , and then incubated in primary antibody diluted in blocking solution overnight at 4°C . The following primary antibodies were used: anti-PV ( PV 25 rabbit polyclonal , 1:500 , Swant ) , or anti-CAMKIIα ( abcam5683 rabbit polyclonal , 1:500 , abcam ) . The following day sections were washed in blocking solution ( 3 washes , 5 min ) , incubated for 1hr at room temperature with secondary antibodies ( Alexa 594 or Alexa 488 goat anti-rabbit IgG; 1:1 , 000 ) , and then washed in PBST ( 4 washes , 10 min ) . Sections were mounted using fluoromout-G ( Southern Biotech ) and confocal or fluorescent images were acquired ( Leica SP5 or Olympus BX43 ) . To quantify viral expression efficiency and specificity , cells in the proximity of injection site were identified in independent fluorescent channels and subsequently scored for colocalization using ImageJ’s cell counter plug-in . Neurons were stimulated by application of continuous light pulse delivered from either blue ( 473 nm , BL473T3-150 , used for ChR2 stimulation ) or green DPSS laser ( 532 nm , GL532T3-300 , Slocs lasers , used for Arch stimulation ) through implanted cannulas . Timing of the light pulse was controlled with microsecond precision via a custom control shutter system , synchronized to the acoustic stimulus delivery . Prior to the start of the experiment , the intensity of blue laser was adjusted to one of three values 0 . 2 , 0 . 5 , or 10 mW/mm2 as measured at the tip of the optic fiber . On average , the lowest light power was sufficient to induce significant reduction in Th ( paired t test , t19 = 2 . 68 , p = 0 . 015 ) . However , in a small fraction of mice ( 6 out of 20 ) , higher power was needed to induce reduction in Th ( 0 . 5 mW/mm2 in 5 mice , and 10 mW/mm2 in 1 mouse ) . The same power was used in auditory discriminative fear conditioning for each subject . Green laser was used at intensity of 10 mW/mm2 , which resulted in similar absolute magnitude of change in spontaneous firing rate over the neuronal population as the lowest level of ChR2 activation ( S3 Fig ) . An additional experiment carried out using 6 subjects ( 2 PV-Arch , 2 PV-ChR2 and 2 CAMKIIα-ChR2 ) demonstrated that photoactivation and suppression of neurons was confined to the AC ( S2 Fig ) . The effect of light on firing rate significantly decayed over distance , but was heterogeneous over cortical depth ( S2 Fig ) . During FC , the mouse was placed in a conditioning cage with a shock floor ( Coulbourn ) inside sound attenuation cubicle ( Med Associates ) , housed in a single-walled acoustic chamber ( Industrial acoustics ) . Throughout conditioning , the cage was illuminated with LED light , the color of which corresponded to the color of laser used for photoactivation of neurons ( blue LED: 470 nm , 170 mW; green LED: 525 nm , 7 mW ) . During LS tests , a custom-made test cage of similar size but different floor and wall pattern and color was used . Auditory stimuli were provided by a free-field magnetic speaker ( Tucker-Davis Technologies ) . Electric shock ( 0 . 5mA , 0 . 5 s ) was delivered by precision animal shocker ( Coulbourn ) . Freezeframe-3 software ( Coulbourn ) was used for stimulus control and analysis of freezing behavior . During the PPI procedure , the mouse was placed in a custom-made tube on the sensor plate ( San Diego Instruments ) and head-fixed using implanted headpost . The speaker , housing , platform and webcam ( Logitech ) were placed in the sound attenuation cubicle ( Med Associates ) , housed in a single-walled acoustic chamber . During tests , the housing was illuminated with LED light , the color of which corresponded to the color of the laser used for photoactivation of neurons . The speaker was positioned above the mouse . The sound delivery apparatus was calibrated using a 1/8-inch condenser microphone ( Brüel&Kjær , Denmark ) positioned at the expected location of the mouse's ear , to deliver each stimulus at 70 dB sound pressure level relative to 20 microPa ( SPL ) . All pure tones presented during training and test sessions were at 70 dB SPL . Seven to ten days after surgery , mice were subjected to at least three consequent days of habituation to experimental setups . During habituation to PPI apparatus , the duration of which gradually increased from 10 to 20 min over 3 d , mice were head fixed and optic fibers connected to cannulas . Following habituation , mice underwent daily PPI testing for frequency discrimination , which lasted for 1–3 d . Following PPI testing , a subset of mice underwent fear conditioning ( FC ) and one day thereafter they were tested for specificity of conditioned fear response . After termination of behavioral experiments , mice were used for electrophysiological recordings . In order to examine whether fear conditioning alters the effect of photoactivation on base firing rate of neurons and their tuning properties , we performed recordings in subgroup of PV-ChR2 mice without subjecting them to fear conditioning ( “naïve” group , n = 6 ) . Comparison of the change in spontaneous and tone-evoked firing rate and sparseness induced by photoactivation between “naïve” group and group that underwent fear conditioning did not reveal significant difference ( S11 Fig ) . Therefore , recording data collected from these groups were pooled . All behavioral experiments were performed during animals’ dark cycle . The measurement of frequency discrimination acuity used a modified PPI of the startle reflex protocol as previously described [3 , 40] . The test measured the magnitude of the ASR to the startle stimulus ( SS ) as a function of the difference in frequency between the background tone and the prepulse tone ( PP ) , which immediately preceded SS . The frequency of the background tone was 15 . 0 kHz . The background tone ( when used ) was presented continuously between the end of SS and the start of PP . The transition between the background tone and PP included 1 ms ramp to avoid clicks . Five frequencies used for PP ( 10 . 2 , 12 . 6 , 13 . 8 , 14 . 7 , and 15 . 0 kHz ) were presented pseudo randomly with 10–20 s ISI , which also varied randomly . Thus , PP differed from the background tone by 0 , 2 , 4 , 8 , 16 and 32% . PP was 80 ms long and was presented right before SS . SS was broadband noise , presented at 100 dB SPL for 20 ms . The magnitude of ASR was measured using a forcesensor plate ( San Diego Instruments ) and defined as the maximum vertical force applied within the 500 ms window following SS minus average baseline activity during 500 ms prior to SS . In each PPI session , 50% of the strongest ASRs for each frequency were averaged and used to calculate PPI: PPI ( % ) =100ASRnoPP−ASRPPASRnoPP where ASRnoPP is the response when PP frequency is equal to the frequency of the background tone ( 15 kHz ) and ASRPP is the response after frequency shift has occurred . ( a ) To assess baseline frequency discrimination mice were subjected to the PPI procedure without photostimulation of neurons . Each test session consisted of 9 startle-only trials , followed by at least 100 pre-pulse trials , followed by one additional startle-only trial . On startle-only trials , background tone was followed directly by SS . On pre-pulse trials , each PP was presented 20 times in quasi-random order with ITI varying randomly between 10 and 20 s . Negative frequency changes were used because mice have been previously shown to be more sensitive to downward frequency shifts[3 , 40] . ( b ) To compare the effect of photoactivation or suppression of PVs on frequency discrimination , mice were subjected to a protocol similar to that described above , but including light delivery though implanted cannulas . On light ‘On’ trials , the laser was presented for 1 s , starting 0 . 5 s before PP onset . On light ‘Off’ trials laser was presented at quasi-random position during ITI . ‘On’ and ‘Off’ trials were shuffled randomly . ( c ) To compare the subjective detectability of 5 experimental tones the background tone was omitted . The session started with 5 startle-only ( no PP presentation ) trials , followed by 50 pre-pulse trials , and terminated by 5 additional startle-only trials . On pre-pulse trials , each PP was presented 10 times in quasi-random order with ITI varying randomly between 10 and 20 s . The amplitude of each tone was then adjusted so that PPI induced by each tone was similar ( S12 Fig ) . The Th was defined as a frequency shift that caused 50% inhibition of the maximum ASR . Th is determined from a parametric fit to a generalized logistic function: PPI=a1+exp ( b+cΔf ) In a standard PPI session , 20 repetitions of each PP were presented ( 100 trials in total ) . However , if either Th was out of the range ( 0 . 5–32% ) or the fit coefficient of the curve ( R2 ) was below 0 . 7 , the mouse underwent an additional 10 repetitions ( 50 trials ) . If Th and fit curve failed to meet the above criteria after 200 trials , the session was excluded from statistical analysis ( 3 out of 61 sessions ) . During FC , following 5 min of silence , 10 tones ( 15 . 0 kHz , 10 . 5 s ) co-terminated with a foot shock ( CS+ ) were presented , at an inter-trial interval ( ITI ) randomly varied between 2 to 6 min . In addition , 10 tones at 11 . 25 kHz ( 10 . 5 s ) , not paired with foot-shock ( CS- ) were presented in random order with 2 min inter-stimulus interval ( ISI ) . Photoactivation and suppression of neurons was performed by delivery of light through implanted cannulas . In one group of mice , photo stimulation started 0 . 5 s before CS+ and CS- onset and co-terminated with the tone ( 11 s total ) . In another group , photo stimulation was terminated 1 s before the tone offset to avoid overlapping with the foot-shock ( 10 s total ) . The LS test consisted of CS+ and three test tones ( 3 . 75 , 7 . 5 , 11 . 25 kHz ) , presented 3 times at 3 min ISI . LS was assayed as the difference in freezing response to CS+ and mean freezing response to three test tones: LS ( % ) =100FCS+−〈Ftest〉FCS+ Where FCS+ is freezing ( % ) during CS+ tone presentation and Ftest is mean freezing during test tones . During conditioning and test sessions , freezing responses were video-recorded and analyzed offline using Freeze Frame software . Freezing responses were judged as complete immobility of the mouse for at least 1 s . Average freezing response during 20 s before the test tones was recorded as baseline , while freezing response during the test tones was recorded as the conditioned response . Subjects that exhibited either very low conditioned freezing to CS+ tone ( <20% , n = 2 ) or very low locomotion throughout the test ( >50% , n = 1 ) were excluded from statistical analysis . During conditioning , photostimulation was presented during CS+ and , in most subjects , terminated 0 . 5 s before the onset of the footshock . However , in a subset of mice ( ChR2: N = 5 , Arch: N = 4 ) , photostimulation overlapped with the footshock ( S13A Fig ) . While overlapping the photostimulation with the footshock affected the freezing response in PV-ChR2 group ( S13B Fig ) , as previously described [43] ) , it did not result in a significant difference in LS ( S13C Fig ) . In PV-Arch group , we did not observe significant effect of the overlap of photostimulation with the footshock on either freezing response ( S13D Fig ) or LS ( S13E Fig ) . Therefore , the two subsets of mice were combined for subsequent analysis within each group . All recordings were carried out inside a double-walled acoustic isolation booth ( Industrial Acoustics ) . Mice were placed in the recording chamber , and a headpost was secured to a custom base , immobilizing the head . Activity of neurons in the primary AC was recorded via a silicon multi-channel probe ( Neuronexus ) , lowered in the area targeting AC via a stereotactic instrument following a durotomy . The electrode tips were arranged in a vertical fashion that permits recording the activity of neurons in different cortical laminae . Electro-physiological data from 32 channels were filtered between 600 and 6000 Hz ( spike responses ) , digitized at 32kHz and stored for offline analysis ( Neuralynx ) . Spikes belonging to single neurons were detected using commercial software ( Plexon ) [79] . Stimulus was delivered via a magnetic speaker ( Tucker-David Technologies ) , calibrated with a Bruel and Kjaer microphone at the point of the subject's ear , to deliver tones at frequencies between 1 and 80 kHz to +- 3 dB [79] . To measure the frequency tuning curves , we presented a train of 50 pure tones of frequencies spaced logarithmically between 1 and 80 kHz , at 8 intensities spaced uniformly between 10 and 80 dB , each tone repeated twice in pseudo-random sequence , counter-balanced for laser presentation . The full stimulus was repeated 5 times . Each tone was 50 ms long , with inter-stimulus interval ( ISI ) of 450 ms . The light-Onset was presented during every other tone , with the onset of 100 ms prior to tone onset , and lasting for 250 ms . The effect of the light-On FR was assessed by as an index of change in FR in light-On and light-Off trials: ΔFR=FRON−FROFFFRON+FROFF The change was computed separately for the spontaneous and tone-evoked firing rate . The spontaneous firing rate ( FRbase ) was computed by averaging FR over 50 ms before tone-Onset across light-On and light-Off trials . The tone-evoked firing rate ( FRtone ) was computed as the average of FR of responses to tones at 60–80 dB SPL at 0–50 ms after tone onset were averaged . To examine frequency selectivity of neurons , sparseness of frequency tuning was computed as: Sparseness=1− ( ∑i=1i=nFRi/n ) 2∑i=1i=nFRi2/n where FRi is tone-evoked response to tone at frequency i , and n is number of frequencies used . To compute the width and BF of tuning , the frequency response function was fitted Gaussian function: FR ( f ) =ae− ( f−fb ) 22σ2 where fb is the BF and σ is the standard deviation of the Gaussian function . The tuning width was measured in octaves as the difference between fb + σ and fb − σ for neurons , for which the Gaussian fit had R2>0 . 4 . Magnitude of neuronal response to tones was defined as the difference between mean spontaneous ( 0–50 ms before tone onset ) and tone-evoked ( 0–50 ms after tone onset ) firing rate and , for each neuron , normalized by setting the peak response magnitude between 0 and 50 ms after tone onset on light-Off trials to 1 . Only responses to tones within 0 . 5 octaves of BF of each neuron were included . To quantify correlation between neuronal responses and behavioral frequency discrimination , normalized tone-evoked response magnitude over all neurons recorded in each mouse was compared to changes in behavioral Th . Only mice with >5 identified single units ( 33 out of 36 mice ) were used for statistical analysis . First , we determined the criteria based on the spike waveform analysis . Putative PV interneurons in PV-ChR2 mice were preselected for waveform analysis if they exhibited a significant ( more than 2-fold ) increase in firing rate in response blue light ( 10 mW/mm2 ) and their spontaneous firing rate exceeded 3 Hz . Waveform analysis showed that spikes of these neurons have relatively low peak to trough amplitude ratio ( <1 . 2 , S1D Fig ) consistently with previous reports [50] . In order to exclude PV interneurons from the pool of neurons used for the analysis of tone-evoked responses , only neurons with peak to trough ratio that exceeded 1 . 2 were used . In addition , putative excitatory cells were identified based on their expected response patterns to sounds and lack of significant activation of the spontaneous firing rate by the laser in PV-ChR2 mice and suppression in PV-Arch mice [50 , 80] . While this subpopulation may still contain inhibitory neurons , the proportion of interneurons recorded was relatively small , as we used silicon electrode probes with relatively low impedance that do not target interneurons [50] . The low impedance of the probes precluded us from conducting a more detailed analysis for fast-spiking versus regular-spiking neurons based on the spike waveform [50] . We constructed a model of the excitatory-inhibitory neuronal circuit based on a firing rate model , based on Wilson-Cowan dynamics [53–55] . The mean activity level of each population was modeled as: dEdt=1τE[−E ( t ) + ( k−r ) S ( jCamK2 ( t ) +jETone ( t ) +Sinh ( jIEI ( t ) ) ] dIdt=1τI[−I ( t ) + ( k−r ) S ( jPV ( t ) +jITone ( t ) +jEIE ( t ) ) ] where E ( t ) is the firing rate of the excitatory population; I ( t ) is the firing rate of the inhibitory population; S ( x ) is the firing transfer function between the combined postsynaptic input and the neuronal firing rate; Sinh ( x ) is the transfer function between the inhibitory firing rate and excitatory postsynaptic current; jEI ( 0 . 2 ) and jIE ( −0 . 2 ) are excitatory–inhibitory and inhibitory–excitatory synaptic weights; jETone ( t ) and jITone ( t ) are tone-evoked input currents to excitatory and inhibitory neurons , respectively , modeled as 50 ms long exponentially decaying inputs of maximum amplitude 3; τ E ( 10 ms ) and τ I ( 10 ms ) are synaptic time constants for excitatory and inhibitory neurons; k and r represent the maximum and minimum firing rates of neurons respectively ( k = 15 , r = 1 ) ; jCamK2 ( t ) is the input to excitatory neurons due to ChR2-driven activation; jPV ( t ) is the input to inhibitory neurons due to either ChR2 ( positive ) or Arch ( negative ) . The optogenetic modulation was modeled as a unitary pulse of 250 ms in duration . We simulated activation of inhibitory neurons by setting jPV ( t ) = 1 , activation of excitatory neurons jCamK2 ( t ) = 1 . 5 , or suppression of inhibitory neurons by setting jPV ( t ) = −1 . The transfer functions is given by: S ( x ) = ( x−a ) ( b−a ) for a < x < b; S ( x ) = 0 for x < a; S ( x ) = 1 for x > b; where for excitatory neurons , a = −2 , b = 1 . 5; for inhibitory neurons , a = 0 , b = 4 . For the inhibitory-to-excitatory inputs , we used a simplified saturating transfer function , Sinh ( x ) , which is the quasistatic solution to a differential equation for the synaptic conductance g with depletion and replenishment given by: dgdt=−gr/Td+ ( g0−g ) /Tr Here , r is the presynaptic firing rate , g is the synaptic conductance , g0 is the maximum conductance , and Td and Tr are the time constants for depletion and replenishment , respectively . The input to the post-synaptic neuron is given by the product gr . Then , Sinh ( x ) =gx1+cxwhere g = 2 , c = 0 . 15 . For visualization , the firing rate of neurons was normalized as in Fig 3A , 3C and 3E , by subtracting the baseline firing rate , and setting the peak of the tone-evoked firing rate to 1 on light-off trials . Because most of behavioral experiments consisted of within-subject repeated measurements , most of the data were analyzed by either two-tailed paired t test or repeated-measures ANOVA using SPSS Statistics ( IBM ) or Matlab ( Mathworks ) . The effect of photoactivation and inactivation of neuronal activity on tuning properties was examined using a one-sample t test . Samples that did not pass Shapiro-Wilk test for normality were compared using Wilcoxon signed rank test . Multiple comparisons were adjusted by Bonferroni correction . Equality of variances was confirmed using Levene’s test .
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Hearing perception relies on our ability to tell apart the spectral content of different sounds , and to learn to use this difference to distinguish behaviorally relevant ( such as dangerous and safe ) sounds . Recently , we demonstrated that the auditory cortex regulates frequency discrimination acuity following associative learning . However , the neuronal circuits that underlie this modulation remain unknown . In the auditory cortex , excitatory neurons serve the dominant function in transmitting information about the sensory world within and across brain areas , whereas inhibitory interneurons carry a range of modulatory functions , shaping the way information is represented and processed . Our study elucidates the function of a specific inhibitory neuronal population in sound encoding and perception . We find that interneurons in the auditory cortex , belonging to a specific class ( parvalbumin-positive ) , modulate frequency selectivity of excitatory neurons , and regulate frequency discrimination acuity and specificity of discriminative auditory associative learning . These results expand our understanding of how specific cortical circuits contribute to innate and learned auditory behavior .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2015
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Bidirectional Regulation of Innate and Learned Behaviors That Rely on Frequency Discrimination by Cortical Inhibitory Neurons
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Dengue virus ( DENV ) nonstructural protein-1 ( NS1 ) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection . Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection . However , it has remained unclear how NS1 becomes associated with the plasma membrane , as it contains no membrane-spanning sequence motif . Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans , we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E . DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells . Moreover , DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells . This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues . Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection .
Dengue hemorrhagic fever and dengue shock syndrome ( DHF/DSS ) are severe and potentially fatal complications of infection by dengue virus ( DENV ) , a mosquito-borne RNA virus of the Flaviviridae family . Globally , DENV infects 25 to 100 million people per year , but the life-threatening complications primarily occur in school-age children [1] . Four serotypes of DENV exist , and DHF/DSS is commonly associated with secondary infection with a different virus serotype [2 , 3] . In the most severe cases , clinical deterioration is characterized by a rapid decline after several days of continuous high fever , thrombocytopenia , and selective vascular leakage at serosal sites [4] . The vascular leakage syndrome results in hemoconcentration , pleural effusions and ascites , and hypotension [4] . An effective strategy for disease prevention or treatment is currently lacking . The pathogenesis of DHF/DSS reflects a complex interplay of the host immune response and the viral determinants of virulence [5–7] . A model of immunopathogenesis has been suggested based on an increased risk of DHF with secondary infection and in children within the first year of life born to DENV-immune mothers [8 , 9] . From this , the hypothesis of antibody-dependent immune enhancement of infection emerged . In support of it , enhancement of DENV infection in monocytes in vitro with pre-illness serum correlates with increased risk of DHF in vivo [9 , 10] , and peak viremia is higher in patients with severe secondary DENV infection [11–13] . Differences in specific genetic determinants among viral isolates [14–16] also modulate virulence , as some DENV strains fail to cause severe disease [17 , 18] . A pathologic cytokine response that occurs after extensive T cell activation also likely contributes to the syndrome [5] . Elevated levels of cytokines including IFN-α , TNF- α , and IL-10 correlate with severe disease [19–24] , and the expansion of cross-reactive low-affinity DENV-specific T cells produces vasoactive cytokines [25–27] . Finally , accumulation of soluble NS1 in serum also correlates with disease severity and is believed to contribute to changes in vascular permeability through antibody-dependent activation of the complement cascade [28] . NS1 is a ∼48-kDa glycoprotein that is absent from the infectious viral particle . NS1 is , however , an essential gene within infected cells , as it functions as a cofactor for viral RNA replication , colocalizing with the double-stranded RNA replicative form [29 , 30] . NS1 is synthesized in an infected cell as a soluble monomer and rapidly dimerizes after post-translational modification in the lumen of the endoplasmic reticulum , with subsequent transport to the cell surface and release into extracellular milieu [31] . In solution , secreted NS1 behaves as a hexamer [32] and accumulates in serum in high amounts ( up to 50 μg/ml ) [11 , 28 , 33] . The mechanism ( s ) by which soluble and cell surface–associated NS1 contributes to flavivirus pathogenesis remains uncertain . It has been proposed to facilitate immune complex formation [28 , 34] , elicit auto-antibodies that react with platelet and extracellular matrix proteins [35 , 36] , cause endothelial cell damage via antibody-dependent complement-mediated cytolysis [35 , 37–39] , directly enhance infection [40] , and attenuate the alternative pathway of complement activation by binding factor H [41] . The mechanism by which NS1 becomes associated with the plasma membrane is poorly understood . The protein lacks amino- or carboxyl-terminal membrane-spanning or anchoring sequences [42] . Although covalent linkage by a glycosylphosphatidylinositol ( GPI ) anchor to the plasma membrane of DENV-infected cells has been suggested [43] , this mechanism has not been confirmed . Also , it does not explain how uninfected , as well as infected cells , accumulate NS1 on their surfaces . In this study , we demonstrate that soluble DENV NS1 attaches to uninfected cells primarily via an interaction with the glycosaminoglycans ( GAG ) heparan sulfate ( HS ) and chondroitin sulfate E ( CS-E ) , and that GAG sulfation is a critical cellular modification necessary for binding . Moreover , soluble DENV NS1 preferentially binds subsets of cells including human microvascular endothelial cells . Our findings suggest that the selective vascular leakage that occurs in severe DENV infection may be related to the relative ability of endothelial cells in different tissues to bind soluble NS1 and be targeted by cross-reactive anti-NS1 antibodies during secondary infection .
Uninfected cells were tested for the ability to bind soluble DENV NS1 . Incubation with either purified DENV NS1 or supernatants from BHK DENV-2 Rep cells resulted in rapid binding of NS1 to many cell types ( Figure 2 and Table 1 ) . Binding of NS1 to Chinese hamster ovarian epithelial ( CHO ) -K1 , Vero , and 4/4 RM4 cells was dose-dependent and saturable , and maximum binding was achieved at a concentration of 20 μg/ml , which is in the range reported in vivo during secondary infection [11 , 28] . DENV NS1 bound to the surface of several types of epithelial and fibroblast transformed cell lines ( BHK , CHO-K1 , Vero , 293T , HepG2 , Hep3B , and L929 ) including those of human and nonhuman origin . NS1 also bound to primary , untransformed cells including keratinocytes ( HaCat , CCD-1102 ) , skin and lung fibroblasts ( Detroit-551 and IMR-90 ) , and freshly isolated tonsillar epithelial cells . As high levels of soluble NS1 are detected in the blood of DHF patients [11 , 28 , 33] , we extended our analysis to human peripheral blood cells . Notably , DENV NS1 failed to bind to the surface of freshly isolated peripheral blood mononuclear cells or erythrocytes . Similar negative results were obtained with purified CD14+ monocytes , CD19+ B lymphocytes , and CD4+ T lymphocytes . Moreover , DENV NS1 only weakly bound to the surface of monocyte-derived macrophages . In contrast to that observed with primary lymphocytes , DENV NS1 bound strongly to the surface of several malignant T cell lines , including Jurkat , H9 , and EL-4 ( Figure 2 and unpublished data ) . As vascular leakage is a hallmark of DHF/DSS , and endothelial cells are believed to be targets of immune-mediated damage , we analyzed the binding of NS1 to human endothelial cells . Interestingly , DENV NS1 bound strongly to human dermal and lung microvascular endothelial cells ( HMEC ) and HMEC-lung blood ( HEMC-LB ) , modestly to aortic endothelial cells , but minimally to primary or immortalized human umbilical vein endothelial cells ( HUVEC or Eahy926 ) . As our data indicated that soluble DENV NS1 bound a subset of uninfected mammalian cells of different lineages , we sought to identify the mechanism of attachment . We hypothesized that NS1 might interact with a highly conserved moiety , such as a GAG . To test this , we compared DENV NS1 binding to wild-type CHO-K1 cells and seven different CHO cell lines ( Table 2 ) that are either defective in GAG biosynthesis or express GAG with distinct structural specificities . CHO-745 cells , which genetically lack xylosyltransferase , the enzyme required for biosynthesis of both HS and CS [46] , showed a 60%–70% reduction ( p < 0 . 0001 ) in binding of DENV NS1 ( Figure 3A and Table 2 ) . Similarly , CHO-M1 cells , which are defective in HS biosynthesis [47] , exhibited a ∼50% reduction ( p = 0 . 02 ) in DENV NS1 binding . However , CHO-H8 cells [48] , which are a variant of CHO-M1 HS-deficient cells overexpressing CS 2-O-sulfotransferase ( 2-OST ) ( producing a 30-fold increase in double sulfated disaccharide residues on CS ) , showed levels of DENV NS1 binding that were similar to CHO-K1 wild-type cells . Thus , both HS and CS sustain cell surface binding to DENV NS1 , at least on CHO cells . To determine if DENV NS1 required modification of HS structures for binding , we tested CHO cell lines that overexpress different 3-O-sulfotransferases ( 3-OST ) ( Figure 3A and Table 2 ) . All 3-OSTs transfer sulfate moieties to the 3-O-position of N-sulfated glucosamine ( GlcNS ) and generate a highly sulfated HS motif with distinct biological properties ( Table 2 ) . CHO-K1 . 5 cells overexpress 3-OST-1 , which adds 3-O-sulfate to GlcNS in a glucuronic acid ( GlcA ) -GlcNS ± 6S sequence context , and facilitate high affinity antithrombin III binding [49] . CHO-K1 . N7 cells overexpress 3-OST-3 , which adds 3-O-sulfate to GlcNS in a GlcA2S/iduronic acid ( IdoA ) 2S-GlcNS ± 6S sequence context , and promote herpes simplex virus ( HSV ) glycoprotein D binding and entry [50] . By contrast , overexpression of 3-OST-5 ( CHO-3-OST-5 cells ) adds 3-O-sulfate to GlcNS in both GlcA-GlcNS ± 6S and GlcA2S/IdoA2S-GlcNS ± 6S sequence contexts . Notably , all CHO cell lines expressing distinct 3-OST showed enhanced DENV NS1 binding by 30%–50% ( p < 0 . 05 ) without a preference for any of the distinct 3-OST-modified HS structures . Thus , unlike other glycoproteins , DENV NS1 prefers highly sulfated HS motifs for binding and does not require specific HS sequences . On CHO cell lines , the specificity of the GAG had a more dominant effect on regulating NS1 binding than the absolute level . Using a previously developed high-pressure liquid chromatography method [51] , we quantified the amount of cell surface HS and CS GAG in different CHO cell lines and compared it to NS1 binding ( Table 2 ) . Although CHO-M1 cells expressed twice the amount of total GAG compared to wild-type CHO-K1 cells , CHO-M1 cells bound NS1 poorly . Thus , the total level of GAG did not directly correlate with NS1 binding . Consistent with this , similar amount of GAG were observed on CHO-K1 and other 3-OST-expressing CHO cell lines , yet 3-OST-expressing CHO cells bound NS1 more strongly . Moreover , Vero cells express ∼3-fold higher levels of HS compared to CHO-K1 cells ( unpublished data ) but bound NS1 less well compared to CHO-K1 cells ( Figure 2 and Table 2 ) . A requirement of GAG for DENV NS1 binding to the cell surface was confirmed independently by enzymatic treatment of BHK and CHO-K1 cells with specific heparin lyases and chondroitinases ( Figure 3B ) . Treatment of BHK or CHO-K1 cells with heparin lyases I , II , and III , which specifically remove HS , reduced DENV NS1 binding ( ∼50%–60% , p < 0 . 05 ) to the levels observed in GAG-negative CHO-745 cells . In contrast , DENV NS1 attachment was not significantly affected by treatment with chondroitinase ABC , which degrades all types of CS [52] . As expected , treatment with both enzymes had no effect on DENV NS1 binding to CHO-745 cells , which lack GAG . Nonetheless , a small amount of NS1 binding to CHO-745 cells was observed , presumably through a subordinate GAG-independent pathway . Overall , the analysis of CHO cell mutants and enzymatic treatments suggested that HS and highly sulfated forms of CS were the primary cell surface GAG that sustained DENV NS1 binding on CHO and BHK cells . As an additional confirmation , we performed competitive binding assays with soluble GAG . Pre-incubation with soluble CS-E or heparin ( HP ) substantially decreased ( ∼50% , p < 0 . 005 ) , in a dose-dependent manner , DENV NS1 binding to BHK cells ( Figure 4A–4C ) . Small , albeit significant inhibition ( ∼15% , p < 0 . 05 ) , was observed even at relatively low concentrations CS-E and HP ( 0 . 01 μg/ml or 0 . 033 nM ) . In contrast , CS-A and -B inhibited NS1 binding only at higher concentrations ( 100 μg/ml or 3 . 3 μM , p < 0 . 05 ) whereas CS-C and -D showed no significant effect ( p > 0 . 2 ) . Similarly , pre-incubation with CS-E and HP reduced DENV NS1 attachment to HMEC cells ( ∼40% , 0 . 01 μg/ml , p < 0 . 01 ) ( Figure 4D–4F ) . In a more direct in vitro binding ELISA , an interaction between DENV NS1 and HS or CS-E was established , whereas only weak or no appreciable binding was detected with CS-A , -B , -C , and -D ( Figure 5 ) . Taken together , these results suggest that NS1 interacts more strongly with HP , HS , and CS-E and more weakly with CS-A and CS-B . CS-A consists of mainly GlcA-GalNAc4S repeating disaccharides , CS-B contains both IdoA-GalNAc4S and GlcA-GalNAc4S as dominant repeating disaccharides , and CS-C consists of mainly GlcA-GalNAc6S repeating disaccharides . CS-D and CS-E are enriched in disulfated disaccharides , GlcA2S-GalNAc6S and GlcA-GalNAc4S6S , respectively . CS-E is unique , as two sulfates are present in the same GalNAc residue compared to other CS , and suggests that clustered sulfates rather than net negative charge on the disaccharide backbone facilitate DENV NS1 binding . The specificity of GAG-protein interactions often depends on the position and degree of sulfation ( reviewed in [53–55] ) . To clarify if GAG sulfation is required for cell binding of DENV NS1 , we treated BHK and CHO-K1 cells with the reversible sulfation inhibitor , sodium chlorate [56] ( Figure 6A and 6B ) . This treatment did not alter cell viability , as analyzed by propidium iodide exclusion ( unpublished data ) . However , binding of DENV NS1 to BHK cells was abolished by the addition of sodium chlorate ( >5 mM ) . In contrast , at comparable doses , DENV NS1 binding to wild-type CHO-K1 was reduced to the level observed with CHO-745 cells lacking GAG . Importantly , the phenotype was reversed by the addition of excess exogenous ( 10 mM ) sodium sulfate ( Figure 6C and 6D ) . Thus , GAG sulfation is required for optimal binding of soluble DENV NS1 to cells . Although our data suggests that soluble DENV NS1 binds to the cell surface of uninfected cells via HS and CS-E , we evaluated if this was the dominant mechanism of attachment of NS1 to DENV-infected cells . Previous studies have suggested that flavivirus NS1 may be displayed on the cell surface of infected cells by a GPI anchor or transmembrane linkage [43] , even though the protein lacks anchor attachment sequences or consensus membrane spanning domains . To evaluate this , BHK cells were infected with DENV-2 and cultured in the presence of high concentrations of soluble HP . No reduction of DENV NS1 surface expression on infected cells was observed ( Figure 7A ) . These results were confirmed by studies with the sulfate inhibitor , sodium chlorate . In contrast to the inhibitory effect of sodium chlorate on soluble NS1 binding ( Figure 6 ) , no reduction of DENV NS1 expression on the surface of infected BHK cells was observed ( Figure 7B ) . Similar studies with BHK DENV-2 Rep cells also showed no change in surface NS1 expression after sodium chlorate treatment ( Figure 7C ) . Finally , in direct contrast to that observed with soluble NS1 and uninfected cells ( Figure 3B ) , treatment of DENV-infected cells with heparin lyases and/or chondroitinase ABC did not decrease surface expression of NS1 ( Figure 7D ) . Thus , NS1 expressed on the surface of infected cells is not modulated by GAG expression and must attach by an independent mechanism . Soluble NS1 has been hypothesized to contribute to DHF pathogenesis by promoting vascular leakage [28 , 34] , which occurs predominantly into pleural and peritoneal cavities [4] . Our cell culture experiments indicated that soluble DENV NS1 binds selectively to subsets of human endothelial cells . We hypothesized , that in vivo , a preferential interaction of DENV NS1 with specific endothelium could contribute to tissue-specific vascular leakage after immune recognition . This mechanism would not require direct DENV infection of endothelial cells , which has been difficult to establish in vivo by pathological criteria [57 , 58] . To assess this , sections of uninfected mouse tissues were incubated with soluble DENV NS1 and analyzed by immunofluorescence and confocal microscopy . Histological analysis was performed on parallel hematoxylin and eosin–stained sections ( unpublished data ) . Specific NS1 binding to endothelial cells lining blood vessels of lung and liver was observed , based on costaining with mAbs against DENV NS1 and the endothelial cell–specific marker CD31 ( Figure 8A and 8B ) . Differences in the pattern of DENV NS1 binding within the same organ were also observed . For example , DENV NS1 bound primarily to endothelial cells lining vessels along the bronchial tree , yet bound poorly to those lining alveolar capillaries . Strong binding of DENV NS1 to the cells lining the outer layer of the adventitia of pulmonary vessels was also apparent ( Figure 8A ) . In the liver , hepatic arteriolar and sinusoidal endothelial cells bound DENV NS1 weakly , whereas those within central veins showed strong binding ( Figure 8B ) . As controls , no appreciable staining was observed if sections were incubated with bovine serum albumin ( BSA ) followed by anti-NS1 mAbs or with purified DENV NS1 and isotype control mAbs . The selectivity of soluble NS1 binding to endothelium in situ was also demonstrated by an absence of NS1 binding to endothelium in the large intestine ( Figure 8C ) and brain ( unpublished data ) of mice . Finally , binding of DENV NS1 to endothelium in situ also was confirmed in human lung tissues ( Figure 9 ) . Next , soluble GAG and DENV NS1 competitive binding assays were performed on lung tissues and images were analyzed by confocal microscopy and Volocity software . Pre-incubation of DENV NS1 with soluble CS-E or HP reduced binding to lung endothelium in situ ( CS-E: ∼40% , p < 0 . 0001; HP: ∼30% , p = 0 . 0002 ) . In contrast , CS-C marginally decreased DENV-NS1 attachment to lung endothelium ( ∼8% , p = 0 . 18 ) . Significant binding of DENV NS1 to serosal surfaces of the lung ( pleura ) and intestine ( peritoneum ) was also observed ( Figure S2A and S2B ) . Based on histological analysis , mesothelial cells were also targets for DENV NS1 binding in the lung and intestine . Binding of DENV NS1 to mesothelial cells was specific as negative results were obtained when the sections were incubated with NS1 followed by isotype control mAbs or without NS1 followed by anti-NS1 mAbs . In competitive binding experiments , soluble HP and CS-E , but not CS-C , also decreased DENV NS1 binding to lung mesothelial cells ( unpublished data ) .
In this study , we demonstrate that soluble DENV NS1 binds to a subset of uninfected cells via interactions with GAG , primarily HS and CS-E . In cell culture , NS1 bound strongly to epithelial cells and fibroblasts , and weakly , if at all , to freshly isolated human peripheral blood leukocytes . Substantial variability was observed in NS1 binding to cultured endothelial cells and endothelium in situ . Experiments with the sulfation inhibitor sodium chlorate established that highly sulfated forms of GAG are required for optimal binding of soluble DENV NS1 . Finally , our experiments suggest that NS1 on the surface of DENV-infected cells is linked primarily by a distinct , GAG-independent mechanism . Two major types of GAGs , HS and CS , are produced by cells in the form of proteoglycans as linear polymers of repeating disaccharides of uronic acids and glucosamines or galactosamines . For HS , sulfation of glucosamine and uronic acid moieties may occur in a clustered manner to generate highly sulfated domains or in a dispersed pattern to generate less sulfated or non-sulfated domains . Moreover , GlcNS , IdoA , and GlcA can be sulfated at multiple positions . Differential sulfation of GAG results in preferential binding of growth factors , cytokines , chemokines , enzymes , extracellular matrix , and other proteins to these structures [59] . Cell type–specific expression of GAG modifying enzymes and proteoglycan core proteins results in the display of unique GAG structures on different types of cells [60] . Consistent with this , our studies demonstrate that DENV NS1 binding to GAG occurs in a cell type–specific manner . The specificity of GAG interactions is determined by the pattern of the disaccharide units , the degree of sulfation , and the spacing of basic amino acid residues in GAG-binding domains of ligands ( reviewed in [53–55] ) . We observed that highly sulfated GAG , including HS , HP , and CS-E , demonstrated the strongest binding for soluble DENV NS1 . The use of mutant CHO lines expressing different levels and forms of GAG established that specific HS and CS structures modified by 2-O and 3-O-sulfotransferases promoted DENV NS1 binding to cell surfaces . These results are analogous , although not identical , to studies with HSV glycoprotein D , which show enhanced cell surface binding when HS is modified by some , but not all 3-OST [50 , 61] . Sulfation was critical for soluble DENV NS1 binding to cell surfaces , especially for BHK cells , as treatment with the sulfate inhibitor sodium chlorate abolished DENV NS1 attachment . Also consistent with our results , CS-E , but not CS-A , -B , or -C , interfered with HSV binding to the target cells [62] . Using radioactive binding or gel mobility shift assays , we observed similarly strong binding between West Nile virus NS1 and HS or CS-E ( L . Zhang , K . Chung , and M . Diamond , unpublished data ) . As binding of soluble DENV NS1 to GAG requires sulfation , it is plausible that the interaction is primarily electrostatic in nature and depends on the relative degree of negative charge on a GAG motif . The major repeating disaccharides in HP are IdoA2S-GlcNS6S ( ∼2 . 7 sulfates/disaccharides ) , whereas the major repeating disaccharides in CS-D and CS-E are GlcA2S-GalNAc6S and GlcA-GalNAc4S6S , respectively ( ∼1 . 7 sulfates/disaccharide ) [62] . Because NS1 preferentially interacts with CS-E and HP compared to CS-D , we hypothesize that NS1 binding may prefer closely spaced sulfates within the same sugar residue . Since HP and CS-E have distinct sugar sequences , the combination of disaccharide unit and position of sulfation on an individual GAG may determine the strength of DENV NS1 binding . The linear sequences XBBXBX and XBBBXXBX ( where B is a basic Arg or Lys amino acid ) are common HP binding motifs in proteins [55] . However , GAG binding sites may not be exclusively defined by linear sequences but also can include conformational epitopes that juxtapose basic amino acids from different segments of a protein [55] . Amino acid sequence analysis of DENV NS1 reveals no apparent canonical GAG binding motifs . Although reverse genetic strategies are planned , the identification of the amino acids involved in GAG recognition may await solution of the NS1 structure . Our data showing that soluble NS1 can bind to the surface of the GAG-deficient line , CHO-745 , albeit at significantly lower levels , suggest that additional molecules also serve as ligands for DENV NS1 attachment . This secondary attachment ligand may be expressed only on subsets of cells as addition of sodium chlorate completely abrogated soluble NS1 binding to BHK cells . Consistent with this , soluble HS and CS-E only partially reduced DENV NS1 binding to HMEC . Thus , different cell types may express multiple ligands for attachment of soluble DENV NS1 . The expression of NS1 on the surface of DENV-infected cells was insensitive to treatments that reduce GAG levels . This also suggests the existence of an alternate mechanism of cell surface NS1 attachment . Some have speculated that a transmembrane form of NS1 exists , although the protein sequence lacks a canonical hydrophobic membrane-spanning domain [42] . Others have postulated that at least some fraction of cell surface NS1 is linked via a GPI anchor [43 , 63] . Based on our data , we hypothesize that DENV NS1 is expressed on cell surfaces by at least two mechanisms: on uninfected cells soluble NS1 binds to the surface via a GAG-dependent and GAG-independent mechanism , whereas on infected cells NS1 attaches via a GAG-independent mechanism , possibly via a membrane or GPI anchor . What remains uncertain , and is a direction for future research , is whether the mechanism of cell surface attachment of NS1 has unique functional consequences , especially in terms of immune recognition or evasion . Our studies demonstrate that soluble DENV NS1 differentially binds to cultured endothelial cells in vitro and endothelium in situ in human and mouse tissues . In contrast , others have shown that intravenous injection of C57BL/6 x SJL mice with high concentrations ( 250 μg/ml ) of soluble DENV-1 NS1 results in binding to and accumulation in hepatocytes but not to other cell types in the liver or other tissues [40] . A possible reason for the disparity in results is that the route of antigen administration may modulate NS1 binding: intravenous injection could prompt rapid first-pass clearance of antigen by the liver . Although further studies are necessary , our data are consistent with specific GAG modification by subsets of endothelial cells in different tissues modulating the level of bound NS1 . Indeed , tissue-specific expression of different isoforms of enzymes in GAG biosynthesis has been reported [64 , 65] . Selective IL-8 binding to endothelial cells has been observed [66] and could be due to subtle differences in the display of GAG on the surface of cells . In favor of this hypothesis , IL-8 selectively binds to subsets of HP and HS [67] . The pathologic mechanism underlying selective vascular leakage at serosal sites during DHF/DSS [4] remains unknown . High levels of intravascular soluble NS1 , as observed in DENV-infected patients , could promote binding and surface expression of NS1 on selective endothelium without a requirement for direct viral infection , which has been difficult to establish histopathologically in fatal DHF cases [57 , 58] . In addition , specific binding to mesothelial cells that line pleura and peritoneum , as observed in our DENV NS1 binding experiments in situ , could contribute to the pleural effusions or ascites that are observed in DHF/DSS patients . Although more experiments are necessary , preferential binding of soluble NS1 to subsets of endothelial and mesothelial cells in vivo could lead to tissue-specific vascular leakage that occurs during severe secondary DENV infection after recognition by anti-NS1 antibodies , immune complex formation , and inflammatory damage [28 , 68] .
CS-A , -B , -C , -D , -E , HP , HS , heparin lyases I , II , and III , chondroitinase ABC , anti-heparin lyase-digested HS ( 3G10 ) , and anti-chondroitinase ABC-digested CS ( 2B6 ) antibodies were all purchased ( Seikagaku ) . DENV-2 NS1–specific monoclonal antibodies ( mAb hybridomas 2G6 , 1A4 , 1B2 , 1F11 , 2E3 , and 2E11 [69] , and unpublished data ) were purified by protein G affinity chromatography . Mouse polyclonal anti-DENV-2 NS1 was produced after BALB/c mice were intraperitoneally immunized three times with purified DENV-2 NS1 ( 10 μg/dose ) at a 2-wk interval . Mice were subsequently treated with pristane followed by injection with myeloma cells to induce ascites formation . Ascites fluid containing anti-NS1 polyclonal antibody was collected , and antibodies were purified by protein-G affinity chromatography . Anti-mouse IgG conjugated with Alexa Fluor 647 , Alexa Fluor 488 , or Cy3 were purchased from Invitrogen . Sodium chlorate and sodium sulfate were obtained commercially ( Sigma ) . The following transformed cell lines were obtained from the ATCC: BHK fibroblasts cells , African green monkey Vero cells , HEK-293T human embryonic kidney carcinoma cells , L929 mouse fibroblasts , IMR-90 human lung fibroblasts , Detroit-550 human skin fibroblasts , CCD-1102 human keratinocytes , HepG2 and Hep3B human hepatocellular carcinoma cells , 4/4RM4 rat lung mesothelial cells , Eahy926 HUVEC , MEG-01 human megakaryoblast cells , Jurkat human leukemic T lymphoblasts , H9 human T lymphoma cells , EL-4 mouse T lymphoma cells , and U937 human myelomonocyte cells . HMEC and keratinocytes ( HaCat ) were gifts ( M . Caparon ) . Wild-type CHO-K1 cells and CHO mutant lines with altered GAG expression ( CHO-745 , CHO-M1 , CHO-H8 , CHO-K1 . 5 , CHO-K1 . N7 , CHO-3OST5–1 , and CHO-PPP6 ) have been described previously [46–49 , 61 , 70] . BHK , Vero , 293T , L929 , and HaCat cells were cultured in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) , 50 mM HEPES , 4 mM L-glutamine , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . IMR-90 , Detroit-551 , HepG2 , and Hep3B were grown in minimum essential Eagle's medium with Earle's BSS , 10% FBS , 1% non-essential amino acids ( NEAA ) , 1 mM sodium pyruvate , 2 mM L-glutamine , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . CCD-1102 cells were grown in keratinocyte serum-free medium supplemented with keratinocyte growth factors ( Invitrogen ) . MEG-01 , U937 , Jurkat , and H9 were cultured in RPMI 1640 medium with 10% FBS , 2 mM L-glutamine , 10 mM HEPES , 1 mM sodium pyruvate , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . EL-4 cells were propagated in Iscove's medium supplemented with 10% FBS , 1% non-essential amino acids , 2 mM L-glutamine , 1 mM sodium pyruvate , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . HMEC were grown in MCDB 131 ( Invitrogen ) supplemented with 10% FBS , 2 mM L-glutamine , 0 . 3% NaHCO3 , 1 μg/ml hydrocortisone ( Sigma ) , 10 ng/ml epidermal growth factor ( Sigma ) , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . All CHO cell lines were grown in Ham's F12 medium containing 10% FBS , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate , except for CHO-30ST5–1 cells , which were cultured in CHO medium supplemented with 400 μg/ml of G418 sulfate ( Cellgro ) . Primary human tonsil epithelial cells were isolated according to a published protocol [71] and cultured in keratinocyte serum-free medium supplemented with keratinocyte growth factor ( Invitrogen ) . Primary HMEC-LB and aortic endothelial cells were purchased from Clonetics ( Cambrex Bio Science ) and maintained according to the manufacturer's protocol . HUVEC were grown in RPMI 1640 containing 10% FBS on 1% gelatin-coated surfaces . Primary human peripheral blood mononuclear cells were isolated from buffy coats obtained from the blood bank or healthy volunteers by Ficoll-Hypaque ( Pharmacia ) density gradient centrifugation . Human CD14+ monocytes , CD19+ B cells , CD4+ T lymphocytes were purified from primary human peripheral blood mononuclear cells via positive selection using antibody-coated magnetic beads ( Miltenyi Biotec ) . To generate macrophages , monocytes were cultured for 7 d in RPMI 1640 supplemented with 50 ng/ml of GM-CSF , 10% FBS , 2 mM glutamine , 1% nonessential amino acids , 1% sodium pyruvate , 100 units/ml penicillin G , and 100 μg/ml streptomycin sulfate . Human platelets were isolated from whole blood of healthy volunteers . After centrifugation at 750 x g for 20 min at 22 °C , platelet-rich plasma was collected and centrifuged at 1 , 200 x g for 15 min at 22 °C to obtain platelets . Human erythrocytes were obtained from healthy donors following a published protocol [72] . All primary cells were cultured for five or fewer passages for the NS1 binding experiments . BHK cells that stably propagate a DENV-2 subgenomic replicon ( BHK DENV-2 Rep cells [45] ) were grown to 80%–90% confluence in DMEM containing 10% FBS and 3 μg/ml puromycin ( Sigma ) . Cell monolayers were washed several times with DMEM and the cells were cultured for another 3 d in serum-free DMEM supplemented with 3 μg/ml puromycin . DENV NS1 secreted in the supernatants of BHK DENV-2 Rep was quantified by NS1 capture ELISA as previously described [28] . Supernatants were collected , centrifuged , pooled , and passed through a 0 . 2-μm filter prior to immunoaffinity chromatography with anti-NS1 mAb 2G6 [28] . Peak elution fractions were combined , diluted 7-fold with 20 mM Tris ( pH 8 . 0 ) , and loaded onto a 1-ml Mono Q ion exchange column ( GE Healthcare ) at a rate of 1 ml/min . The protein was eluted with a linear salt gradient ( 0–1 M NaCl ) over 20-column volumes at 1 ml/min . Purity and immunoreactivity of DENV NS1 were confirmed by SDS-PAGE with silver staining and western blot . Concentrations of purified protein were determined by the bicinchoninic acid assay . Purified DENV NS1 from DENV-infected mammalian cells was obtained as previously described [28] . Adherent cells were removed from tissue culture plates after incubation with an EDTA solution ( 4 mM EDTA plus 10% FBS in PBS ) . Cells ( 5 × 105 ) in suspension were incubated on ice for 1 h with 100 μl of purified DENV NS1 at indicated concentrations or 300 μl of serum-free supernatants from BHK DENV-2 Rep cells . After washing once with 3 ml of medium , 50 μl of DENV-2 NS1 specific mAb 2G6 ( 25 μg/ml ) or an isotype-matched negative control mAb was added to the cells and incubated on ice for 45 min . After subsequent washing , bound primary mAbs were detected after 30-min incubation with a 1:500 dilution of Alexa Fluor 647-conjugated anti-mouse IgG ( Invitrogen ) . Of note , experiments were also performed in parallel with serial washing steps and no significant difference in binding or background was observed . Propidium iodide ( 0 . 2 mg/ml ) was added immediately before flow cytometry to exclude dead cells . Cell suspensions were incubated with DENV NS1 in the presence or absence of varying concentrations of soluble GAG for 1 h on ice . Bound DENV NS1 was detected after antibody staining as described above and analyzed by flow cytometry . Cell suspensions were centrifuged at 200 x g for 2 min and were resuspended in PBS containing 0 . 1% BSA . Heparin lyases I , II , III ( 0 . 06 U/ml final concentration ) or chondroitinase ABC ( 0 . 1 U/ml final concentration ) was added to a 50-μl of cell suspension and incubated at 37 °C in a shaking incubator for 1 h . After three washes with cold DMEM , the cells were incubated sequentially with DENV NS1 and DENV NS1 mAbs and analyzed by flow cytometry . Different types of CHO cells were cultured as described above . CHO cells were rinsed and detached from cell culture dishes after incubation in PBS/EDTA for 15 min . Cells ( 107 ) were treated with 2 ml trypsin-EDTA ( 1X solution , Mediatech ) at 37 °C for 10 min , which released cell surface proteoglycans . These were collected and used for cell surface GAG isolation and quantification by high-pressure liquid chromatography as reported previously [51] . Maxi-Sorp microtiter plates ( Nalge Nunc International ) were adsorbed with soluble GAG ( 1 mg/ml in PBS ) at 4 °C overnight . After four washes with PBS ( 300 μl/well ) , nonspecific binding sites were blocked with 1% heat-inactivated ( 65 °C , 15 min ) BSA in PBS for 2 h at 37 °C and followed by five washes with PBS . Clarified serum-free supernatants from BHK or BHK DENV-2 Rep cells ( 100 μl diluted 1:1 in PBS ) were added to each well and incubated for 2 h at room temperature . Plates were then washed five times with PBS containing 0 . 05% Tween-20 followed by a 1-h incubation at room temperature with 100 μl of purified DENV NS1 specific mAb mixtures ( 1A4 , 1B2 , 1F11 , 2G6 , and 2E3; 1 μg/ml of each in PBS containing 0 . 1% BSA ) . After washing , biotinylated goat anti-mouse IgG ( 1 μg/ml ) and horseradish peroxidase-conjugated streptavidin were added sequentially for 1-h incubations at room temperature . After six final washes with PBS , signal was detected by adding 150 μl of TMB substrate ( DakoCytomation ) and 50 μl of 0 . 1 N H2SO4 stop solution to each well . Plates were evaluated at 450 nm on a 96-well plate reader ( Genios Pro; Tecan Instruments ) . Sulfation was inhibited by sodium chlorate treatment [56] . BHK and CHO cells were cultured in sulfate-free Joklik Modification Minimum Essential Medium Eagle ( Sigma ) or Ham's F12 medium ( Tissue culture support center , Washington University ) supplemented with 10% dialyzed FBS ( Sigma ) containing different concentrations ( 1–75 mM ) of sodium chlorate . In some experiments , 10 mM sodium sulfate was added to replenish sulfate to the cells . After overnight culture , cells were processed for DENV NS1 binding as described above . BHK cells ( 1 . 6 × 105/well ) were seeded onto 12-well tissue culture plate ( Costar ) . 24 h later , cells were infected with DENV-2 ( strain 16681 ) at a multiplicity of infection ( MOI ) of 3 . After a 2-h incubation at 37 °C , cell monolayers were extensively washed and cultured in DMEM containing 4% FBS . In some experiments , DENV-infected cells were cultured in medium containing 1 , 10 , or 100 μg/ml of HP or CS-C or 25 mM sodium chlorate . At specific intervals post-infection , cells were harvested from culture plates with an EDTA solution ( 4 mM EDTA plus 10% FBS in PBS ) . DENV NS1 on the surface was determined by incubation of 5 × 105 cells with anti-DENV-2 NS1 mAb clone 2G6 or mouse polyclonal anti-DENV-2 NS1 ( 20 μg/ml final concentration ) for 1 h at 4 °C . After three washes , cells were incubated with Alexa Fluor 647-conjugated anti-mouse IgG ( 1:500 ) and analyzed by flow cytometry . In some experiments , DENV-infected cells were treated with a mix of heparin lyases I , II , III or chondroitinase ABC prior to detection of surface NS1 . Human lung tissue: The protocol to obtain human lung tissues from patients was approved by the Institutional Review Board at the Faculty of Medicine Siriraj Hospital , Mahidol University . A human lung , surgically removed with informed consent from a patient with lung cancer , was evaluated macroscopically for cancer-free regions . Selected tissues , cut from the central part of the cancer-free region of the lung , were cryoprotected in 30% sucrose for generation of 6- to 7-μm frozen sections . Cryosections were thawed , fixed in 4% paraformaldehyde ( Sigma ) in PBS at room temperature for 10 min , and washed extensively with PBS . Tissues were incubated with purified DENV NS1 or BSA at 20 μg/ml for 1 h at room temperature and followed by three washes with 0 . 1% BSA in PBS 5 min each . Sections were incubated with a mixture of mAbs ( 1A4 , 1F11 , 2G6 , 1B2; 20 μg/ml final concentration ) against DENV NS1 . A mixture of IgG1 ( MOPC-21 , Sigma ) and IgG2a ( UPC-10 , Sigma ) at 20 μg/ml was used as isotype control Abs . After three washes , sections were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody ( Invitrogen ) and followed by 1-h incubation at room temperature with rabbit anti-human CD31 ( PECAM ) ( Santa Cruz Biotechnology ) at the dilution of 1:10 . Sections were washed three times and incubated with Cy3 conjugated donkey anti-rabbit IgG ( Jackson Immuno Research Laboratories ) . Nuclear staining was achieved by incubation for 15 min with 1:100 dilution of Hoechst dye ( Invitrogen ) and visualized using a Zeiss 510 Meta LSM confocal microscope . Mouse tissues: To obtain tissues from mice , 4- to 6-wk-old uninfected C57BL/6 mice were anesthesized with ketamine and xylazine and perfused with 20 ml of PBS . Tissues were dissected and cryoprotected in 30% sucrose for generation of frozen sections . Serial 6-μm cryosections were air-dried for 1 h at room temperature followed by washing with PBS . Sections were fixed with 50% acetone in PBS for 10 min on ice , incubated with PBS containing 2 M NaCl ( pH 7 . 4 ) for 10 min at room temperature , and blocked in PBS containing 0 . 5% BSA for 1 h at room temperature or overnight at 4 °C . Subsequently , sections were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature followed by incubation with a mixture of anti-NS1-mAbs ( 1A4 , 1F11 , 2G6 , 1B2; 10 μg/ml final concentration ) or isotype control mAbs ( 10 μg/ml ) diluted in PBS containing 0 . 5% BSA . After three washes , sections were incubated with Cy3 conjugated with goat anti-mouse IgG ( Zymed ) at the dilution of 1:200 . Co-staining with endothelial cell marker , CD31 ( PECAM ) , was accomplished by a 1-h incubation with rat anti-mouse PECAM-1 ( CD31 ) ( BD Pharmingen ) at a dilution of 1:250 followed by incubation with a 1:500 dilution of secondary Alexa Fluor 488 conjugated anti-rat IgG ( Invitrogen ) . Nuclear staining was achieved by incubation for 15 min with 1:2 , 500 dilution of TO-Pro-3 ( Invitrogen ) . Sections were mounted using fluorescent mounting medium ( Vector Laboratories ) and analyzed using a Zeiss 510 Meta LSM confocal microscope . For soluble GAGs and DENV NS1 competitive binding experiments , serum-free supernatants from BHK DENV-2 Rep were mixed with soluble CS-C , CS-E , or HP ( 3 . 3 μM final concentration ) prior to incubation with sections of mouse lung . The sections were further processed for DENV NS1 and CD31 staining as described above . 50–60 images of each condition were captured by confocal microscopy , and fluorescent intensity of each image was analyzed using the Volocity software ( Improvision ) . Regions that stained positively for CD31 ( intensity greater than 427 arbitrary units on the intensity scale of 0–4 , 095 ) were selected for analysis . Mean intensity of DENV NS1 binding was calculated for all measured regions . For analysis of DENV NS1 binding to pleura , regions were manually selected based on anatomical sites at the surface of the lung . Datasets were compared by a two-tailed , unpaired t test . Multiple comparisons were performed using an ANOVA test . Statistical significance was achieved when p-values were < 0 . 05 . Data analysis was performed using Prism software ( GraphPad ) .
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Dengue virus ( DENV ) is a mosquito-transmitted virus that infects humans and has become a global emerging infectious disease threat . Four serotypes of DENV exist , and the most severe cases are associated with secondary infection with a different virus serotype . Clinical deterioration is characterized by bleeding and selective vascular leakage from endothelium in specific tissue sites . An increased understanding of how DENV proteins contribute to this phenotype is vital to developing novel vaccines and identifying individuals at risk for severe disease . DENV nonstructural protein-1 ( NS1 ) is one such protein: during infection , it is secreted and accumulates in the supernatant and on the surface of cells . In this study , we demonstrate that soluble DENV NS1 attaches to subsets of cells , including some but not all endothelial cells , primarily via an interaction with specific glycosaminoglycans ( heparan sulfate and chondroitin sulfate E ) . This was confirmed in tissue binding studies as DENV NS1 bound to lung and liver but not intestine or brain endothelium . Our findings suggest that the selective vascular leakage that occurs in severe DENV infection may be related to the relative ability of endothelial cells in different tissues to bind soluble NS1 and to be targeted by cross-reactive anti-NS1 antibodies during secondary infection .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"viruses",
"infectious",
"diseases",
"virology",
"in",
"vitro",
"immunology",
"microbiology",
"eukaryotes",
"mus",
"(mouse)",
"homo",
"(human)"
] |
2007
|
Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E
|
Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization . However , factors regulating tissue-specific insulator function have not yet been discovered . Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila . Mutation of shep improves gypsy-dependent enhancer blocking , indicating a role as a negative regulator of insulator activity . Unlike ubiquitously expressed core gypsy insulator proteins , Shep is highly expressed in the central nervous system ( CNS ) with lower expression in other tissues . We developed a novel , quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue . Additionally , mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues . Consistent with negative regulatory activity , ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes . Taken together , these data reveal a novel , tissue-specific mode of regulation of a chromatin insulator .
Chromatin insulators are DNA-protein complexes that influence eukaryotic gene expression by organizing the genome into distinct transcriptional domains . Functionally conserved from Drosophila to humans , insulators regulate interactions between regulatory elements such as enhancers and promoters and demarcate silent and active chromatin regions ( for review , see [1] ) . Chromatin insulators are thought to exert effects on gene expression by constraining the topology of chromatin and facilitating the formation of intra- and inter-chromosomal looping ( for review , see [2] ) . These higher order interactions can vary between cell types , thereby facilitating tissue-specific transcriptional output . Drosophila harbor several distinct classes of chromatin insulators , including the well studied gypsy insulator , also known as the Suppressor of Hairy wing ( Su ( Hw ) ) insulator . The zinc-finger DNA-binding protein , Su ( Hw ) , recognizes a particular motif , imparting specificity to the gypsy insulator . In addition to Su ( Hw ) , the core gypsy insulator complex contains Centrosomal protein 190 ( CP190 ) , which also harbors a zinc finger domain , and the non-DNA-binding protein , Modifier of mdg4 2 . 2 ( Mod ( mdg4 ) 2 . 2 ) . These core proteins are required for gypsy insulator activity [3]–[7] . Both CP190 and Mod ( mdg4 ) 2 . 2 contain broad complex , tramtrack , bric-a-brac ( BTB ) dimerization domains that have been suggested to mediate insulator-insulator interactions and facilitate the formation of long range insulator-mediated loops along the chromatin fiber [4] , [8] . Specialized nuclear arrangement of gypsy insulator complexes correlates tightly with insulator function . The gypsy insulator proteins bind to thousands of sites throughout the genome with more than half of Su ( Hw ) binding sites occurring in intergenic regions and a large number of sites located within introns [9] , [10] . Consistent with a role in boundary formation , Su ( Hw ) sites are positively correlated with both Lamin-associated domains and boundaries between transcriptionally active and silent chromatin [10] , [11] . It has been shown that gypsy insulator proteins coalesce at a small number of foci in diploid nuclei , termed insulator bodies , which have been proposed to act either as hubs of higher order chromatin domains [8] or storage sites for insulator proteins [12] . Importantly , mutation of certain insulator components results in impaired insulator activity coincident with diffuse or smaller , more numerous insulator bodies [4] , [8] , [12]–[14] . However , formation of insulator bodies is not sufficient for gypsy insulator activity [15] , [16] , and a detailed mechanistic understanding of insulator bodies is still lacking . Nevertheless , the tight correlation between gypsy insulator function and insulator body localization suggests an important role for these structures . Finally , in addition to a variety of accessory proteins [17]–[19] , a role for RNA in insulator function and insulator body organization was suggested based on RNA-dependent protein interaction with insulator complexes [20] . Genome-wide studies indicate that the locations of insulator protein binding sites are mainly consistent across different cell types but that insulator-dependent looping configurations may dictate differences in gene expression . In Drosophila , it has been shown that external stimuli can alter chromatin association of CP190 , possibly leading to a change in chromatin looping [21] . Recent large-scale chromatin conformation capture ( 3C ) -based studies have implicated insulator protein binding sites as key contact points mediating looping throughout the genome [22]–[25] . In several studies across species , specific chromatin conformations are observed in loci that produce tissue- or cell-type specific transcripts [26]–[32] . Whether insulators either establish tissue-specific chromatin organization or maintain configurations established via transcription is unclear . Furthermore , factors that control tissue-specific insulator-dependent chromatin organization remain unknown . This study identifies a CNS enriched , RNA recognition motif ( RRM ) containing protein , Alan Shepard ( Shep ) , as the first tissue-specific regulator of gypsy insulator activity and insulator body localization . We show that Shep interacts directly with Mod ( mdg4 ) 2 . 2 and Su ( Hw ) and also associates with gypsy insulator proteins in vivo . Using a novel quantitative , tissue-specific insulator assay , we find that Shep negatively regulates gypsy insulator activity in the CNS . In addition , mutation of Shep improves compromised insulator function and insulator body formation . Finally , genome-wide localization in the CNS-derived BG3 cell line reveals enrichment of overlap between Shep and Mod ( mdg4 ) 2 . 2 but less frequent than expected overlap among Shep , Su ( Hw ) and Mod ( mdg4 ) 2 . 2 together . These data suggest that gypsy chromatin insulator function can be regulated in a tissue-specific manner .
The putative RNA-binding protein Shep was identified as a novel interaction partner of the gypsy insulator complex . Shep , encoded by the alan shepard locus , was found by yeast two-hybrid screening as a strong interactor of Mod ( mdg4 ) 2 . 2 [17; M . Capelson and V . Corces , personal communication] . The shep gene was named based on its identification in a gravitaxis screen [33] and is predicted computationally and suggested by EST data to produce four different protein isoforms with distinct N-terminal domains that share a mostly common C-terminal region bearing two highly conserved , tandemly arranged RNA recognition motifs ( RRMs; Figure 1A ) . Isoforms B/D and E contain an additional 10 amino acid linker between the RRM domains , and all isoforms except B/D contain a 7 amino acid stretch at the C-terminus . Unlike core gypsy insulator proteins , Shep is conserved between flies and vertebrates ( data not shown ) . We confirmed the Mod ( mdg4 ) 2 . 2-Shep physical interaction in vitro using recombinant proteins . GST-fusions of Shep isoforms A , B/D , and E ( Figure 1B–1C , lanes 4–6 ) in comparison to GST-Su ( Hw ) as a positive control ( lane 3 ) and GST alone as a negative control ( lane 2 ) were isolated from bacterial extracts and tested for their ability to interact with purified recombinant His-Mod ( mdg4 ) 2 . 2 . His-Mod ( mdg4 ) 2 . 2 is detected in the bound fraction in association with Su ( Hw ) and each Shep isoform but not GST alone , indicating a direct protein-protein interaction between Shep and Mod ( mdg4 ) 2 . 2 . Similarly , we found that Shep also can interact directly with Su ( Hw ) . GST-fusions of Shep isoforms A , B/D , and E ( Figure 1D–1E , lanes 4–6 ) in comparison to GST alone ( lane 2 ) and positive control , GST-Mod ( mdg4 ) 2 . 2 ( lane 3 ) , were tested for their ability to interact with purified recombinant His-Su ( Hw ) . His-Su ( Hw ) is detected in the bound fraction in association with Mod ( mdg4 ) 2 . 2 and each Shep isoform but not GST alone . For both Su ( Hw ) and Mod ( mdg4 ) 2 . 2 binding assays , a near 1∶1 molar binding ratio between insulator proteins and Shep was observed , similar to the ratios observed between Mod ( mdg4 ) 2 . 2 and Su ( Hw ) in both binding experiments . These data provide evidence for direct protein interaction between Shep and Mod ( mdg4 ) 2 . 2 as well as between Shep and Su ( Hw ) . Shep polyclonal antisera were generated using the common C-terminal region downstream of the RRMs allowing detection of all isoforms . Multiple bands are detected in larval extracts by Western blotting , and isoforms A , B/D , C and E were inferred by predicted molecular weights of 68 , 60 , 45 , and 44 kDa , respectively ( Figure 2A , lane 1 ) . All bands are depleted upon shep RNAi hairpin knockdown , which targets all isoforms ( lane 2 ) , indicating antibody specificity . When the shepEY04794 allele , which contains a UAS sequence upstream of the shep C and E promoter , is induced ubiquitously using Gal4 , the 45 kDa doublet is enriched over wildtype , identifying these two bands as isoforms C and E ( lane 3 ) . Finally , homozygous P-element insertion in shepKG10149 predicted to disrupt translation of isoform A causes specific loss of the largest band ( lane 4 ) . By process of elimination , isoform B/D corresponds to the apparent 60 kDa band . We used our specific Shep antisera to test whether gypsy insulator proteins associate with Shep in vivo by coimmunoprecipitation . When Shep complexes are immunoprecipitated from embryo nuclear extracts using Shep or control preimmune antisera , Shep is efficiently purified with the specific antibody ( Figure 2B ) . Furthermore , a fraction of total gypsy insulator proteins CP190 , Su ( Hw ) and Mod ( mdg4 ) 2 . 2 are detected in the bound fraction in association with Shep . The Polycomb Group ( PcG ) proteins , Pc and E ( z ) are not purified in the bound fraction , indicating specificity of the interaction between Shep and insulator proteins ( Figure S1 ) . Therefore , these data demonstrate that Shep interacts by direct protein interactions with Mod ( mdg4 ) 2 . 2 and Su ( Hw ) in vitro and associates with gypsy insulator proteins in vivo . Direct physical interaction between Shep and gypsy insulator proteins prompted us to examine the functional relationship between shep and the gypsy insulator . We first obtained and characterized shep alleles bearing either P-element insertions or FRT-derived deletions independently generated from seven different genetic backgrounds [Figure 3A]; [ Table 1; 34 , 35] . To determine whether these alleles are loss-of-function , we performed quantitative RT-PCR for total and specific shep isoform transcript levels and observed decreases in larvae hemizygous for shep or containing homozygous shep P-element insertions ( data not shown ) . Furthermore , four different homozygous P-element insertions result in loss of Shep protein , two greatly reducing all isoforms ( Figure 3B , lanes 2–3 ) and two eliminating isoform A ( lanes 7–8 ) . No changes in Shep protein were observed when P-element alleles are heterozygous ( data not shown ) , suggesting that these mutations are recessive . Additionally , Df ( 3L ) Exel6104 transheterozygous deficiency combinations are viable and retain isoforms C and E , suggesting that isoforms A and B/D are not essential ( Figure 3C , lanes 6–7 ) . Other transheterozygous combinations of deficiencies or homozygous deficiencies cause lethality ( Table 1 ) , but due to deletion of neighboring essential genes , we cannot determine whether shep itself is essential for viability using these alleles . Importantly , no change in CP190 , Su ( Hw ) or Mod ( mdg4 ) 2 . 2 protein levels is observed in shep mutants relative to wildtype levels ( Figure 3B–3C ) . These data show that P-element insertions and deficiencies decrease Shep protein levels and likely constitute loss-of-function alleles . We observed that mod ( mdg4 ) mutants are particularly sensitive to shep expression levels . Homozygous shep P-element insertion alleles are viable in a wildtype background; however , in combination with mod ( mdg4 ) u1 , which is fully viable but null for the mod ( mdg4 ) 2 . 2 isoform , homozygous shep mutants displaying reduced Shep protein specifically exhibit strongly reduced viability ( Table 1 ) . We observed lethality in late pupal development and pharate adults; only 9 . 2% of shepBG00836 and 23% of shepd05714 mod ( mdg4 ) u1 double mutant pupae survive to adulthood . Synthetic lethality was also observed for shep mutant alleles in combination with the mod ( mdg4 ) T6 loss-of-function point mutation , confirming the genetic interaction . Moreover , overexpression of the shepEY04794 allele containing a UAS insertion or the Shep E isoform from a transgenic copy inserted on a different chromosome using the Act5C::Gal4 driver causes complete inviability of adult flies in the mod ( mdg4 ) u1 background but not in wild type . In contrast , overexpression of the Shep E isoform harboring point mutations in the RRM domain designed to disrupt RNA-binding activity but not protein folding does not cause lethality in mod ( mdg4 ) u1 flies despite both versions of Shep E protein being expressed at the same levels in wildtype flies ( data not shown ) . The apparent sensitivity of mod ( mdg4 ) u1 null mutants to alterations in Shep levels is consistent with direct physical interactions between Shep and insulator proteins and further suggests an antagonistic functional relationship between Mod ( mdg4 ) 2 . 2 and Shep , likely requiring Shep RNA-binding activity . In order to assess whether shep loss-of-function affects insulator activity in vivo , we examined the phenotypes of two well-characterized gypsy-dependent alleles , y2 and ct6 . These alleles result from gypsy retrotransposon insertion between the upstream body enhancer and promoter of y or between the upstream distal wing margin enhancer and promoter of ct [36] . These insertions block enhancer function , resulting in loss of abdominal cuticle pigmentation or misshapen wing margin , respectively . In an otherwise wildtype background , shep P-element alleles and deficiencies produce no decrease in enhancer blocking activity at y2 or ct6 ( data not shown ) , and since y2 and ct6 are fully active for enhancer blocking , an increase in insulator activity cannot be assessed . In order to sensitize the assay , y2 and ct6 were examined in the presence of the mod ( mdg4 ) u1 mutation . This mutation disrupts insulator function and allows partial restoration of enhancer-promoter communication . The gypsy-dependent phenotypes in homozygous shep P-element alleles in the mod ( mdg4 ) u1 background were scored for ct6 on a scale of 0–4 with increasing severity of phenotype . Approximately half of male mod ( mdg4 ) u1 wings display a score of zero ( Figure 4A ) . In contrast , for eight of ten homozygous P-element and all heterozygous deficiency alleles of shep , we observed positive effects on enhancer blocking activity at ct6 in the mod ( mdg4 ) u1 background ( Figure 4A , Table 1 , Table S1 ) , indicating increased gypsy insulator activity . For shepBG00836 and shepd05714 mod ( mdg4 ) u1 double homozygous mutants , only escapers could be scored due to synthetic lethality . Similar changes in insulator phenotypes were observed for shep hemizygous mutations ( Figure 4B ) but not heterozygous mutations in the mod ( mdg4 ) u1 background ( data not shown ) , indicating that these shep mutations are recessive with respect to insulator activity . To verify that the P-element insertion alleles are loss-of-function for enhancer blocking activity , the insulator phenotypes of each shep P-element allele crossed to each deficiency were examined . We found that insulator phenotypes and synthetic lethality remained the same or insulator function was slightly increased compared to homozygous P-elements , except when shepBG00836 and shepd05714 are transheterozygous with Df ( 3L ) Exel6104 ( Table 1 ) . In these cases , synthetic lethality is rescued , corresponding to elevated isoform C and E transcript and protein levels likely due to artificial juxtaposition of the C and E promoter to a cis-regulatory element from a partially deleted upstream gene or mini-w+of the original P-element remaining after FRT excision ( data not shown ) . Nevertheless , insulator activity of these shepBG00836 and shepd05714 transheterozygous mutants is improved compared to mod ( mdg4 ) u1 , confirming that shepBG00836 and shepd05714 are loss-of-function alleles ( Figure 4B ) . We determined that shep P-element mutants in the mod ( mdg4 ) u1 background do not affect the phenotype of ctn , caused by insertion of a roo transposable element ( data not shown ) . This result suggests that the effect of shep on ct6 is due to changes in gypsy insulator activity and not direct regulation of ct expression . Importantly , since shep mutants affect insulator activity in mod ( mdg4 ) u1 null mutants , it likely that , in vivo , Shep can interact with Su ( Hw ) in the absence of Mod ( mdg4 ) 2 . 2 . Overall , these data indicate that the wildtype function of Shep is to negatively regulate gypsy insulator activity . In contrast to positive effects on ct6 , shep mutations in the mod ( mdg4 ) u1 background do not affect y2 . The phenotype of y2 remained unchanged by mutation or deletion of shep in the mod ( mdg4 ) u1 background ( Figure 4C , data not shown ) . The specific effect at ct6 but not y2 in shep mutants raises the possibility that shep negatively regulates a subset of gypsy insulators . In order to determine how Shep regulates insulator function and in what contexts , we examined the distribution of Shep in late stage wildtype embryos . We find that Shep protein is enriched in the embryonic CNS including the brain and ventral nerve cord , areas that are also positive for the neuron-specific protein Elav ( Figure 5A ) . The overlap between Shep and Elav is partial in that Shep is also expressed in glial cells . Shep levels are low but detectable in non-CNS tissues; likewise , microarray expression data from various developmental stages are consistent with our results [37] . In the third instar larval stage , higher overall protein levels are detected in the brain compared to eye , leg , or wing imaginal discs or salivary glands by Western blotting ( Figure 5B ) as well as immunofluorescence ( data not shown ) . These data demonstrate that Shep is a CNS-enriched protein at both embryonic and larval stages . In order to examine whether Shep affects insulator complexes in a tissue-specific manner , we examined the localization of insulator bodies in the presence and absence of Shep in larval brain compared to non-CNS cell types . Wild type , mod ( mdg4 ) u1 and double mutant shepBG00836 , mod ( mdg4 ) u1 whole mount larval brain and imaginal disc tissues were stained using antibodies directed against CP190 . Because the brain contains heterogeneous cell types , we focused on peripheral cells in the medulla of the brain lobe in which 1–2 insulator bodies are visible in the nucleus per focal plane . In mod ( mdg4 ) u1 mutants , insulator bodies are disrupted in all tissues including the brain , resulting in an increased number of foci compared to wild type ( Figure 5C ) . In shepBG00836 , mod ( mdg4 ) u1 double mutants , insulator body localization in the brain reverts to a wildtype appearance ( observed in 8 of 9 experiments ) . The same effect is also observed in perineurial glia of the outer cell layer surrounding the brain hemispheres ( data not shown ) . In contrast , peripheral cells of the eye and leg imaginal discs , which display low Shep expression , insulator bodies are indistinguishable in shepBG00836 , mod ( mdg4 ) u1 compared to mod ( mdg4 ) u1 mutants . We also did not observe differences in CP190 localization in peripheral cells of the wing imaginal disc; however , insulator bodies in all genotypes are less prominent in this tissue type ( data not shown ) . Additional shep mutants examined , shepKG10149 , shepe00306 , shepBG00655a , and shepBG02613 , display similar effects ( data not shown ) . Restoration of mislocalized insulator bodies when shep levels are reduced in the brain but not non-CNS tissue suggests a tissue-specific role for Shep in disrupting insulator activity . In order to determine whether Shep affects insulator activity in the CNS , we developed a versatile barrier assay that allows quantification of gypsy insulator activity using identical reporters in essentially any tissue of interest . This assay relies on three transgenes: the transcriptional reporter UAS-luciferase inserted into a defined attP landing site , either insulated by flanking Su ( Hw ) binding sites or non-insulated [38]; a Gal4-inducible dsRNA hairpin construct for knockdown of a gene of interest [39]; and a tissue-specific Gal4 driver . This system allows for directly comparable quantification of luciferase activity in the insulated or non-insulated context in the presence or absence of a protein of interest . Use of the Gal4 system allows interrogation of a specific subset of cells for both the reporter as well as the hairpin knockdown within an otherwise wildtype organism , which is not easily achieved using standard genetic manipulation of existing mutants . We used luciferase reporter constructs inserted into attP3 on the X chromosome [40] , which display extremely low basal expression unless insulated ( Figure 5E–5G ) relative to other attP insertion sites tested [38] . Insulator-dependent expression at attP3 is likely due to its positioning within a PcG repressed region ( Figure S2 ) . Addition of insulators flanking the UAS-luciferase reporter likely stops the spread of repressive chromatin , allowing for measurable activity . Due to high variability of expression among individuals , luciferase levels were measured in individual whole third instar larvae , and values for each population ( n≥12 ) were compared by one-way ANOVA . As proof of principle , ubiquitously expressed Act5C::Gal4 induces high luciferase activity in insulated compared to non-insulated lines ( Figure 5E ) . As expected , su ( Hw ) knockdown causes a drastic reduction in both Su ( Hw ) protein ( Figure 5D , lanes 3 and 7 ) and luciferase activity in insulated but not non-insulated lines ( Figure 5E ) , indicating that luciferase expression directly reports Su ( Hw ) -mediated insulation . In contrast , upon shep knockdown ( Figure 5D , lanes 4 and 8 ) an increase in luciferase activity is observed for the insulated line ( p = 0 . 0055 , Tukey's HSD post hoc test ) , indicating an increase in insulator activity ( Figure 5E ) . Therefore , Shep negatively influences both gypsy-dependent barrier and enhancer blocking activities . Since ubiquitous knockdown of shep could report an increase in insulator activity in any or all tissues , CNS-specific Gal4 expression was utilized to quantitatively address whether shep affects gypsy insulator activity in the CNS . Localized Gal4 expression in the CNS with l ( 3 ) 31-1::Gal4 induces luciferase to a lower level than ubiquitous Gal4 due to its restricted expression pattern ( Figure 5F ) . Upon su ( Hw ) knockdown in the CNS , luciferase expression returns to non-insulated levels . In contrast , when shep is knocked down , a marginally significant increase in luciferase levels is observed ( p = 0 . 053 ) , demonstrating that shep negatively affects insulator activity in the CNS . Finally , we tested whether Shep affects barrier activity in muscle cells , a tissue type that expresses low levels of Shep . Muscle-specific Mef2::Gal4 induces high levels of luciferase activity; accordingly , su ( Hw ) knockdown results in a dramatic decrease in luciferase activity ( Figure 5G ) . In contrast , shep knockdown in muscle tissue has no significant effect compared to Mef2::Gal4 alone ( p = 0 . 99 ) , demonstrating that shep does not play a substantial role in insulator activity in muscle tissue . However , ectopic overexpression of Shep C and E using shepEY04794 in muscle tissue is sufficient to result in decreased insulator activity ( p = 2 . 2×10−5 ) . Therefore , in muscle cells , artificially reaching a certain threshold of Shep protein expression reduces insulator activity . This quantitative and tissue-specific insulator assay further supports a role for Shep as a negative regulator of gypsy insulator activity . In order to determine the extent to which Shep colocalizes with insulator proteins , we mapped the genome-wide chromatin association profiles of Su ( Hw ) , Mod ( mdg4 ) 2 . 2 , and Shep by ChIP-seq in the BG3 larval CNS-derived cell line . Using previously characterized Su ( Hw ) and Mod ( mdg4 ) 2 . 2 antibodies [16] , [41] , [42] as well as our specific Shep antisera ( see methods ) , we observe sharp peaks of Su ( Hw ) , Mod ( mdg4 ) 2 . 2 , and Shep , as well as broader peaks of Shep signal ( Figure 6A–6B ) . Using the SPP algorithm [43] at a 1% false discovery rate ( FDR ) , we detected 4099 Su ( Hw ) peaks , 1575 Mod ( mdg4 ) 2 . 2 peaks , and 4443 Shep peaks ( Figure 6C ) , numbers in agreement with previous studies of Su ( Hw ) and Mod ( mdg4 ) 2 . 2 binding profiles in various cell types [9] , [10] , [42] . Similar to previous studies [10] , [42] , [44] , the majority of Mod ( mdg4 ) 2 . 2 sites overlap with Su ( Hw ) , and strong enrichment of overlap is observed compared to random expectation ( Figure 6D ) . As expected , Su ( Hw ) is found mostly in inter- and intragenic regions [9] , [10] , [44] ( Figure 6C ) . In contrast , Shep binding is mainly observed over genes , with 65% of Shep peaks falling in transcription start sites ( TSSs ) . An intermediate distribution pattern is observed for Mod ( mdg4 ) 2 . 2 . Given that Shep can interact directly with either Su ( Hw ) or Mod ( mdg4 ) 2 . 2 and copurifies with a fraction of total gypsy insulator core proteins , we expected a substantial degree of overlap between Shep and either Su ( Hw ) or Mod ( mdg4 ) 2 . 2 . Indeed , nearly half of Mod ( mdg4 ) 2 . 2 sites overlap with Shep , and 16% of Shep sites overlap with Mod ( mdg4 ) 2 . 2 ( Figure 6E ) . The observed overlap between Shep and Mod ( mdg4 ) 2 . 2 is greater than random expectation ( Figure 6D ) . In contrast , no enrichment is observed for colocalization between Shep and Su ( Hw ) . Nevertheless , nearly one quarter of Shep binding sites overlap with either Su ( Hw ) or Mod ( mdg4 ) 2 . 2 ( Figure 6E ) , supporting the notion that a substantial fraction of chromatin-associated Shep harbors insulator-related activity . Although expressed at low levels in salivary glands , Shep localization in polytene chromosomes also shows partial overlap between Shep and gypsy insulator proteins ( Figure S3 ) . Chromatin association of Shep at non-gypsy insulator sites could reflect alternate unknown functions of Shep or a gypsy insulator-independent means of recruitment . We next compared Shep genome-wide localization with that of a variety of chromatin-associated factors and histone modification marks in BG3 cells . Enrichment scores for two-way overlaps between all factors were calculated , and unsupervised hierarchical clustering was performed ( Figure S4 ) . This analysis reveals high similarity of binding profiles of the insulator proteins Su ( Hw ) , Mod ( mdg4 ) 2 . 2 , CP190 , and CTCF ( Figure 6D ) . In contrast , Shep genome-wide localization most closely resembles factors associated with active transcription such as RNA polymerase II . Analysis of Shep sites not overlapping with either Su ( Hw ) or Mod ( mdg4 ) 2 . 2 also overlap significantly with active transcription marks . Consistent with our comparative analysis , Shep localization is likewise observed at highly transcribed puff regions of polytene chromosomes ( Figure S3 ) . Interestingly , Shep genome-wide localization also displays similarity to that of Chromator , a protein recently implicated as a boundary factor potentially capable of organizing physical chromatin domains [25] and also overlaps significantly with CP190 and BEAF ( Figure 6D ) . Consistent with Shep functioning as a negative regulator of gypsy insulator activity , we noted a significantly lower than expected frequency of three-way overlap among Shep , Su ( Hw ) and Mod ( mdg4 ) 2 . 2 . In fact , the three factors are only observed together at 271 sites ( Figure 6E ) . Considering the 1403 Mod ( mdg4 ) 2 . 2 sites that colocalize with either Su ( Hw ) or Shep , this degree of three-way overlap is lower than expected by chance ( p<1×10−4 , permutation test; p = 2 . 2×10−16 , hypergeometric test ) . The same results are obtained when this analysis is performed on Su ( Hw ) sites that overlap with either Mod ( mdg4 ) 2 . 2 and Shep as well as the Shep sites that overlap with either Su ( Hw ) or Mod ( mdg4 ) 2 . 2 ( see methods ) . Taken together , these results indicate substantial colocalization of Shep with Mod ( mdg4 ) 2 . 2 but limited three-way overlap among Shep and both gypsy insulator proteins .
Shep acts as a tissue-specific negative regulator of gypsy insulator function and insulator body localization . Shep localization is most enriched in the CNS at both embryonic and larval stages; however , it is also expressed at lower levels in additional tissues . Although we have demonstrated that Shep functions in the CNS , Shep can also repress enhancer blocking activity in the wing and could possibly affect insulator activity in other tissues . For example , ubiquitous reduction of Shep levels strongly improves overall barrier activity , suggesting that tissues outside of the CNS may also harbor Shep activity . Nonetheless , Shep does not appear to function in all tissues; knockdown of Shep does not affect barrier activity in muscle tissue , no changes in insulator body localization are observed in eye or leg tissue of shep mutants , and no effect is observed for y2 enhancer blocking in pigment cells of shep mutants . Interestingly , when Shep is overexpressed in muscle tissue , reduction of barrier activity is observed , suggesting that a certain threshold of Shep protein is needed to repress insulator activity . Since Shep protein can be detected at least at low levels in all tissues tested thus far , it is unlikely that the mere presence of Shep protein is sufficient to disrupt gypsy insulator activity . It remains to be determined what other cofactors , such as proteins or RNAs , may contribute to Shep activity . Shep may negatively regulate insulator activity by interfering with insulator protein interactions required for their activity . ChIP-seq analyses shows that the genome-wide binding profile of Shep in CNS-derived BG3 cells overlaps substantially with that of Mod ( mdg4 ) 2 . 2 but not extensively with both Su ( Hw ) and Mod ( mdg4 ) 2 . 2 combined . Lack of three-way overlap is not entirely unexpected given that Shep is a negative regulator of gypsy insulator activities . Shep coimmunoprecipitation experiments copurify only a small fraction of total insulator proteins present in nuclear extracts , suggesting that Shep-insulator complexes are not abundant or not stable in vivo . Since Shep can bind either Mod ( mdg4 ) 2 . 2 or Su ( Hw ) in vitro at a 1∶1 ratio , Shep binding could compete with direct interaction between Mod ( mdg4 ) 2 . 2 and Su ( Hw ) or their interactions with other factors such as CP190 . Moreover , our finding that mod ( mdg4 ) mutants are highly sensitive to Shep dosage suggests an antagonistic functional relationship between Mod ( mdg4 ) 2 . 2 and Shep . Specifically , Shep may negatively regulate higher order insulator-insulator complex interactions , which appear to be mediated by direct interaction between Mod ( mdg4 ) 2 . 2 and CP190 [4] . Insulator body localization in larval brains of shep , mod ( mdg4 ) u1 mutants reverts back to a wildtype pattern compared to compromised mod ( mdg4 ) u1 mutants , perhaps indicating that the normal function of Shep may be to prevent larger insulator complexes from forming in these cell types . Our results are consistent with the possibility that Shep promotes tissue-specific chromatin configurations by modulating insulator complexes . While differential occupancy of insulator proteins at their respective binding sites may play a role in regulating certain loci [21] , occupancy throughout the genome does not differ extensively between cell types [9] , [45] . Therefore , alternate mechanisms to control insulator activity likely exist . Shep activity could prevent insulator-insulator contacts otherwise present in tissues that do not express shep , resulting in relief of enhancer blocking or repression by silencers . Interestingly , shep was identified as a regulator of complex behavioral traits in screens for altered sensory-motor responsiveness to gravity [33] and aggressive behavior [46] , suggesting the possibility that regulation of an insulator-based mechanism could exist to effect changes in neurological function . Given that Shep is an RRM-containing protein , RNA-binding may contribute to the ability of Shep to associate with insulator complexes in vivo . Shep RRMs are highly conserved , and lethality caused by Shep overexpression in the mod ( mdg4 ) mutant background is not observed when the RRMs are mutated . This result suggests that Shep RRMs may be functional with respect to insulator activity . One possibility is that the specific RNA bound by Shep could affect targeting of Shep to insulator sites . Another not mutually exclusive prospect is that Shep is recruited to chromatin cotranscriptionally by binding nascent transcripts . It will be important to determine in future studies if Shep binds RNA while in complex with gypsy insulator proteins as well as the identities of Shep and insulator-associated RNA . Our results point to a novel role for Shep and possibly RNA to regulate insulator activity in a tissue-specific manner .
Stocks were raised at 25°C on standard cornmeal medium . Shep P-element insertion alleles , shep deficiencies , Act5C::Gal4 , Mef2::Gal4 , and l ( 3 ) 31-1::Gal4 were obtained from the Bloomington and Exelixis Stock Centers . Lines expressing su ( Hw ) ( 10724 GD ) or shep dsRNA ( 37863 GD ) were obtained from the Vienna Drosophila RNAi Center . UAS::luciferase constructs were inserted into the attP3 landing site [38] . The ct6 phenotype was scored in flies on the first day after eclosion . For all genotypes , males show a more severe wing notching phenotype than females . The y2 phenotype was scored in flies aged for 1 d at 25°C . Larvae for luciferase insulator assays and whole mount immunofluorescence were raised at 25°C . Larvae for polytene chromosome staining were raised at 18°C . Embryos aged 0–24 h for nuclear extracts and immunofluorescence were collected from a population cage as described [20] . Anterior thirds of larvae were used for Western blotting . Coding regions of FlyBase annotated Shep isoforms RA , RB/RD , and RE were amplified by PCR from Trizol ( Invitrogen ) extracted , Oregon R embryonic cRNA that was oligo-dT primed and reverse transcribed by Superscript III ( Invitrogen ) . The isoform RA clone obtained differs from FlyBase annotations in 2 locations where either a downstream splice site was used , as in isoforms B , D , and E , resulting in 12 extra amino acids and an additional unannotated exon was included resulting in an additional 11 amino acids; RRM domains remain intact in this isoform A variant . The shep , su ( Hw ) , and mod ( mdg4 ) 2 . 2 cDNAs were inserted into pENTR/D-TOPO and recombined into pDEST 15 ( Invitrogen ) to generate N-terminal GST fusion constructs . All plasmids were sequenced for verification . Expression of GST , GST-Shep , GST-Su ( Hw ) , GST-Mod ( mdg4 ) 2 . 2 , His-Su ( Hw ) , and His-Mod ( mdg4 ) 2 . 2 was induced in E . coli BL21 cells by 1 mM Isopropyl β-D-1-thiogalactopyranoside at 37° C . Proteins were purified under native conditions by affinity using Glutathione-Agarose ( Pierce ) or Ni-NTA-Agarose ( Qiagen ) . 2 µg immobilized GST or GST fusion proteins were incubated with 35 µg soluble His-Mod ( mdg4 ) 2 . 2 in PBSMT ( 137 mM NaCl , 2 . 7 mM KCl , 10 mM NaH2PO4 , 1 . 8 mM KH2PO4 , 250 mM MgCl2 , 0 . 3% Triton X-100 supplemented with Complete protease inhibitors ( Roche ) , 1 mM PMSF , 1 mM DTT , and 100 mg/mL BSA ) in a volume of 350 µL . Binding reactions were carried out at 4°C rotating for 2 h . Unbound protein was removed , and beads were washed 5 times in PBSMT . Bound His-Mod ( mdg4 ) 2 . 2 was eluted in sample buffer , separated by SDS-PAGE , and detected by Western blotting . His-Su ( Hw ) binding reactions were carried out in the same manner except in HBSM ( 50 mM HEPES , pH 6 . 7; 150 mM NaCl; 5 mM KCl; 2 . 5 mM MgCl2 ) supplemented with 0 . 3% Triton-X 100 , 0 . 2 M KCl , protease inhibitors , 1 mM PMSF , 1 mM DTT , and 100 mg/mL BSA . 6X-His-Shep isoform E ( AA169–368 ) , CP190 antigen [4] , Su ( Hw ) antigen [41] and Mod ( mdg4 ) 2 . 2 antigen [47] were expressed in BL21 cells , affinity purified by Ni-NTA-agarose ( Qiagen ) according to the manufacturer's protocol under denaturing conditions and used to immunize rabbits and guinea pigs using standard procedures ( Covance Research Products ) . For Western blotting , guinea pig α-Shep serum was used at 1∶2000 , guinea pig α-CP190 was used at 1∶10 , 000 , guinea pig α-Mod ( mdg4 ) 2 . 2 [41] was used at 1∶1000 , guinea pig α-Su ( Hw ) [41] was used at 1∶7500 , α-Pc [32] was used at 1∶1000 , α-E ( z ) [48] was used at 1∶1000 , and α-Pep [49] was used at 1∶1000 . For insulator body staining , rabbit α-CP190 [4] was used at 1∶30 , 000 . The monoclonal α-Elav9AF89 was obtained from the Developmental Studies Hybridoma Bank and used at 1∶1000 , and guinea pig α-Shep serum was used at 1∶200 for IF . Nuclei from 20 g of embryos were prepared as described [20] . Nuclei were lysed in 4 mL HBSM supplemented with 0 . 3% TritonX-100 ( HBSMT ) , complete protease inhibitors and 1 mM PMSF by dounce homogenization with the B pestle . Extracts were cleared of insoluble material by centrifugation , and half of the supernatant was incubated with pre-immune serum and half with α-Shep serum pre-conjugated to protein A sepharose . IPs were carried out for 1 h at 4°C , rotating . Unbound protein was removed and beads were washed 4 X in HBSMT and 1 X in HBSM . Bound protein was eluted in sample buffer , separated by SDS-PAGE , and detected by Western blotting . Polytene chromosome spreads were prepared as described previously [8] . Brains and imaginal discs were dissected from at least 5 larvae of each genotype per experiment and stained as described previously [32] . Chromosomes and discs were imaged using a Leica DM5000B epifluorescent microscope and captured using OpenLab software . Indirect immunofluorescence of mixed stage Oregon R embryos was carried out as described [50] , [51] . Blocked embryos were incubated rotating with primary antibodies overnight at 4°C and secondary antibodies for 2 h at 37°C . After washing , embryos were incubated in DAPI and mounted in 2 . 5% DABCO ( Sigma ) in 70% glycerol . Embryos were imaged on a Zeiss 510 confocal microscope . Females homozygous for attP3::UAS-luciferase transgenes were crossed to Gal4 expressing males; luciferase in individual F1 male larvae was quantified . Any homozygous lethal Gal4 lines were selected against GFP expressing balancer chromosomes . Larvae were collected on dry ice and stored at −80°C until use , at which time they were homogenized in 30 µL Glo Lysis buffer ( Promega ) and incubated at room temperature for 10 min . Debris was cleared from extracts by centrifugation , and 20 µL soluble material was dispensed into opaque 96-well plates; the same volume of luciferase reagent ( Promega ) was added to each well , and plates were incubated in the dark for 10 min . Light emission was quantified using a Spectramax II Gemini EM plate reader ( Molecular Devices ) . Luciferase values were normalized to total protein determined by Bradford assay carried out in parallel . Luciferase values between genotype populations were log transformed to obtain a normal distribution and compared by one-way ANOVA . Tukey HSD post hoc tests were used to determine pairwise p values between genotypes . For further information including additional Gal4 lines tested , see Text S1 . BG3-c2 cells were grown in S2 medium ( Sigma ) supplemented with 10% fetal calf serum and 10 µg/mL insulin . Cells were maintained in monolayer at 25°C . Cells were fixed in 1% formaldehyde added directly to cells in culture medium for 10 min at RT with gentle agitation; formaldehyde was quenched by addition of glycine to 0 . 125 M with gentle agitation for 5 min at RT . 5×106 to 107 cells were used per IP . Cells were pelleted at 400 rcf and washed twice in ice cold PBS . Cells were resuspended in 1 mL ice cold cell lysis buffer ( 5 mM PIPES , pH 8 , 85 mM KCl , 0 . 5% NP-40 ) supplemented with protease inhibitors , and nuclei were released by Dounce homogenization with the B pestle and pelleted by centrifugation at 9190 rcf for 5 min at 4°C . Nuclei and chromatin were further processed as described [41] . Chromatin was fragmented to an average size of 300 bp by sonication and validated by agarose gel electrophoresis . Sequencing libraries were prepared according to the standard Illumina ChIP-seq protocol . Highly similar profiles were obtained with two independent α-Shep antibodies; therefore , the antibody ( guinea pig ) displaying the highest signal to noise ratio was utilized for subsequent analyses . Rabbit α-Su ( Hw ) [16] and rabbit α-Mod ( mdg4 ) 2 . 2 [42] were used for ChIP-seq . Highly similar profiles were obtained with two independent α-Mod ( mdg4 ) 2 . 2 antibodies [41]; therefore , the antibody displaying the highest signal to noise ratio was utilized for subsequent analyses . Libraries were constructed with TruSeq adapters and sequenced on an Illumina HiSeq multiplexed in a single lane . For directed ChIP , quantitative PCR was performed as previously reported [32] .
|
Mounting evidence in human , mouse , and Drosophila demonstrates a role for the DNA–protein complexes known as chromatin insulators in orchestrating three-dimensional genome organization . Several genes that are only expressed in specific cell types display distinct chromatin configurations correlated with expression status . Recent evidence shows that chromatin insulators play a role in defining tissue-specific chromatin conformation; however , tissue-specific factors that may modulate insulator activity remain unknown . Here we identify a putative RNA–binding protein , Shep , which is expressed most highly in the CNS and interacts directly with insulator complexes . We developed a novel quantitative , tissue-specific insulator assay and found that Shep negatively regulates insulator activity in the CNS . We also find that mutation of shep alters insulator complex nuclear localization in the brain but not other tissues . Finally , we mapped Shep and gypsy insulator protein localization throughout the genome and found that Shep colocalizes with one individual insulator protein but less often than expected with an intact insulator complex . These data suggest that Shep negatively influences insulator activity in a tissue-specific manner .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"animal",
"models",
"drosophila",
"melanogaster",
"model",
"organisms",
"molecular",
"cell",
"biology",
"cell",
"biology",
"chromosome",
"biology",
"genetics",
"molecular",
"genetics",
"biology",
"genomics",
"chromatin",
"genetics",
"and",
"genomics",
"gene",
"function"
] |
2012
|
Tissue-Specific Regulation of Chromatin Insulator Function
|
In the wake of the West African Ebola virus disease ( EVD ) outbreak of 2014–2016 , thousands of EVD survivors began to manifest a constellation of systemic and ophthalmic sequelae . Besides systemic arthralgias , myalgias , and abdominal pain , patients were developing uveitis , a spectrum of inflammatory eye disease leading to eye pain , redness , and vision loss . To investigate this emerging eye disease , resources and equipment were needed to promptly evaluate this sight-threatening condition , particularly given our identification of Ebola virus in the ocular fluid of an EVD survivor during disease convalescence . A collaborative effort involving ophthalmologists , infectious disease specialists , eye care nurses , and physician leadership at Eternal Love Winning Africa ( ELWA ) Hospital in Liberia led to the development of a unique screening eye clinic for EVD survivors to screen , treat , and refer patients for more definitive care . Medications , resources , and equipment were procured from a variety of sources including discount websites , donations , purchasing with humanitarian discounts , and limited retail to develop a screening eye clinic and rapidly perform detailed ophthalmologic exams . Findings were documented in 96 EVD survivors to inform public health officials and eye care providers of the emerging disease process . Personal protective equipment was tailored to the environment and implications of EBOV persistence within intraocular fluid . A screening eye clinic was feasible and effective for the rapid screening , care , and referral of EVD survivors with uveitis and retinal disease . Patients were screened promptly for an initial assessment of the disease process , which has informed other efforts within West Africa related to immediate patient care needs and our collective understanding of EVD sequelae . Further attention is needed to understand the pathogensis and treatment of ophthalmic sequelae given recent EVD outbreaks in West Africa and ongoing outbreak within Democratic Republic of Congo .
The West African Ebola virus disease ( EVD ) outbreak from 2014–2016 was of historic magnitude with over 28 , 600 cases and 11 , 300 deaths predominantly within the highest transmission countries of Liberia , Sierra Leone and Guinea . [1] This has resulted in the largest cohort of EVD survivors in history with thousands of EVD survivors requiring medical care . Following survival from acute EVD , Ebola survivors are at significant risk of systemic and ocular sequelae . [2] These sequleae include arthritis , arthralgias , headache , hair loss , abdominal pain , and uveitis . Knowledge of the ophthalmic complications identified in EVD survivors prior to this most recent outbreak was limited given the size of prior outbreaks dispersed throughout central Africa . [3] As reports of eye disease emerged via communications with providers in West Africa , in addition to the repatriation of health care workers ( HCWs ) from West Africa to the United States following Ebola virus ( EBOV ) infection[4 , 5] , the clinical imperative for rapid screening evaluation of EVD survivors and assessment of the prevalence of eye disease , clinical spectrum , and treatment algorithms became increasingly evident . Specifically , after our team cared for a repatriated United States health care worker ( HCW ) with aggressive , sight-threatening panuveitis associated with heterochromia , scleritis , and refractory hypotony following recovery from life-threatening , critical illness due to EVD , there were concerns for ophthalmic sequelae with potentially vision-threatening consequences throughout West Africa . [4 , 6] Another United States HCW had also developed severe uveitis requiring oral corticosteroids to avert severe vision impairment . [5] In addition , the finding of replicating Ebola virus in the ocular fluid raised the emerging public health concern of possible infectivity of ocular fluid if eye care providers performed invasive procedures on EVD survivors . [4] With increasing anecdotal reports of sight-threatening eye disease emerging from West Africa and concerns about undiagnosed and untreated eye disease at the Eternal Love Winning Africa ( ELWA ) EVD survivor clinic , the administrative and clinical leadership of ELWA Hospital , in partnership with clinicians from Emory Eye Center , responded to the emerging reports of ocular symptoms and eye disease in EVD survivors via the development of a screening eye clinic . This eye clinic , entitled the ‘Quiet Eye West Africa Project’ was rapidly implemented to assess the prevalence of eye disease , degree and burden of vision impairment , and resource requirements for specific ophthalmologic needs . In this report , we describe the detailed personnel requirements , ophthalmic equipment and supply procurement strategy , screening eye clinic design and flow that could be utilized in the screening setting , and a modular design of the clinic for clinical evaluation and teaching of basic components of the eye exam to in-country staff . In addition , infection control precautions and personal protective equipment guided by our initial assessment of EBOV by RT-PCR in both intra- and extraocular fluid8 are described for the screening of survivors in the EVD outbreak setting . Given the recent and ongoing outbreaks in the Democratic Republic of Congo ( DRC ) , there likely will be an ongoing need to understand the resources , equipment , expertise and systems needed for EVD survivor care beyond the critical importance of acute EVD care in resource limited settings .
Eternal Love Winning Africa Hospital is a health facility founded by Serving in Mission ( SIM ) in 1965 located in Paynesville City , Monrovia , Liberia . Faith-based organizations ( Samaritan’s Purse and SIM ) developed and operated the ELWA ( ELWA-1 and 2 ) Ebola Treatment Units ( ETUs ) from June to August 2014 with the support of Medecins sans Frontieres-Belgium ( MSF-B ) . [9] ELWA-1 and 2 cared for 69 patients from June to August 2014 and 53 ( 77% ) were confirmed cases . [9] An EVD Survivor Clinic was initiated in January 2015 as a response to the emerging EVD systemic sequelae increasingly observed in survivors . EVD Survivor services provided include general adult and pediatric medicine , surgical consultation , rheumatology , psychosocial counseling , prenatal care , imaging , and laboratory studies . In-country Liberian personnel requirements required at ELWA Hospital during development of the eye clinic included registration ( 2 individuals ) , interpreters/ medical assistants ( 4 ) , and primary care physicians ( 2 ) involved in direct EVD patient care in an Ebola Treatment Unit ( ETU ) setting . Emory University and United States HCWs included three ophthalmologists and one infectious disease provider familiar with EVD survivor sequelae and EVD patient care in an ETU setting . In addition , engagement with the Ministry of Health and Sanitation Liberia was important for ongoing discussion of the patient findings , determination of resources needed for clinical ophthalmic care , and temporary Liberian medical licensure for physicians involved in EVD survivor ophthalmic care . EVD survivors who were receiving ongoing care at ELWA Hospital Survivor Clinic were offered an ophthalmic evaluation during the screening program period at the time of their general medical care evaluation . In addition , EVD survivor associations were engaged by ELWA clinicians regarding the screening effort prior to the eye care program so that they could receive an evaluation , care , or referral as needed . The presence of an eye clinic for screening , treatment , and referral was also directly communicated with existing EVD survivor clinics in Monrovia ( e . g . Médecins Sans Frontières ) for patient evaluation . A supply list for the ophthalmic clinic was generated to include all clinical equipment required for the eye exam ( Snellen visual acuity charts , Tumbling “E” chart , penlights , Tonopen for intraocular pressure assessment and disposable Tonopen tip covers , portable slit lamp , indirect ophthalmoscopes with condensing lens and appropriate voltage converters; Table 1 ) . Because of the uncertain availability of topical medications required to treat uveitis and uveitis sequelae , as well as other treatable ocular diseases ( i . e . ocular hypertension ) , supply lists were generated for topical medications required for clinical examination ( i . e . topical proparacaine , 2 . 5% phenylephrine , 1% tropicamide ) , as well as for therapeutic use for ocular inflammatory disease ( i . e . topical corticosteroid , topical cycloplegic agents ) . Multiple sources were researched for ophthalmic equipment and medications to limit the overall cost; these sources included industry donations , discount websites ( eBay , Amazon ) and international ophthalmic distributors where discounts could be utilized for the purchase of ophthalmic equipment . In addition , humanitarian pricing was solicited for capital equipment to minimize funding requirements for procurement . While donations were of benefit for the near-term goals of the project , longer term strategies for medication procurement including supply chain , cost , and source of revenue are considerations , as donations rely on supportive industry sponsorship , availability , and timing of donations from industry partnerships . Clinical forms were designed to capture systemic sequelae relevant to EVD survivors as well as a complete ophthalmic examinaztion including ocular vitals ( visual acuity , intraocular pressure , extraocular motility , confrontational visual fields , and pupil exam ) , slit lamp examination , and dilated fundus exam ( Fig 1 ) . Standardized descriptors of uveitis and complications of uveitis according to Standardization of Uveitis Nomenclature . iPhone devices were utilized with the slit lamp eyepiece for anterior segment photography and iPhone devices with 28-diopter condensing lenses were utilized for fundus photography for optic nerve , macular lesions , and mid-peripheral retinal lesions ( Fig 2 ) . A conference room with a large table and two separate patient examination rooms were utilized and partitioned into task-specific stations including 1 ) history-taking 2 ) pupillary evaluation , ocular motility , visual field testing , intraocular pressure , and dilation 3 ) slit lamp examination , indirect ophthalmoscopy , photography , and counseling including description of medication regimens , referral to other providers , and follow-up visit ( Fig 1 ) . Patients checked in at a registration desk with HCWs wearing PPE that included a face shield , fluid impervious gown and gloves . EVD survivor certificates were verified at the registration desk . Forehead temperature with an infrared thermometer was measured . Patients with temperatures less than 38 degrees Celsius ( 100 . 4 degrees Farenheit ) were evaluated . Ophthalmic examination was deferred in patients with a fever or any systemic signs of EVD ( fevers , rigors , myalgias , diarrhea , vomiting ) until complete medical evaluation could be performed . HCWs in the screening eye care facility wore fluid-impervious gowns and gloves . Strict handwashing precautions were performed with 0 . 05% chlorine and all equipment ( portable slit lamp , Tonopen ) was disinfected with alcohol between patients . At the time the clinic was conducted , limited information was available on the kinetics of EBOV in the tear film , specifically whether the tear film was fully clear of EBOV . EBOV persistence in tear film has been documented at 33 days post acute infection . [10] For this reason , a protective gown and gloves were utilized to avoid any potential skin exposure , as well as strict hand disinfection between patients . During the West African EVD outbreak , there was heightened travel regulatory approval to ensure safety of the HCWs and the public in the rare event of EBOV infection . Emory University physicians underwent several tiers of university regulatory approval including Department Chair , Dean , Chief Executive Officer of The Emory Clinic , and the Emory University Executive Travel Safety Committee ( Table 2 ) . After returning from West Africa , HCWs underwent 21-day temperature and EVD symptom monitoring according to Georgia Department of Public Health , Centers for Disease Control and Prevention , and Emory University Hospital guidance . Travel medical and evacuation insurance in the event of acute illness was also purchased for United States-based HCWs .
With thorough supply lists designed for ophthalmic examination in a resource-limited setting , multiple sources were utilized for procurement and judicious resource use , as summarized in Table 1 . Ten different sources were utilized to procure supplies and medications utilizing a combination of cost reduction from distributors , donations where possible , and retail when necessary or products were unavailable at a reduced cost . Industry donations included topical corticosteroids , ocular hypotensives and cycloplegic agents to treat acute uveitis . Two slit lamps were utilized for patient evaluation including a portable slit lamp and free standing slit lamp , which was deconstructed , transported following breakdown of its components into a suitcase and rebuilt for clinical use . iPhone photography through the slit lamp oculars and iPhone photography with a 28-diopter condensing lens were utilized for documentation of anterior segment and posterior segment pathology . The estimated value of medications , equipment , and supplies for the screening eye clinic was $94 , 800 and all materials were procured for $11 , 895 , a discount of approximately 87% of the total value of goods utilizing multiple methods of procurement , donations through the generosity of various entities ( i . e . clinic , industry foundation ) , as well as humanitarian discount pricing when possible . Components of the clinic design included modular stations for 1 ) history , review of systems , and general physical examination 2 ) visual acuity measurement 3 ) pupil , motility , visual fields , intraocular pressure measurement and dilation 4 ) slit lamp and indirect ophthalmoscopy , photography , and counseling . These clinical stations provided opportunities for both clinical care and instruction of the basic eye exam for in-country HCWs and were recorded on standardized case report forms for documentation and follow-up ( Appendix ) . In particular , patients were evaluated for findings suggestive of uveitis . Specific complications of uveitis assessed included posterior synechiae , band keratopathy , and cataract . The use of indirect ophthalmoscopy and hands-on experience with in-country eye care provider allowed the evaluation of patients for retinal and optic nerve disease . Following the clinic setup and operations in April 2015 , the ophthalmic clinic , equipment and medications remained available for ELWA hospital physicians and staff for patient follow-up examination and treatment . In addition , EVD survivors who were not specifically assessed during clinic operations could be evaluated thereafter . Based on this setup , 96 EVD survivors were successfully examined , treated or referred when appropriate . [11] To ensure appropriate transfer of care and follow-up , patients who needed ongoing follow-up were discussed with physicians permanently stationed at ELWA Hospital . Of the EVD survivors screened , EVD-associated uveitis was identified in 21 survivors and EVD-associated optic neuropathy was observed in 4 patients . Patients with uveitis were found to have vision impairment in 60% of eyes , while severe vision impairment ( i . e . World Health Organization criteria for severe vision impairment , 20/400 or poorer ) in approximately 40% of individuals . Our screening clinic setup was also sufficient to classify EVD survivors who were screened into anterior , posterior , and panuveitis classificatons of uveitis . Visual acuity qualified as severe visual impairment by World Health Organization criteria in 38 . 5% of affected eyes . [11] Our findings are in agreement with recent literature on the burden and vision impact of eye disease in EVD survivors who developed uveitis as a sequelae of their acute EVD infection . [12 , 13] Based on these initial experiences in Liberia and developing a screening eye clinic in partnership with ELWA Hospital , the portability , structure , and equipment of the ophthalmologic exam and consultation setup was discussed with the World Health Organization , Ministry of Health and Sanitation Sierra Leone National Eye Program and partnering non-governmental organizations including Partners in Health , and Médecins sans Frontières . Over 2 , 700 EVD survivors in Sierra Leone have been screened through the National Eye Program with clinical form development , treatment protocols and guidance on uveitis prevalence and disease burden . [7] Information attained from early experiences in Liberia developed in collaboration with ELWA Hospital were communicated with WHO and CDC officials involved in the EVD outbreak response as attention shifted from the acute outbreak to EVD survivor care . [2 , 14]
In summary , through a collaboration with United States-based ophthalmologists and leadership of ELWA Hospital , in conjunction with eye care specialists from the Ministry of Health , Liberia we were able to develop and rapidly implement a screening eye clinic for EVD survivors . The procurement of equipment through a variety of sources , modular clinic design , and attention to PPE appropriate for the ophthalmic examination setting were necessary to provide efficient care , referral , and safety for patients and health care providers . Lessons learned from this clinic were communicated to other countries with thousands of EVD survivors and underscore the importance of ongoing understanding of eye disease in EVD survivors , particularly given the ongoing outbreak in eastern DRC .
|
The West African Ebola virus disease ( EVD ) outbreak from 2014–2016 resulted in thousands of EVD survivors at-risk for uveitis , an inflammatory eye condition with the potential for vision impairment or blindness if not promptly diagnosed and treated . Given the equipment and resource limitations within West Africa , particularly for subspecialty eye care , a screening eye clinic was developed at ELWA Hospital in Liberia to promptly screen , treat , and refer patients with eye disease . The implementation of a screening eye clinic in collaboration with leadership at ELWA Hospital was of immediate relevance given our limited understanding of this eye disease prior to the recent EVD outbreak . Resourceful procurement of medications and equipment , as well as the development of modular systems within an eye clinic setting , facilitated patient examination , teaching , and documentation of this spectrum of ophthalmic illness . Development of a screening eye clinic and its rapid implementation were needed to improve our collective undersanding of the spectrum of uveitis as a disease sequelae of EVD , relevant for recent and future outbreaks .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[] |
2019
|
Development of a screening eye clinic for Ebola virus disease survivors: Lessons learned and rapid implementation at ELWA Hospital in Monrovia, Liberia 2015
|
The Helicobacter pylori cag pathogenicity island ( cagPAI ) encodes a type IV secretion system . Humans infected with cagPAI–carrying H . pylori are at increased risk for sequelae such as gastric cancer . Housekeeping genes in H . pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI , which transports the bacterial oncogene CagA into host cells , has not been systematically investigated . Here we compared the complete cagPAI sequences for 38 representative isolates from all known H . pylori biogeographic populations . Their gene content and gene order were highly conserved . The phylogeny of most cagPAI genes was similar to that of housekeeping genes , indicating that the cagPAI was probably acquired only once by H . pylori , and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa . Most isolates induced IL-8 release in gastric epithelial cells , indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements . More than one third of cagPAI genes , in particular those encoding cell-surface exposed proteins , showed signatures of diversifying ( Darwinian ) selection at more than 5% of codons . Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins ( e . g . HP0522 , HP0535 ) . One of these , HP0535 , is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages , similar to cagA . Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins , including several whose function is still unknown .
Helicobacter pylori persistently infects more than one half of all humans , and can cause ulcer disease , gastric cancer , and MALT lymphoma [1] . The H . pylori cag pathogenicity island ( cagPAI ) is an intriguing virulence module of this obligate host-associated bacterium [2]–[4] . H . pylori strains that possess a functional cagPAI are particularly frequently associated with severe sequelae , notably gastric atrophy and cancer [4]–[7] . The cagPAI is ∼37 kb long , and contains ∼28 genes [3] . These genes encode multiple structural components of a bacterial type IV secretion system ( t4ss ) as well as the 128 kDa effector protein , CagA [7] . After H . pylori has adhered to a host cell , the Cag t4ss translocates CagA into that cell . CagA is subsequently phosphorylated by host cell kinases and interacts with multiple targets ( e . g . SHP-2 , Grb2 , FAK ) , profoundly altering host cellular functions [8] , [9] . The alterations induced by the cagPAI are thought to ultimately contribute to malignant transformation [4] , [10] , and CagA has been designated a bacterial oncoprotein [11] . H . pylori has a high mutation rate , which has resulted in extensive genetic diversity [12] , and also recombines frequently with other H . pylori [13] . H . pylori isolates have been subdivided into distinct biogeographic populations and subpopulations with specific geographical distributions that reflect ancient human migrations [14]–[16] . The global population structure of H . pylori is now well understood based on multilocus haplotypes from seven housekeeping genes . However , very little is known about the biogeographic variation of virulence factors , such as the cagPAI , nor has the impact of genetic variation on disease outcome and host adaptation been adequately addressed . Previous analyses on the basis of comparative genome hybridization have demonstrated marked differences between biogeographic populations with respect to the cagPAI [17] . Microarray analysis of 56 globally representative strains of H . pylori revealed that the cagPAI was present in almost all strains from some biogeographic populations and subpopulations in Africa and Asia , while it was variably present in other populations [17] . The cagPAI was lacking in all isolates of hpAfrica2 , which is distantly related to the other populations [17] . Currently , nine complete cagPAI sequences are publicly available [2] , [18]–[22] , whose isolates belong to hpEurope ( 7 sequences ) , hspWAfrica ( 1 ) and hspEAsia ( 1 ) ( see Results ) , and no sequence data is available for the cagPAI in the other six populations and subpopulations where the cagPAI is present . Here we analyze complete cagPAI sequences from 38 isolates representing all known H . pylori populations and subpopulations and compare their genetic polymorphisms with measures of functional expression . Our data show that the cagPAI has shared a long evolutionary history with the H . pylori core genome , and displays a remarkable global conservation of gene content , structure and function , with minor exceptions . We provide evidence that the cagPAI was acquired by ancestral H . pylori in a single event that occurred before modern humans migrated out of Africa . Sequence comparisons identified domains in multiple components of the t4ss that are likely to be under diversifying selection , and these findings can guide future research into the function of t4ss components .
In order to define the occurrence of the cagPAI in H . pylori , we screened a globally representative collection of H . pylori isolates from 53 different geographical or ethnic sources [15] , [16] ( Figure 1 ) . 877 isolates were tested for the presence of the cagPAI by a PCR approach . Strains were classified as cagPAI-positive if we succeeded in separate PCR amplifications for the 5′ and 3′ ends of the cagPAI , or as cagPAI-negative if we succeeded in amplifying an empty site with primers from the flanking regions . The cagPAI was present in at least 95% of strains assigned to the hpAfrica1 ( hspWAfrica plus hspSAfrica ) , hpEastAsia ( hspEAsia , hspMaori ) and hpAsia2 populations . In contrast , none of the hpAfrica2 strains possessed the cagPAI , and it was only variably present in strains from the populations hpEurope ( 225/330 strains; 58% ) , hpNEAfrica ( 58/72: 81% ) , and hpSahul ( 32/49; 65% ) or the hspAmerind subpopulation of hpEastAsia ( 5/18; 28% ) . Based on their multilocus sequence typing ( MLST ) haplotypes , seven strains with published cagPAI sequences belong to the hpEurope population ( NCTC11638 from Australia [2]; 26695 from England [18]; and DU23 , DU52 , Ca52 , Ca73 [20] and HPAG1 [21] from Sweden ) . J99 from the U . S . A . [22] belongs to hpAfrica1 , and F32 [19] from Japan belongs to the hspEAsia population of hpEastAsia . None of these published cagPAI sequences were from strains of the hpNEAfrica , hpSahul , or hpAsia2 populations , from the hpEastAsia subpopulations hspAmerind or hspMaori , or from the hpAfrica1 subpopulation hspSAfrica , although those populations are also potentially important for our understanding of the evolutionary history of H . pylori . We therefore selected 29 strains from our global strain collection to supplement these nine published cagPAI sequences and provide a globally representative sample of cagPAI diversity ( Figure 1 ) . These strains included all known biogeographic populations , except for the cag-negative hpAfrica2 . The entire cagPAI , approximately 37 kilobasepairs in length , was sequenced and annotated from each of the 29 strains , either after shot-gun cloning of overlapping long-range PCR products or via direct amplification of multiple , smaller PCR products . The 38 complete cagPAI sequences were compared by pairwise sequence alignments and by a multiple alignment in Kodon relative to the cagPAI from J99 used as a scaffold sequence ( Figure 2 ) . The general pattern of gene content and gene order ( signifying macrodiversity ) was similar in most sequences , with only limited variation due to changed synteny or deletions . Synteny changes resulted from genomic rearrangements , horizontal genetic exchange ( e . g . replacement of HP0521 by HP0521b ) , possibly in conjunction with IS ( insertion sequence ) element insertion , or gene inversions , such as for HP0535 . Insertions , deletions , point mutations , frameshift mutations or disruption through insertion elements ( Figure S1 ) were also observed in some of the cagPAI sequences , some of which should have resulted in pseudogenes . We therefore tested all strains for their ability to induce interleukin-8 ( IL-8 ) in gastric epithelial cells ( Figure 2 , Figure 3 ) , as an indicator of PAI function [23] . Most of the strains containing a cagPAI were able to induce IL-8 , indicating that many of the mutations did not drastically reduce the general function of the cagPAI ( Table 1 ) . Most new mutations are deleterious , whether associated with single nucleotide polymorphisms , mobile elements or genomic rearrangements , and will be removed by purifying selection . However , mutations without a drastic effect on fitness , so-called neutral or nearly neutral mutations , can remain as rare variants within a population for long time periods . The vast majority of such mutations remain at low frequency until they are ( usually ) lost due to genetic drift . Rare neutral mutations can become more frequent over time , or even become fixed , also due to genetic drift [24] . Still other mutations are under positive selection . These rapidly become frequent or fixed due to Darwinian selection . In isolated clonal populations , Muller's ratchet can even result in some deleterious mutations rising to high frequency [25] and the same is true of extreme bottlenecks , which can fix deleterious mutations immediately . These basic evolutionary principles indicate that the demographies of rare versus frequent mutations differ and should be examined separately . A number of frequent cagPAI macrodiversity variants were found , some of which were present in all isolates of at least one sub-population , or almost all isolates ( Table 1 ) . These included insertion events due to one of three variants of IS606 [26] or of a mini-IS605 insertion [27] , [28] , an inversion of gene HP0535 plus its flanking non-coding DNA , a deletion of either the complete HP0521 ORF ( Δ2; Figure 2 ) or part of that ORF , or the replacement of HP0521 by the unrelated ORF HP0521B ( Figure 2 , Table 1 ) . Additionally , most of the 3′ ( right ) half of the cagPAI is lacking in all three hspAmerind strains due to one of two similar 11 . 2 kb deletions with distinct 3′ ends ( Δ4 , Δ5; Figure 2 ) . These large deletions terminate within HP0546 , and are associated with a second ( intergenic ) deletion of 410 bp or a 620 bp deletion that terminates within the N-terminal part of HP0547 ( cagA ) . In strains V225 and HUI1769 , a copy of the deleted segment plus the HP0546 and HP0547 ORFs have translocated to a separate , currently unidentified , location of the chromosome , leaving a shortened version of HP0546 at the original location ( Figure 2 ) . It is interesting to note that IL-8 induction was not eliminated by any of these frequent mutations ( Figure 2 , Figure 3 , Table 1 ) , suggesting that they are not deleterious to cagPAI function , and might be neutral or even under positive selection . Rare variants were present in only one or two strains , are probably transient , and will tend to disappear during genetic drift [29] . The rare variants included frameshift mutations in multiple ORFs within three single isolates ( CC42C , HPAG1 and L72 ) and IS elements ( mini-IS605 , IS605 , IS606 , IS607 or IS608 [26] ) that have integrated at distinct locations in 7 other isolates ( Table 1; Figure S1 ) . Our dataset consisted of only 38 isolates , and it was possible that these rare mutations might be more widely distributed . We therefore screened 95 other globally representative strains for the presence of IS605 , IS606 , IS607 or IS608 at those locations , but only identified two additional strains with IS element insertions , one each for IS605 ( MOR3055 – hspWAfrica ) and IS607 ( BASQ9523 – hpEurope ) ( data not shown ) . Thus , strains carrying these particular insertion mutations really are rare . We also found two rare , distinct genomic rearrangements ( Table 1 ) . One of these was in strain NCTC11638 from Australia and has been reported previously [2] . It splits the cagPAI between ORFs HP0534 and HP0535 into two segments , one of which is translocated elsewhere in the genome , and is distinct from the split of the cagPAI in the hspAmerind strains . Previous analyses identified the same rearrangement in 4/40 strains from Italy [2] , but it was not found in any of the other 38 cagPAI sequences analyzed here nor in any of the 95 other , globally representative strains that we investigated by PCR . The other rearrangement separated HP0547 ( cagA ) through HP0549 plus flanking DNA from the rest of the cagPAI . It has been previously described for two hpEurope strains from Sweden and one from Australia [20] . We found the same pattern in a fourth hpEurope strain isolated in Palestine ( PAL3414 ) . Both of these rearrangements were present in less than 5% of isolates . The 17 rare mutations were identified in a total of 12 isolates . Only three of those , CC42C , HUI1692 and L72 , did not induce IL-8 , indicating that the majority of the rare sequence changes also did not cause a severe loss of cagPAI function . This observation is compatible with most of the rare mutations being selective neutral or near-neutral . Three overlapping small deletions ( Δ1 , Δ2 , Δ3 ) that removed the HP521 ORF were found in all but one hpEastAsia isolate , one hpEurope isolate and the hpSahul strain ( Figure 2; Table 1 ) , but those did not abolish cagPAI function ( see above ) . Eight other deletions were found in four individual strains ( Figure 2 ) . Two of these isolates were unable to induce IL-8: CC42C ( hspSAfrica ) contains multiple frameshift mutations and an insertion of IS606 as well as deletion Δ11 , which removes part of cagA ( HP547 ) . Δ4 and Δ6 deleted half of the cagPAI in hspAmerind strain HUI1692 . The cagPAI is clearly decaying in both CC42C and HUI1692 . In contrast , although deletions Δ5 and Δ7–Δ10 also removed large parts of the cagPAI in hspAmerind strains V225 and HUI1769 , these deletions occurred in a segment that has been duplicated to a separate location ( see above ) and these two isolates remain able to induce IL-8 . Thus , with one exception ( Δ1 ) , these deletions are rare and seem to be associated with accelerated decay of non-functional cagPAI genes . In addition , the cagPAI in non IL-8-inducing strain L72 also contained one frameshift and one premature stop codon in a coding region , and seems to be undergoing decay . Darwinian selection for variation in coding regions can also be exerted at the nucleotide or protein level . We therefore analyzed sequence polymorphisms ( microdiversity ) in individual cagPAI genes for traces of such selection ( Materials and Methods ) . Similar to housekeeping genes [30] , almost all alleles of each cagPAI ORF were unique to one isolate among the 38 strains . Exceptionally , we identified duplicates of a single allelic sequence in six genes; in each case , the strains possessing the duplicate alleles were from a common population ( Table S4 ) . Occasional duplicate alleles within populations have also been described for housekeeping genes [30] and are considered to represent homologous recombination . Again , similar to housekeeping genes , most cagPAI genes seemed to be under purifying selection because their Ka/Ks ratios were ≤0 . 2 ( Table 2 ) . However , five genes ( HP0534-0535 , HP0538 , HP0546-0547 ) showed signs of positive or diversifying selection because their overall Ka/Ks ratios were greater than 0 . 2; of these , cagA ( HP0547 ) had the highest proportion of non-synonymous polymorphisms ( Ka/Ks = 0 . 45 ) . However , Ka/Ks ratios are relatively insensitive indicators of Darwinian selection , which can act at the level of single protein epitopes or conformational domains . We therefore used a Bayesian method ( PAML/CODEML [31] ) to search MLST and cagPAI genes for codons that might be under diversifying selection ( indicated by ω >1 ) . Only two of the seven MLST housekeeping genes ( trpC , yphC ) contained an appreciable frequency ( 3 . 9%; 5 . 3% ) of codons with posterior probabilities of ω >1 being above 0 . 95 ( Table 2 ) . In contrast , >5 . 3% of the codons matched this criterion in 10 of the 28 cagPAI ORFs ( Table 2 ) , including four of the five ORFs with high overall Ka/Ks ratios ( HP0535 , HP0538 , HP0546 , HP0547 ) . We also tested eleven cagPAI ORFs , including nine with high frequencies of codons under selection according to PAML , and two with lower frequencies ( HP0524 , HP0525 ) with a second Bayesian program , OmegaMap [32] , [33] , which unlike PAML also takes into account the occurrence of recombination ( ρ ) between different alleles ( Table S5 ) . OmegaMap detected fewer codons with high probabilities of positive selection , but the codons that it identified often overlapped with codons that had been identified as being under positive selection by PAML ( Table S5 ) . Finally , we employed a sliding window along codons of PAML posterior probabilities of ω to identify clusters of sites with signs of diversifying selection ( Figure 4 ) . The combination of three forms of analysis ( criteria: Ka/Ks >0 . 2 , or likelihood of at least 95% for ω >1 in ≥5 . 3% of codons , or at least two clusters of two or more adjacent amino acids ( aa ) predicted under diversifying selection in PAML ) identified 13 cagPAI genes that are likely to have evolved under diversifying selection: HP0520 , HP0522 , HP0523 , HP0527 , HP0528 , HP0534 , HP0535 , HP0536 , HP0538 , HP0539 , HP0540 , HP0546 and HP0547 . Of these , functions or structural contributions are known only for HP0523 ( virB1 ) , HP0527 ( virB10 ) , HP0539 ( virB5 ) , HP0546 ( virB2 ) and HP0547 ( cagA ) [7] , [34]–[38] . The percentage of codons with high likelihood of positive selection was highest in cagA ( 26 . 9% ) , followed by cagY ( 15 . 5% ) and a gene of unknown function , cagQ ( HP0535; 9 . 9% ) ( Table 2 ) . In addition to a high frequency of putative codons under diversifying selection , HP0527 ( cagY ) and HP0547 ( cagA ) also exhibited variable gene lengths . This was due to variable numbers of repetitive modules within the genes , as previously reported [35] , [39] . In the CagA protein , the number of phosphorylation sites ( C-terminal EPIYA repeat motifs ) differed , as did the types of these repeats ( Figure 3 ) . As previously described [39] , the third EPIYA motif of CagA was type D in most ( 13/17 ) Asian strains whereas type D was not found in isolates from any other population . This reflected the preponderance of type D EPIYA in isolates assigned to the hpEastAsia and hpAsia2 populations . If the EPIYA type D motif were ancestral in Asian populations , this finding might reflect horizontal acquisition of cagA by the four exceptional Asian strains from Western strains . Homologous recombination involving the cagPAI has also been reported in isolates from Mestizos in Peru [40] and might reflect selection due to functional differences that are related to ethnic specificity . We next asked whether the phylogeny of cagPAI genes was similar to that of housekeeping genes . Concatenated sequences of the cagPAI genes yielded a tree ( Figure 5B ) that is very similar to the tree based on a concatenate of the seven MLST housekeeping genes ( Figure 5A ) . Similarly , matrices of pairwise genetic distances of the concatenated cagPAI genes were highly correlated with corresponding matrices of pairwise distances of concatenated housekeeping genes ( R = 0 . 65 , p<0 . 001 ) ( Figure 5C ) . These data show that 42% of the variance among cagPAI genes can be attributed to a linear relationship with housekeeping genes . The correlations for individual cagPAI genes ranged from R = 0 . 17 to R = 0 . 74 ( Table 2 ) . While most cagPAI genes thus fell into the range observed for the individual housekeeping genes ( 0 . 46 to 0 . 69 ) , the correlations were lower for particular cagPAI genes ( e . g . cagL , R = 0 . 17 ) , which might reflect selection and/or recombination between cagPAIs from different bacterial populations . These observations indicate a generally similar genealogy of cagPAI and housekeeping genes , which would imply that the cagPAI has accompanied H . pylori since before human migrations out of Africa some 60 , 000 years ago [17] . In agreement , the genetic diversity of the cagPAI genes per population decreased significantly with distance from Northeast Africa ( data not shown ) . Only five of the strains tested here were not able to induce IL-8 ( Figure 3 ) . The same five strains did not translocate CagA into AGS cells , a second marker of t4ss function ( Figure 3B ) . For three of the five strains ( CC42 , L72 and HUI1692 ) , a lack of function can be explained by sequence features of coding sequence ( CDS ) decay . The cagPAI of CC42C contains multiple pseudogenes , some of which are crucial for t4ss function [3] . Half of the cagPAI including numerous essential t4ss genes is lacking in strain HUI1692 . For strain L72 , a point mutation results in a premature stop codon in gene HP0530 , which is essential for t4ss function . In contrast , the cagPAI sequences did not offer obvious explanations for the lack of induction of IL-8 by strains M49 and D3a . We therefore investigated the transcript abundance of all 14 genes involved in IL-8 induction and of cagA for 28 sequenced strains as well as for the reference strains 26695A and J99 ( Figure 3C; Table S3 ) . The inability of strain M49 to induce IL-8 can be accounted for by very low transcript levels for 7/15 cagPAI genes ( Figure 3C; Table S3 ) ; the cause of this low transcription is unknown . However , we are unable to explain the inability of strain D3a to induce IL-8 , because it was not impaired in cagPAI transcription ( Table S3 ) . We are also not readily able to explain the considerable variation of transcript levels among the other strains that did induce IL-8 ( Table S3 ) , except that it did not correlate with the macrodiversity patterns described above ( data not shown ) . Similar to the variable transcript levels , the levels of IL-8 induction also varied dramatically ( Figure 3 ) . This variation did not correlate with strain assignments to biogeographic populations or with the type and number of EPIYA motifs within CagA ( Figure 3A; [39] ) . Nor did they correlate with quantitative values for adhesion of the strains to AGS or MKN28 gastric epithelial cells ( data not shown ) .
It has previously been noted from limited samples that different populations of H . pylori differ in the frequency of possession of the cagPAI [14] , [17] . Our data on 877 isolates from all known H . pylori populations and subpopulations provide unambiguous evidence for this variability . Carriage of the cagPAI varies from almost universal presence in hpEastAsia and hpAfrica1 through intermediate presence ( hpEurope ) to complete absence ( hpAfrica2 ) ( Figure 1 ) . The cagPAI is also absent in the related species H . acinonychis [17] , which resulted from a host jump from humans to large felines [41] . The absence of the cagPAI from hpAfrica2 and H . acinonychis has been interpreted as the ancestral state , i . e . H . pylori acquired this genomic island by horizontal gene transfer from an unknown source after H . pylori had established itself in humans [17] . But when was it acquired , and on how many occasions ? The data presented here indicate that the cagPAI was only acquired once because its microdiversity correlated with microdiversity within housekeeping genes ( Figure 5 ) . That acquisition was prior to 60 , 000 years ago , the time when H . pylori accompanied modern humans during their migrations “out of Africa” [16] , because cagPAI sequence microdiversity diminished with distance from North East Africa . An important implication of this conclusion is that , with the exception of hpAfrica2 , the variable presence of the cagPAI in H . pylori populations usually reflects secondary loss , rather than inheritance of the ancestral virgin state . Previous analyses have shown that strains that circulate within the same communities , and even within the same stomach , can be mixed in respect to possession of the cagPAI [30] . This observation indicates that cag positive bacteria do not outcompete cag negative bacteria in all environments . Nevertheless , our data support the inference [17] that a functional cagPAI provides a fitness advantage to H . pylori in most human populations: macrodiversity variants that inactivated t4ss function through deletions or insertion of IS elements were rare , whereas macrodiversity variants that were frequent did not affect t4ss function . For instance , shortening , complete loss or replacement ( by HP0521b ) of gene HP0521 was observed in almost all populations but this did not reduce cagPAI functionality , suggesting that this gene is not important for t4ss functions . Similarly , the genetic organization of the cagPAI was in general strongly conserved , and insertion elements did not play a decisive evolutionary role for the cagPAIs , unlike previous conclusions [2] . Even separation of the cagPAI in two parts did not lead to loss of function , except when a deletion was involved . High variation at the level of sequence microdiversity was found along the cagPAI , but this is also true of housekeeping genes , and might possibly result from the high frequencies of mutation and recombination in H . pylori [14] , [16] . However , unlike most housekeeping genes , multiple cagPAI ORFs showed signs of Darwinian diversifying selection , as indicated by higher Ka/Ks values and codon-based analyses , which identified specific amino acids or regions of particularly high non-synonymous diversity in 13 cagPAI genes ( Figure 4 , Table 2 ) . In the following we attempt to interpret these measures of selection by mapping them onto known components including structural features of the t4ss encoded by the cagPAI . Seventeen of the cagPAI genes are essential for the known t4ss functions ( IL-8 induction , CagA translocation [3] ) , of which 12 have been characterized in structural or functional terms ( virB1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 and virD4 orthologs , cagA ) . In Figure 6 , we present a schematic structural model of the cagPAI t4ss apparatus including all known structural Cag proteins plus the effector CagA . Different shades of grey indicate the proportion of amino acids which are likely to have undergone diversifying selection according to PAML . The translocated effector protein CagA ( HP0547 ) , which interacts with various host proteins [42] , had the highest proportion of such amino acids of the entire cagPAI . These were distributed along its entire length , suggesting functional adaptation or modulation . CagA binds to host cell integrins [42] and is translocated into host cells by the cagPAI t4ss . Within the host cell , individual domains of CagA interact with intracellular proteins such as SH-2 proteins and protein kinases ( e . g . Src , Abl [19] , MARK2/PAR1b kinase family [7] , [9] ) . These interactions render it potentially subject to diversifying or positive selection due to host polymorphisms which could even result in modified host protein interactions . A prominent example of amino acid diversity noted previously are the EPIYA motifs in the C-terminal half of CagA , which differ between Asian ( hpAsia2; hpEastAsia ) ( type D ) and all other populations [43] . The D type EPIYA repeat binds SHP-2 phosphatase more avidly than other types [19] . A clear bipartite “Eastern”/“Western” separation in the present global dataset was not only observed in phylogenetic trees based on the C-terminal half of CagA containing the divergent EPIYA repeat motifs , but also in its less well-characterized N-terminal moiety . Interestingly , CagA from the ancient and isolated hpSahul population [15] localised in between the Eastern and Western type CagA clusters ( not shown ) . The global strain selection provided further evidence of functional adaptation in a different CagA motif . Recently , structural analyses of a second CagA subdomain ( CM domain , aa 885 to 1005 ) in complex with its interaction partner from the human host , the cellular kinase MARK2 , were performed [44] . This analysis revealed the crucial contribution of specific residues in CagA ( MKI motif; [44] ) to the physical interaction with the kinase . The short CagA peptide that could be mapped in the cocrystal ( Phe948–Lys961 ) is characterized in our strain collection by high amino acid variability ( Figure 7A and 7B ) . Superposition of the amino acids under selection ( according to PAML ) onto the structure of the peptide [44] revealed that all but five of the 14 amino acids in this MARK2 binding domain of CagA have a high posterior probability of being under diversifying selection ( Figure 7A ) . Interestingly , Arg952 and Val956 , which both strongly influence MARK2 binding [44] , have a likelihood of 1 . 0 and 0 . 81 , respectively , of being under positive selection whereas two other MARK2 binding residues , Leu950 and Leu959 , were not under diversifying selection . This result suggests that , although some specific MARK2 binding sites in CagA do have a lower propensity of being under positive selection , the binding strength of CagA to MARK2 can still be influenced by H . pylori protein variation , indicative of functional fine-tuning . These predicted functional implications of global variation in the MKI motif are in agreement with an earlier study by Lu et al . [9] who observed differences in CagA PAR1b binding and function when they exchanged two Western and Eastern phylogeographic variants of the CagA MARK2/PAR1b binding region within CagA chimeras . We therefore expect that other regions of CagA that are under selection ( Figure 4 ) also warrant detailed structural and functional analyses . The observed CagA diversity , which is proposed to allow functional fine-tuning , may not only be associated with different host ethnicities but also with niche-dependent intrahost diversification during long-term colonization ( e . g . stomach antrum versus corpus ) [45] , [46] . A prior general comparison of component diversity in type III and IV secretion systems from different bacterial species [47] found that core structural proteins located in the bacterial cytoplasm or the inner membrane exhibit significantly lower diversity than do structural proteins exposed on the surface of the bacteria or secreted effector proteins [47] . Two well-characterized cag genes whose gene products are exposed on the cell surface have experienced strong selection: cagY ( HP0527 ) , which encodes a VirB10 ortholog that is a structural component of the cagPAI t4ss [36] , and cagC ( HP0546 ) , which encodes a VirB2 pilin subunit ortholog [35] , [38] . CagY is under selection due to host antibodies and/or direct host interactions [35] , [36] . In cagC , those codons with the highest likelihood of diversifying selection ( amino acids 21 to 42; Table S5 ) overlap with codons forming surface-exposed and highly strain-specific epitopes in the N-terminus of mature CagC [38] . The virB2 ( HP0546 ) and virB5 ( HP0539 ) orthologs of the cagPAI show signatures of diversifying selection in the present study; they encode surface-exposed pilin and pilus tip structural components of the Cag apparatus [48] and their sequence homology with functionally related VirB2 and VirB5 proteins from other bacteria is so low that they had to be identified by non-sequence-based approaches [37] , [38] . We also find that 9 other cagPAI genes are under diversifying selection but their function is largely unclear . These include HP0520 , HP0522 ( part of the Cag outer membrane subcomplex [49] ) , HP0523 ( cagγ; proposed to code for a virB1 orthologous peptidyglycan hydrolase [34] , [50] ) , HP0528 ( virB9 ) , HP0534 , HP0535 , HP0536 , HP0538 ( encodes a membrane protein [50] , [51] ) , and HP0540 [52] . Of these , HP0535 exhibits extensive non-synonymous variation and a clear bipartite Eastern-Western subdivision , similar to cagA . This gene is not involved in IL-8 induction or CagA translocation and is not predicted to possess a signal peptide . It may be a non-canonical secreted protein ( score of 0 . 48 by SecretomeP ) . Based on the signs of selection and high diversity , we hypothesize that the HP0535-encoded protein interacts closely with CagA or is a novel effector protein that is translocated into host cells by the Cag t4ss . Of the other genes under diversifying selection whose function is unknown , HP0520 might be a non-canonical secreted protein because its SecretomeP score was also high ( 0 . 92 ) . In contrast to the genes just described , genes encoding cagPAI proteins that are not thought to be exposed on the bacterial surface [3] should be subject to purifying selection . In agreement with this expectation , other cagPAI genes including virD4 ( HP0524 ) and virB11 ( HP0525 ) orthologs [36] , [50] , displayed lower non-synonymous diversity and fewer codons under positive selection ( Figure 6; Table S5 ) . In conclusion , the present work reports a genetic and functional approach within a global population genetic perspective to study diversity in a complex secretion system . This comprehensive library of data allowed the identification of genes with a high probability of having undergone diversifying selection . cagPAI genetic diversity is accompanied by modulations in functionality , but rarely by complete loss of function . Functional modulation of the t4ss appears to be an important feature in vivo and is predicted to rely not only on protein diversification but also on strain-dependent transcript level diversity in the cagPAI . These data will be a resource for future research on the biological roles and variable host interactions of individual cagPAI proteins . It will also foster research on the phylogeographic variability and evolution of determinants of host interaction in other microbes . The diversity in this dataset will also be useful to evaluating predictions by recent evolutionary models based on the structure of proteins , such as neutral networks of protein folds [53] , [54] ) , which might be able to distinguish selection processes that favor structural versus functional conservation .
Bacterial isolates and sequences of seven housekeeping gene fragments ( atpA , efp , mutY , ppa , trpC , ureI , yphC ) have been described previously [13] , [16] , [55] . Strains were checked for the presence of the cagPAI by PCR , amplifying the 5′ ( Primers O2872 + O2902 ) and 3′ ( O2899 + O3326 ) flanking regions , or for absence ( empty site ) ( primers O2872 + O3326 ) . Primer sequences are provided in Table S1 . Strains were chosen to represent all currently defined H . pylori populations possessing the cagPAI ( Figure 1 , Figure 2 ) . The complete cagPAI was amplified for sequencing as two overlapping long range PCR products of ∼20 kb each with primers O2903 + O3048 and O3047 + O2904 ( Table S1 ) , respectively in 50 µl reactions with the EXL long range polymerase kit ( Stratagene ) using the following conditions: bacterial DNA 20 ng , Primers 20 µM each , 6 µl of 2mM dNTPs , 5 µl Buffer 1 , 1 µl stabilizing solution , 1 µl EXL Polymerase , H2O to 50 µl . An initial denaturation for 1 min at 94°C was followed by 30 cycles of 45 sec at 94°C , 1 min at 65°C and 17 min 30 sec at 68°C . Long range PCR fragments were subjected to shotgun cloning . DNA fragments ranging from 0 . 8 to 1 . 2 kb were end repaired and cloned into the pGEM T-Easy vector ( Promega ) , inserts were sequenced to 10-fold coverage by MWG Biotech . Alternatively , the cagPAIs were amplified as overlapping PCR products of ∼5 kb each with additional primers listed in Table S1 ( primer combinations available on request ) and sequenced with an extended set of primers ( Table S1 ) by gene walking . The cagPAI sequence of strain PNGhigh85 was obtained by shotgun 454 sequencing of the whole genome ( unpublished ) . Sequences were assembled with Gap4 ( Staden Package , GCG Wisconsin ) . The individual cagPAI sequences have been submitted to the EMBL Nucleotide Sequence Database ( accession numbers FR666825 - FR666857 ) . Details for RNA preparation and RT-PCR are given in Text S1 . RT-PCR primers and cycling conditions for transcript analyses of the cagPAIs are listed in Table S2 . CDSs were annotated in ACT and in KODON ( Applied Maths BVBA , Sint-Martens-Latem , Belgium ) , automatic multiple sequence alignment of individual cagPAI genes was performed in BIONUMERICS ( Applied Maths BVBA , Sint-Martens-Latem , Belgium ) and corrected manually after visual inspection , where necessary . Sequence comparison and graphical output of multiple complete cagPAI sequences was performed in KODON . We only included one of eleven cagPAI sequences ( F32 ) available from Japanese strains [19] because information is lacking on the phylogeographic population assignment of the remaining 10 strains . Pairwise genetic distances , phylogenetic trees and FST were calculated in MEGA3 [56] and in Arlequin [57] , respectively . Pairwise geographic distances and distance from North East Africa ( Addis Ababa , Ethiopia ) , as well as confidence intervals were calculated as previously described [16] . For analyses of increasing diversity with geographic distance from East Africa , the dataset was stripped of recent migrants [16] which resulted in the use of 33 out of the 37 cagPAI sequences . Pseudogenes were excluded from the dataset in all phylogenetic analyses . Ks/Ka ratios were determined in DnaSP4 . 0 [58] and SWAAP , including a sliding window analysis . The number and location of potential codons under selection ( ω ) in each cagPAI gene were determined using the program CODEML in PAML 3 . 15 [59] , implementing a sliding windows graphic representation . This software calculates the ratio of maximum likelihood of different evolutionary algorithms ( models ) for each codon ( site ) of a coding sequence to be under positive selection ( ω>1 ) , followed by Naive Empirical Bayes ( NEB ) and Bayes Empirical Bayes ( BEB ) analyses of posterior probabilities . Sites with a posterior probability P>0 . 95 by the CODEML codon substitution models M3 ( discrete ) or M8 ( beta and ω ) of ω>1 were considered as being under positive or diversifying selection . The likelihood of codons under diversifying selection in the presence of recombination was further analyzed using OmegaMap ( V 0 . 5; [32] ) . This software uses a Bayesian modeling algorithm to calculate the probability of codons to evolve under diversifying selection ( ω>1 ) in the presence of recombination ( ρ ) . By explicitly modeling recombination , this method has a low rate to detect false positives . The settings used in the program were: norders = 100 , thinning = 100 , rhoprior = inverse , omegaprior = inverse , block length = 3 and 100 , 000 or 250 , 000 iterations . 5 , 000 iterations were deduced after each calculation as the burn-in phase . The model type used for both ω and ρ was “variable” . Three repetitions of the calculations with different settings were initially performed for control genes of defined structural properties and where some information is available about their function ( e . g . HP0546 ) , to exclude high variations in the calculations due to inadequate settings . Pseudogenes were excluded from the dataset . Fragments of the housekeeping genes atpA , efp , mutY , ppa , trpC , ureI , and yphC were amplified and both strands were sequenced from independent PCR products as described [55] . Alternatively , comparable sequences were extracted from the published genomes ( 26695 , HPAG1 , J99 ) . These sequences were assigned to populations and subpopulations by STRUCTURE [14] . IL-8 induction assay using the human gastric epithelial carcinoma cell line AGS ( isolated from adenocarcinoma from a Caucasian patient ) was performed for all strains of the sequencing project . Strain 26695A [60] was used as a reference . Cells were cultured in RPMI 1640 medium ( buffered with 25 mM HEPES , supplemented with 10% heat-inactivated fetal bovine serum ( medium and serum: Biochrom , Berlin , Germany ) . Details for bacterial culture conditions are given in Text S1 . Cell infection experiments for IL-8 secretion measurement were performed on subconfluent cell layers ( 70%–90% confluence ) in 24-well tissue culture plates . Cells were washed three times and preincubated in fresh medium with serum for 30 min prior to infection . By the addition of exponentially growing bacteria that were resuspended in cell culture medium ( RPMI 1640 , 25 mM HEPES , 10% heat-inactivated serum ) , the infection was started ( MOI of 50 ) . To synchronize the infection , the incubation plates were centrifuged at 500 x g , 20°C , for 3 min . The coincubation was carried out for 20 h . Non-infected cells ( mock coincubated ) were used as negative control . Supernatants were harvested , cleared of cell debris by centifugation , immediately frozen and stored at −20°C until use . Release of IL-8 into the cell supernatants was quantified by using BD OptEIA IL-8 enzyme-linked immunosorbent assay kit ( BD Pharmingen; San Diego , USA ) according to the company's instructions , using appropriate dilutions . The assays were performed in triplicate and the means and standard deviations of at least six independent coincubations were calculated . Adherence of the strains was tested in a high throughput assay , but no correlation was found between adherence and the IL-8 induction ( data not shown ) . To study CagA translocation , AGS cells were cultured in six-well plates and infected with H . pylori at a multiplicity of infection ( MOI ) of 100 . After 4 h of coincubaction , non-adherent bacteria were removed by washing twice with PBS-Dulbecco ( pH 7 . 4; Biochrom , Berlin , Germany ) . Cells were harvested with a cell scraper and resuspended in 1 ml PBS ( pH = 7 . 4; Biochrom , Berlin , Germany ) . After centrifugation ( 250 x g , 4°C , 5 min ) , cells were resuspended in 300 µl of modified RIPA buffer ( 20 mM Tris-HCl [pH 7 . 5] , 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 1% Triton X-100 , 2 . 5 mM sodium pyrophosphate , 1 mM β-glycerol phosphate , 1 mM sodium orthovanadate , 1 protease inhibitor tablet per 10 ml buffer ( Complete , Roche , Mannheim , Germany ) , 1 mM PMSF ) . During lysis , cells were incubated on ice for 30 min . Lysates were cleared by centrifugation ( 10 min , 21 , 900 x g , 4° ) and the pellets were carefully separated from the supernatants . The pellet fraction was resuspended in 100 µl RIPA buffer and the fractions were immediately frozen at −80°C . To determine the amount of protein , a BCA protein assay was performed using the BCA Protein Assay kit ( Pierce , Rockford , IL , USA ) according to the manufacturer's instructions . Equal amounts of cleared cell lysates ( see above; corresponding to 10 µg of protein ) of infected cells were resuspended in 5 x SDS loading buffer ( 0 . 31M Tris-HCl , pH6 . 8 , 37 . 5% glycerol , 10% SDS , 0 . 05% bromophenol blue , 20% β-mercaptoethanol ) and boiled for 10 min . For determination of molecular mass , BenchMark pre-stained Protein Ladder ( Invitrogen , Karlsruhe , Germany ) was used . Samples were separated on 10 . 4% denaturing SDS-polyacrylamide gels and transferred to nitrocellulose membranes ( Protran BA 85 , Whatman , Dassel , Germany ) by semi-dry blotting . Membranes were blocked with 5% non-fat dried milk in TBS-T ( 20 mM Tris-HCl , 13 . 7 mM NaCl , 0 . 1% Tween 20 , pH 7 . 4 ) for 1 h and subsequently incubated with specific primary antibody . Anti-CagA-antibody ( Rabbit anti-H . pylori Cag antigen IgG fraction [polyclonal] , Austral Biologicals , San Ramon , USA ) was used at a dilution of 1/1 , 000 for the detection of CagA protein . To detect phosphorylated CagA , PY99-antibody ( Santa Cruz Biotechnology , Heidelberg , Germany ) was used ( dilution 1/250 ) . Goat-anti-Rabbit-HRP antibody ( dilution 1/10 , 000 , Jackson Immunoresearch Laboratories , Suffolk , Great Britain ) or Goat-anti-mouse-HRP-antibody ( dilution 1/5 , 000 , Dianova , Hamburg , Germany ) were used as secondary antibodies . Signal detection was performed with Enhanced SuperSignal West chemiluminescence substrate ( Pierce , Rockford , IL , USA ) , and detection was on X-ray film ( Hyperfilm , Amersham Biosciences , Buckinghamshire , UK ) .
|
Most humans are infected with Helicobacter pylori . The H . pylori cag pathogenicity island ( cagPAI ) encodes a secretion apparatus that can translocate the CagA protein into host cells . Humans infected with cagPAI–carrying H . pylori are at increased risk of severe disease , including gastric cancer . We analyzed the nucleotide sequences and functional diversity of the cagPAI in a globally representative collection of isolates . Complete cagPAI sequences were obtained for 29 strains from all known H . pylori biogeographic populations . The gene content and arrangement of the cagPAI and its function were highly conserved . Diversity in most cag genes consisted in large part of synonymous polymorphisms . However some genes—in particular those that encode proteins predicted to be secreted or located on the outside of the bacterial cell—had particularly high frequencies of non-synonymous polymorphisms , suggesting that they were under diversifying selection . Our study provides evidence that the cagPAI was only acquired once and provides an important resource that can guide future research on the biological roles and host interactions of cagPAI proteins , including several whose function is still unknown .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"and",
"genomics/microbial",
"evolution",
"and",
"genomics",
"microbiology/cellular",
"microbiology",
"and",
"pathogenesis",
"infectious",
"diseases/gastrointestinal",
"infections"
] |
2010
|
A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island
|
Throughout most of the mammalian genome , genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues . At metastable epialleles ( MEs ) , conversely , epigenotype is established stochastically in the early embryo then maintained in differentiated lineages , resulting in dramatic and systemic interindividual variation in epigenetic regulation . In the mouse , maternal nutrition affects this process , with permanent phenotypic consequences for the offspring . MEs have not previously been identified in humans . Here , using an innovative 2-tissue parallel epigenomic screen , we identified putative MEs in the human genome . In autopsy samples , we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages . Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci , suggesting that their epigenetic state is established stochastically . We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians , who experience dramatic seasonal fluctuations in nutritional status . DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season , providing the first evidence of a permanent , systemic effect of periconceptional environment on human epigenotype . At MEs , epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA . MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease .
Epigenetic mechanisms maintain mitotically heritable differences in gene expression potential without alterations in DNA sequence [1] , enabling the diverse cell types of multicellular organisms to stably regulate appropriate patterns of gene expression . The established role of epigenetic mechanisms in cancer and various developmental syndromes has spurred increasing interest in the role of epigenetic dysregulation in a broad range of human diseases including neurological disorders , cardiovascular disease , diabetes , and obesity . A major obstacle to studying epigenetics and human disease , however , is the inherent tissue specificity of epigenetic regulation . In studies of genetic epidemiology , DNA from peripheral blood can be used to assay for a genetic variant present throughout the body . Conversely , epigenetic regulation [2] , [3] ( and hence dysregulation ) may be tissue- and cell-type specific [4] , [5]; in many cases , therefore , epigenetic information present in easily obtainable biopsy samples will not provide insights into the epigenetic etiology of disease . Another major obstacle is that interindividual epigenetic variation may often be a consequence of genetic variation [6] , making it difficult to disentangle epigenetic and genetic causes of disease . Hence , genomic loci at which systemic interindividual epigenetic variation occurs independently of genotype would offer major opportunities to advance our understanding of epigenetics and human disease . Such loci have been identified in the mouse; at murine metastable epialleles ( MEs ) epigenetic regulation is established stochastically in the early embryo then maintained in all germ-layer lineages , resulting in dramatic and systemic interindividual variation in locus-specific epigenetic regulation . Murine MEs cause obvious phenotypic variation among genetically identical mice . For example , the Agouti viable yellow ( Avy ) ME affects the expression of the Agouti gene which regulates fur pigmentation; isogenic mice heterozygous for Avy range from yellow to mottled to brown [7] . Similarly , the Axin Fused ( AxinFu ) ME confers epigenetic stochasticity upon Axin , resulting in interindividual variation in tail kinking among isogenic AxinFu heterozygous mice [8] . Rather than affecting fur color or tail development , however , MEs in the human genome could affect individual susceptibility to various diseases . Indeed , because agouti protein binds antagonistically to the melanocortin 4 receptor in the hypothalamus [9] , yellow Avy/a mice become hyperphagic and obese , illustrating how epigenetic dysregulation at MEs can result in metabolic disease . Maternal nutrition and other environmental exposures before and during pregnancy influence the stochastic establishment of epigenetic regulation at murine MEs , with permanent phenotypic consequences [10]–[12] . Hence , if MEs can be identified in humans , they would not only facilitate an advanced understanding of the role of epigenetics in human disease , but also provide excellent candidate loci at which to test epigenetic pathways in the developmental origins hypothesis , which proposes that early environmental influences affect developmental mechanisms , causing permanent metabolic changes that affect risk of adult disease [4] , [13] . Of various interacting epigenetic mechanisms including cytosine methylation in DNA , covalent histone modifications , and autoregulatory DNA binding proteins , DNA methylation is recognized as the most stable epigenetic mark [14] , making it a prime candidate to mediate the life-long epigenetic changes postulated in the developmental origins paradigm [5] , [13] . Here , we have designed an innovative epigenomic screen based upon the epigenetic characteristics of murine MEs , and have screened for MEs in the human genome . We provide evidence that MEs do exist in humans . At the loci we identified , systemic interindividual variation in DNA methylation was confirmed in autopsy samples , and stochastic establishment of epigenotype was supported by epigenetic discordance within monozygotic ( MZ ) twin pairs . Further , by studying children conceived during different seasons in rural Gambia we show that , as in mice , developmental establishment of DNA methylation at such sites is responsive to maternal environment around the time of conception .
We devised a human genome-scale screening approach based on a definitive characteristic of murine MEs: systemic interindividual variation in DNA methylation [11] , [12] . Genomic DNA from peripheral blood leukocytes ( PBL ) and hair follicles ( HF ) ( mesodermal and ectodermal lineages , respectively ) of 8 healthy Caucasian adults was screened for interindividual differences in DNA methylation by methylation-specific amplification microarray [15] ( MSAM ) . We employed a parallel , 2-tissue interindividual cohybridization design: the same four interindividual comparisons ( matched for age and sex ) were performed in both PBL and HF DNA ( Figure 1 ) . Consistent with previous studies [2] , [16] , most CpG sites assayed did not show measurable interindividual differences in methylation ( Table S1 ) . Moreover , interindividual differences were more often observed in a single tissue than in both tissues ( Table S1 ) . Nonetheless , our approach identified 107 genomic loci exhibiting concordant interindividual MSAM differences in both tissues ( Table S2A ) . MSAM is based upon serial digestion of genomic DNA with the methylation sensitive/insensitive isoschizomers SmaI and XmaI; our screen could therefore detect genetic variation in addition to systemic epigenetic variation . Indeed , initial attempts to validate several candidates by bisulfite pyrosequencing failed to detect differences in DNA methylation and instead identified single nucleotide polymorphisms ( SNPs ) within SmaI/XmaI sites . We bioinformatically annotated all potentially informative human SmaI/XmaI intervals with a known SNP within either SmaI/XmaI site ( CCCGGG ) . SNPs that introduce a SmaI/XmaI site within a consensus SmaI/XmaI interval also could affect the MSAM signal , and were likewise annotated . Of 107 SmaI/XmaI intervals originally identified in our screen , 34 were associated with SmaI/XmaI SNPs , a significant over-representation ( P = 1 . 3×10−8 ) . After excluding these ( Table S2B ) , we observed that the remaining 73 candidate MEs tended to localize in subtelomeric regions ( Figure S1A ) . Given the propensity for copy number variation in subtelomeric regions [17] , we identified all potentially informative SmaI/XmaI intervals located within known human copy number variants and segmental duplications . Nearly half ( 35 ) of the remaining 73 candidate MEs were located within these genetically variable regions , many more than expected by chance ( P = 2 . 1×10−23 ) . After excluding these ( Table S2C ) , the subtelomeric localization was eliminated ( Figure S1B ) . Excluding all candidate SmaI/XmaI intervals associated with known SNPs , copy number variants , and segmental duplications is extremely conservative , and likely excludes MSAM hits that are in fact caused by interindividual variation in DNA methylation . Indeed , 2 hits in which interindividual DNA methylation differences had already been validated before we performed the bioinformatic filtering were among the affected intervals: the interval at ZNF696 is associated with a SmaI/XmaI SNP , and that at FLJ20433 is within a copy number variant . These 2 loci were retained in the final list of candidate MEs , bringing the number to 40 . Of 13 we analyzed by bisulfite pyrosequencing , interindividual variation in PBL and HF DNA methylation was confirmed in 8 . ( Failure to validate could be caused by uncharacterized SNPs and CNVs , inability to assay both of the informative SmaI/XmaI sites , or low overall methylation levels . ) Our screen was performed using DNA from Caucasians , using 2 tissues that can be sampled relatively non-invasively . To verify concordance across tissues derived from all 3 germ layers , and determine if interindividual epigenetic variation at candidate MEs is conserved across genetically divergent populations , post-mortem liver , kidney and brain tissue was obtained from 8 Vietnamese motor vehicle accident victims ( healthy donors ) . Of the 8 genomic regions with confirmed interindividual variation in DNA methylation in the Caucasian PBL and HF samples , 5 ( BOLA3 , FLJ20433 , PAX8 , SLITRK1 , and ZFYVE28 ) showed interindividual variation that was highly correlated among liver , kidney , and brain in the Asian sample ( Figure 2A–2E , and Table S3 ) . ( SLITRK1 was exceptional in that methylation in brain did not correlate with that in liver and kidney ( Figure 2E ) . This is potentially analogous to the murine AxinFu ME , at which DNA methylation in tail differs from that in all other tissues [11] . ) For comparison , we similarly analyzed regions within IGF2 , GNASAS , and IL10 , at which DNA methylation in PBL DNA has been associated with early famine exposure [18] , [19] . Although substantial interindividual variation in DNA methylation was confirmed at these loci , not a single statistically significant inter-tissue correlation was found ( Figure 2F–2H , and Table S3 ) . To identify specific genomic characteristics that may confer the special epigenetic behavior of these loci , we bioinformatically compared 6 kb windows encompassing the 40 putative ME SmaI/XmaI intervals and 5000 ‘control’ intervals on the array . We assessed several characteristics of associated CpG islands , as well as the distribution of various classes of tranposable elements ( Figures S2 , S3 , S4 , S5 , S6 , S7 , S8 , S9 ) . The only significant finding was in the distribution of long-terminal repeat ( LTR ) retrotransposons; these were depleted at and distributed symmetrically around control intervals , but preferentially localized downstream of putative ME intervals ( P = 0 . 001 ) ( Figure S7 ) . Although clearly insufficient to explain epigenetic metastability , this finding is noteworthy in that nearly all known murine MEs are associated with intracisternal A particle LTR-retrotransposons [20] , [21] . Our aim was to identify interindividual epigenetic variation that occurs stochastically; the multiple-tissue screening approach could , however , also detect epigenetic variation associated with genetic variation [6] , [22] , [23] . Indeed , while performing pyrosequencing validation of one candidate ME , ZNF696 , a proximal SNP was identified that explained most of the interindividual variation in methylation ( Figure S10 ) . To attempt to rule out such effects , one could map the genomic region flanking each candidate ME to identify haplotype blocks correlated with methylation status . But effects of genetic variation on DNA methylation can occur in cis over tens or even hundreds of kb [23] , [24] , or in trans [25] . By genetic mapping alone , therefore , it is virtually impossible to exclude that the systemic interindividual epigenetic variation at these select loci is attributable to genetic variation . Epigenetic discordance within pairs of MZ twins would provide support that interindividual epigenetic variation at our candidate MEs is truly stochastic . We measured DNA methylation at BOLA3 , FLJ20433 , and PAX8 in buccal DNA from 23 pairs of MZ twins ( Figure S11 ) . At PAX8 , although there was significant inter-twin correlation , about half of the variance in DNA methylation was not shared by co-twins . At BOLA3 and FLJ20433 there was no inter-twin correlation . These data provide evidence that the interindividual epigenetic variation at our candidate MEs is not genetically mediated . Another way to determine whether the epigenetic variation at these loci is truly stochastic is to test for an early environmental effect . Unlike interindividual epigenetic variation that is secondary to genetic variation , the stochastic epigenetic variation at bona fide MEs can be influenced by maternal nutrition during early embryonic development [10]–[12] . Demonstrating an effect of periconceptional nutrition on DNA methylation at the identified genomic regions would therefore provide further support that they are MEs . The rural villagers in West Kiang , the Gambia are subsistence farmers whose nutritional status varies dramatically by season . During the rainy season ( July–November ) depletion of food stores from the previous harvest , combined with an intense agricultural workload , causes negative energy balance and consequent effects on reproductive outcomes [26] . Relative to the dry season , average birth weight during the rainy season is 200–300 g lower and the incidence of small for gestational age infants is doubled [27] . Importantly , seasonal effects on fetal development persist to affect adult mortality in this population [28] , but the underlying biologic mechanisms remain unknown . To test the hypothesis that periconceptional nutrition affects developmental establishment of DNA methylation at candidate MEs , we compared DNA methylation in peripheral blood leukocytes ( PBL ) of Gambian children conceived during either the dry or the rainy season . Effects of seasonality vary from year to year; we therefore used retrospective birth weight data to identify 1991 , 1994 , 1995 , 1997 , and 1998 as years with strong effects of seasonality ( Figure S12 ) . Individuals conceived during August–September ( rainy season ) were compared with those conceived during March–May ( dry season ) , matching for sex and year of conception ( n = 30/season ) . Blood was collected from the children at age 8 . 9±0 . 5 years ( mean ± sem ) ; age at blood collection did not differ between the season of conception groups . Preliminary analyses of the DNA methylation data showed a highly significant ( season of conception ) × ( year of conception ) interaction ( P = 0 . 005 ) , indicating that the effect of seasonality was not consistent in all years . Examining the effects in each year indicated that 1997 was an outlier . Excluding individuals conceived in 1997 eliminated the ( season of conception ) × ( year of conception ) interaction ( P = 0 . 17 ) and left n = 25 individuals per season , representing four years ( 1991 , 1994 , 1995 , and 1998 ) in subsequent analyses . Since maternal supplementation with dietary methyl donors increases DNA methylation at MEs in murine offspring [10]–[12] , we anticipated that DNA methylation would be reduced in individuals conceived during the nutritionally challenged rainy season . We found the opposite . At all 5 putative MEs , DNA methylation was significantly higher among individuals conceived during the rainy season ( Figure 3A ) . The overall effect of season of conception on DNA methylation at the 5 MEs combined was highly significant ( P = 0 . 0001 ) . ( Detailed statistical analyses provided in Text S1 . ) Unlike persistent changes in DNA methylation associated with periconceptional famine exposure [18] , [19] the effect sizes at the genomic regions we identified were not subtle; rainy season conception was associated with absolute methylation increments of over 10% at both PAX8 and ZFYVE28 ( Figure 3A ) . To determine if the association of season of conception with DNA methylation might be due to chance genetic differences between the groups ( such as , for example , differences in one carbon metabolism ) , we compared DNA methylation at generic LINE1 elements ( an indicator of genome-wide methylation [29] ) and the same 3 ‘control’ genes studied in the Asian sample ( IGF2 , GNASAS , and IL10 ) . Contrary to large studies which have associated early famine exposure with subtle persistent changes in DNA methylation at IGF2 , GNASAS , and IL10 [18] , [19] , we found no effect of season of conception in the non-ME control regions , either singly or combined ( Figure 3B ) , indicating that developmental establishment of DNA methylation at MEs is exceptionally sensitive to maternal environment . The overall effect of season of conception at these putative MEs is especially compelling given that each individual's DNA methylation at one was generally not predictive of methylation at others ( Table S4 ) , meaning that stochasticity at these genomic regions is not coordinated . Underscoring the broad relevance of these findings , the genomic loci we identified exhibit similar epigenetic behavior across genetically distinct human populations ( Figure S13 ) indicating that they are ancestral features of the human genome .
Murine MEs have attracted extensive study because of their mysterious ability to cause dramatic phenotypic variation among isogenic animals [7] , [21] , [30] , [31] . Viewed by some as an epigenetic oddity , however , their relevance to humans has been questioned [32] . Here , we have for the first time identified elements that are likely to be human MEs , which are characterized by stochastic and systemic interindividual epigenetic variation . These loci exhibit similar interindividual variation in DNA methylation across tissues derived from all 3 germ layers of the early embryo , indicating setting of epigenotype prior to gastrulation . Epigenetic discordance at these genomic loci within MZ twin pairs indicates that establishment of their epigenetic state is determined not genetically , but stochastically . Further , as at murine MEs [10]–[12] , developmental establishment of epigenotype at these loci is exquisitely sensitive to maternal periconceptional environment . Interindividual epigenetic variation that is both systemic and stochastic has not been previously documented in humans . In many cases human interindividual epigenetic variation has been found to be caused by genetic variation [6] , [22] , [23] . Recent studies of MZ twin pairs have identified epigenetic differences that occur independent of genetic variation [33] , [34] , but since those differences were studied only in specific tissues it is not clear if they occur systemically . Our results suggest that interindividual epigenetic variation is more often tissue-specific than systemic . Only about half of the SmaI/XmaI intervals showing interindividual variation in PBL , and 15% of those showing interindividual variation in HF , exhibited consistent interindividual variation in both tissues ( Table S1 ) . A key issue is whether establishment of epigenotype at the loci we have identified is truly stochastic . One might argue that the systemic interindividual differences in DNA methylation could be caused by genetic variation , but two pieces of evidence suggest otherwise: the epigenetic discordance within MZ twin pairs , and the effect of season of conception . We must , however , note some caveats . Since we studied DNA methylation in only one tissue from MZ twins , we can not definitively say the observed MZ twin discordance arose in the very early embryo . Future studies should examine ME methylation in MZ twins using DNA from multiple tissues representing the three embryonic germ layers . Likewise , in the Gambian studies , we studied DNA methylation in only one tissue . Hence , although the most parsimonious interpretation of the season of conception effect on PBL DNA methylation is an environmental influence on the early embryo , other interpretations are plausible . For example , if 3 months of age ( i . e . 1 year after conception ) is a critical window for developmental epigenetics in PBL , there could be a seasonal influence on these processes . Alternatively one could postulate reverse causality , whereby physiological changes induced by seasonal influences on development lead to secondary alterations in DNA methylation . Studying the effect of season of conception on ME DNA methylation in multiple tissues ( which is currently underway ) will test both of these alternative hypotheses . It is unlikely that postnatal seasonal effects or secondary effects of altered physiology would induce similar epigenetic changes in diverse tissues . Although DNA methylation at the PAX8 ME was significantly correlated within MZ twin pairs ( Figure S11 ) , this does not necessarily indicate a genetic effect on epigenotype . If setting of epigenotype at MEs occurs prior to blastocyst cleavage during MZ twinning , both members of an MZ twin pair could carry concordant epigenetic states at MEs , despite stochastic establishment . Given the different timing of blastocyst cleavage in dichorionic vs . monochorionic MZ twins , examining ME DNA methylation among these different subtypes of MZ twin pairs may prove informative . The identification of human MEs should advance the study of epigenetics and human disease . Because individually-variable DNA methylation at these loci exhibits little tissue-specificity , epigenetic dysregulation in pathophysiologically relevant tissues such as thyroid and brain , for example , can be inferred from PBL DNA . Indeed , among the putative MEs we identified are genes implicated in hypothyroidism ( PAX8 ) [35] , and Tourette's syndrome ( SLITRK1 ) [36] . Such sites therefore represent excellent candidate loci for future studies of epigenetic epidemiology which will utilize existing DNA sample collections to explore associations between epigenetic variation and human disease . Moreover , given their epigenetic lability to early environmental influences , human MEs may enable the elaboration of mechanistic pathways linking early environment to later risk of disease [4] , [37] . To the extent that epigenetic variation at MEs is associated with diseases such as cardiovascular disease , type-2 diabetes , and obesity , we may better understand how early nutrition and other environmental exposures predict adult risk of these diseases [5] . By no means should it be inferred that MEs are the sole genomic substrate for early environmental influences on epigenetic regulation . Extensive data from animal [38] , [39] and human studies [18] , [19] indicate that environmental factors affect epigenetic processes over a broad range of developmental periods , with long-term consequences . Stochastic establishment of epigenotype at MEs , however , does appear to be particularly sensitive to periconceptional environment . For example , by studying 60 famine-exposed humans and their unexposed same sex-siblings , Heijmans et al detected persistent effects of periconceptional famine exposure on PBL DNA methylation at the IGF2 DMR [18] , GNASAS , and IL10 [19] . Here , we show that seasonal variation in periconceptional nutrition – likely a milder perturbation – induced significant changes in DNA methylation at all 5 putative MEs studied , but not at the IGF2 DMR , GNASAS , or IL10 . Moreover , unlike epigenetic changes that occur only in specific tissues , environmentally-induced epigenetic changes at MEs affect the entire body , and are therefore more likely relevant to human physiology and disease . Enormous interest in transgenerational epigenetic inheritance has recently been stimulated by provocative data indicating that environmental influences during development might affect the health of subsequent generations [40] , [41] . Since transgenerational epigenetic inheritance is known to occur at murine MEs [7] , [30] , it is logical to consider whether MEs may likewise provide opportunities to understand non-genetic inheritance in humans . Our findings raise additional questions for future study . First , what causes epigenetic metastability ? The stochastic establishment of epigenotype at MEs must fundamentally be a consequence of the genetic sequence in these genomic regions . Indeed , known murine MEs result from transposition of retrotransposons in or nearby genes [21] . But among the genomic loci identified here , no obvious genetic signature of epigenetic metastability was detected . Since our screen was limited to genomic regions containing multiple SmaI/XmaI sites , we detected only a subset of human MEs . Our parallel , 2-tissue screening approach is , however , adaptable to various epigenomic platforms and should enable the identification of many more human MEs . It may then be possible to gain a better understanding of the molecular basis of epigenetic metastability . Second , we still know very little about exactly how maternal nutrition before and during pregnancy affects establishment of epigenotype at MEs . Contrary to our expectations , Gambian individuals conceived in the nutritionally challenged rainy season gained DNA methylation at these putative MEs , emphasizing that our original conjecture that hunger would be associated with a functionally-limiting methyl donor deficiency was overly simplistic . In light of earlier findings that maternal blood folate levels paradoxically increase during the rainy season in the Gambia [42] ( potentially due to increased consumption of leafy vegetables ) , our data suggest that rather than energy intake , availability of one-carbon donors is of key importance . Studies in mouse models and humans are currently underway to improve our understanding of how maternal dietary and other environmental exposures ( e . g . insecticides [43] or naturally occurring toxins [44] ) affect developmental epigenetics in the preimplantation embryo . In summary , we have provided strong evidence that stochastic establishment of epigenetic regulation occurs at specific human genomic loci , resulting in interindividual epigenetic variation that affects tissues from all 3 germ layers and persists to adulthood . We have shown that seasonally variable maternal periconceptional exposures affect this stochastic process . Systemic and persistent epigenetic imprints at these loci are likely to be found among diverse human populations that experience seasonal variation in nutritional sufficiency [27] , [45] , [46] or other environmental exposures during early embryonic development .
MSAM was performed as previously described [15] , using a starting quantity of 0 . 5 µg genomic DNA . MSA products from 2 individuals were differentially dye-labeled and cohybridized to a custom 4×44k array . Array probes were within potentially informative SmaI/XmaI intervals ( 60–1500 bp ) and were selected from Agilent's proximal promoter and CpG island probe libraries ( Agilent Technologies , Santa Clara , CA , USA ) . The 43 , 222 probes on the array cover 19 , 187 SmaI/XmaI intervals ( average 2 . 3 probes/interval ) . Genomic coordinates are based upon hg18 ( NCBI Build 36 . 1 ) . Relevant details of the microarray experiment , including experimental design , microarray probe listing , and hybridization data sets are available in the GEO database ( http://www . ncbi . nlm . nih . gov/geo/ ) ( accession # GSE19823 ) . Four 2-individual MSAM comparisons ( Table S5 ) were performed using PBL DNA , and the same four 2-individual comparisons were performed using HF DNA ( incorporating a dye swap ) ( Table 1 ) . The analysis was performed at the level of SmaI/XmaI interval; average and median signal intensity , signal ratio , and P value of all probes within each SmaI/XmaI interval were calculated . Candidate MEs were identified as follows . For a given paired comparison ( say comparison A ) we selected all SmaI/XmaI intervals with both an average A1/A2 signal ratio >1 . 8 or <0 . 556 and median P<0 . 0002 in both PBL and HF . All candidates identified in this manner were further filtered to eliminate those in which the 2 tissues showed discordant interindividual ratios in any of the other pairwise comparisons; only SmaI/XmaI intervals for which the ratio of the PBL:HF signal ratios was >0 . 445 and <2 . 25 for all comparisons ( A , B , C , D ) were retained . ( If there is no tissue-specificity in DNA methylation , this ‘ratio of ratios’ equals 1 . The maximum departure from this we allowed ( 2 . 25 ) corresponds to a signal ratio of 1 . 5 in one tissue and 0 . 667 in the other . ) This procedure resulted in the 107 candidate MEs listed in Table S2A . Site-specific analysis of CpG methylation was performed by bisulfite pyrosequencing . Genomic DNA ( 0 . 5–2 µg ) was bisulfite modified [38] and pyrosequencing was performed as previously described [50] . The quantitative performance of each pyrosequencing assay was verified by measuring methylation standards comprised of known proportions of unmethylated ( whole genome-amplified ) and fully methylated ( SssI-treated ) genomic DNA [50] . For initial validation of interindividual variation at candidate SmaI/XmaI intervals , DNA methylation was , whenever possible , measured at both SmaI/XmaI sites . Subsequent characterization ( measurements in other populations , etc ) was performed in the vicinity of the SmaI/XmaI site showing the greatest interindividual variation . We assessed interindividual variation in the Caucasian samples at 13 of the 40 candidate MEs identified in the MSAM screen: AK098581 , AXIN2 , BOLA3 , FLJ20433 , ITPKB , MN1 , PAX8 , RCC1 , SLITRK1 , SOX10 , ZNF561 , ZNF696 , and ZFYVE28 ( primers listed in Table S9 ) . In 5 of these ( AXIN2 , ITPKB , MN1 , RCC1 , and SOX10 ) the pyrosequencing assays failed to confirm interindividual variation in DNA methylation . ZNF696 was excluded because it exhibited interindividual variation in methylation that was mostly explained by genetic variation at a neighboring SNP ( Figure S10 ) . At the remaining 7 loci we examined tissue-specificity of interindividual variation in the Asian liver , kidney , and brain samples . Five ( BOLA3 , FLJ20433 , PAX8 , SLITRK1 , and ZFYVE28 ) exhibited significant inter-tissue correlations in DNA methylation consistent with MEs . Two that did not ( AK098581 and ZNF561 ) were excluded from further consideration . We selected 48 autosomal SNPs with previously demonstrated high reliability for genotyping on the Illumina platform and high minor allele frequency ( MAF<0 . 3 ) in the Yoruban HapMap population ( as the best surrogate we had for the Malawi population ) . SNPs were selected to be physically distant from each other . These were genotyped on all of the Malawian twin samples . PREST ( Pedigree RElationship Statistical Test ) was used to estimate the probability of the putative twins sharing 0 , 1 , or 2 alleles IBD ( p0 , p1 , p2 ) based on the pairwise analysis of the 48 SNP markers , and the kinship coefficient estimated as phi = 0 . 25*p1+0 . 5*p2 . In the absence of genotyping error , true MZ twins are expected to have p0 = p1 = 0 and phi = 0 . 5 . Relative enrichment of candidate ME SmaI/XmaI intervals associated with SmaI/XmaI SNPs , CNVs , and segmental duplications was analyzed by chi-square tests . Analysis of CGIs and repetitive elements in the vicinity of MEs and control intervals was performed by analysis of variance ( ANOVA ) ( Proc GLM , SAS Version 9 . 2 ) . Inter-tissue correlations in interindividual variation in methylation were assessed by Pearson correlation analysis ( Proc CORR , SAS ) . A REML multifactorial ANOVA ( JMP Version 8 . 0 ) was used to assess factors affecting average methylation in the Gambian season of conception analysis . Methylation was measured multiple times within each individual for each locus and averaged , for a total of 539 averaged observations . Individual and locus were assessed as random factors , with locus nested within locus type ( ME or control ) . Methylation was arcsine transformed to improve normality . Normality was assessed by Shapiro-Wilk Tests for each sample combination of gene and season of conception ( 18 combinations consisting of 30 individuals each ) . All samples were statistically indistinguishable from normal distributions , after sequential Bonferroni correction for carrying out 18 simultaneous tests . One interaction , ( season of conception ) × ( locus type ) was investigated as an a priori test .
|
There is growing interest in the possibility that interindividual epigenetic variation plays an important role in a broad range of human diseases . The tissue-specificity of epigenetic regulation , however , will in many cases make it difficult to obtain the appropriate tissues in which to perform large-scale studies linking epigenetic dysregulation to disease . We have used an innovative two-tissue DNA methylation screen to identify genomic regions that exhibit interindividual epigenetic variation which occurs systemically—i . e . similarly in all tissues . Such regions—called metastable epialleles—have previously been identified in mice because they cause visible phenotypic variation amongst genetically identical individuals . Indeed , we found that even monozygotic twins show substantial epigenetic discordance at these loci . Further , we show that , as in mice , establishment of DNA methylation at these putative human metastable epialleles is labile to maternal environment around the time of conception . Metastable epialleles should facilitate an improved understanding both of the role of interindividual epigenetic variation in human disease and of the effects of early environment on the establishment of human epigenotype .
|
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"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"and",
"genomics/epigenetics",
"molecular",
"biology/dna",
"methylation",
"nutrition"
] |
2010
|
Season of Conception in Rural Gambia Affects DNA Methylation at Putative Human Metastable Epialleles
|
Phosphoinositide-3-kinases have been shown to be involved in influenza virus pathogenesis . They are targeted directly by virus proteins and are essential for efficient viral replication in infected lung epithelial cells . However , to date the role of PI3K signaling in influenza infection in vivo has not been thoroughly addressed . Here we show that one of the PI3K subunits , p110γ , is in fact critically required for mediating the host’s antiviral response . PI3Kγ deficient animals exhibit a delayed viral clearance and increased morbidity during respiratory infection with influenza virus . We demonstrate that p110γ is required for the generation and maintenance of potent antiviral CD8+ T cell responses through the developmental regulation of pulmonary cross-presenting CD103+ dendritic cells under homeostatic and inflammatory conditions . The defect in lung dendritic cells leads to deficient CD8+ T cell priming , which is associated with higher viral titers and more severe disease course during the infection . We thus identify PI3Kγ as a novel key host protective factor in influenza virus infection and shed light on an unappreciated layer of complexity concerning the role of PI3K signaling in this context .
Phosphoinositide 3-kinases ( PI3K ) are classified into three main groups ( class I , class II and class III ) according to sequence homology of the catalytic subunit and their substrate specificity [1] . Class I PI3K are further divided into class IA and class IB . Class IA PI3K form dimers consisting of either one of the catalytic subunits p110α , p110β or p110δ , and the common regulatory subunit p85 [2] [3] [4] [5] . They typically act downstream of receptor tyrosine kinases and are important regulators of cell growth , division and survival [6] . In contrast , class IB PI3K ( also termed PI3Kγ ) comprises only one catalytic subunit , p110γ , which associates with the regulatory subunits p101 or p84 [7] [8] [9] [10] [11] . PI3Kγ signals downstream of G-protein coupled receptors ( GPCR ) such as chemokine receptors or receptor tyrosine kinases [12] . Both class IA and PI3Kγ can be activated by ras [13] [14] . Classes II and III PI3K are ubiquitously expressed and mainly involved in regulation of protein trafficking and cell homeostasis . PI3Kγ on the other hand is preferentially expressed in hematopoietic cells , although expression was also shown in peribronchial epithelial cells , the endothelium , the brain and the heart [15] [16] . Several groups have addressed the role of PI3Kγ in immune responses using specific inhibitors or p110γ-deficient mice . Neutrophils and macrophages , which are p110γ-deficient , exhibit reduced migration in vitro in response to chemotactic stimuli such as IL-8 and MIP-1α as well as the GPCR agonists C5a and fMLP [17] . Consistently , in vivo recruitment of neutrophils and macrophages to inflamed peritoneum is severely impaired in p110γ-/- animals upon peritoneal infection with Listeria monocytogenes [18] . In addition to the defects observed in innate immune cells , PI3Kγ-deficiency results in impaired adaptive immune responses . PI3Kγ-signaling in conjunction with PI3Kδ , plays a minor role in thymocyte as well as B cell development and the absence of PI3Kγ leads to a small reduction of peripheral CD4+ but not CD8+ T cells [19] . Addressing the migration capacity of lymphocytes , it was shown that PI3Kγ is superfluous for T cell homing in steady-state conditions [20] . Under inflammatory conditions however , PI3Kγ-/- mice display a reduced recruitment of CD8+ T cells . Peritoneal infection with Vaccinia virus or Lymphocytic Choriomeningitis virus infection into the footpads result in decreased numbers of CD8+ T cells at the site of inflammation in PI3Kγ-/- mice [21] [22] . In both studies , PI3Kγ-/- CD8+ T cells exert normal effector functions in terms of Interferon-γ ( IFN-γ ) production and cytoxicity . In contrast to T cells , B cells develop normally in PI3Kγ-/- mice and do not show any deficiency in migration [20] . More recently , we could show that PI3Kγ is required for development of lung CD11b+ DC and CD103+ DCs in particular by regulation of signaling downstream of Flt3 , while it is dispensable for DC development in many other tissues [23] . In line with this data , several reports have revealed a central role for PI3Kγ in murine models of human immune-mediated inflammatory diseases such as rheumatoid arthritis and airway inflammation as well as autoimmune diseases such as systemic lupus [24] [25] [26] . Therefore , PI3Kγ is considered a promising target for the treatment of inflammatory disorders [27] . Using chemical inhibitors such as Wortmannin , PI3K family members or effectors downstream such as Akt kinases were shown to be required by influenza virus for infection of lung epithelial cells in vitro [28] [29] , in particular through interactions with the viral protein NS1 [30] . Furthermore , Influenza virus strains carrying mutations rendering them unable to activate PI3K signaling were shown to lead to attenuated infection in vitro and in vivo [30] . However , the importance of PI3K signaling for host defense as well as the specific roles of individual PI3K subunits for influenza virus infection in vivo , remain poorly understood . In this context PI3Kγ itself has not received much attention in the context of influenza infection . Given the defects in innate and adaptive immunity in PI3Kγ-deficient mice and its potential direct involvement in influenza virus pathogenesis , we investigated the role of PI3Kγ-signaling upon infection with influenza virus in vivo . We found that PI3Kγ-deficiency led to greatly enhanced susceptibility to influenza virus infection due to delayed viral clearance . This was caused by impaired T cell priming by lung resident dendritic cells due to a pre-existing developmental deficiency in the lung dendritic cell compartment of PI3Kγ-deficient animals . We thus describe a novel role of PI3Kγ in regulating host responses against respiratory viral infections .
To address the role of p110γ in Influenza A virus infection ( IAV ) in vivo we infected p110γ kinase–dead ( p110γ-KD ) animals with a sub-lethal dose of the highly pathogenic strain IAV PR8 . These animals carry an inactivating mutation in the kinase domain of p110γ and thus allow us to delineate the role of p110γ kinase function during IAV infection in vivo . Monitoring weight and temperature loss over time , we observed a much more severe disease course in p110γ-KD animals as opposed to WT controls with a more pronounced temperature and weight loss ( Fig 1A and 1B ) . Furthermore , at a fourfold higher dose ( i . e . 200 pfu ) , half of the p110γ-KD mice succumbed to infection by day 15 , while 100% of WT animals survived ( Fig 1C ) . The enhanced morbidity observed in p110γ-KD mice infected with 50 pfu was paralleled by a delayed viral clearance at later time-points of infection , where p110γ-KD animals had higher viral titers at day 9 and 11 p . i , while there was no difference at day 5 p . i ( Fig 1D ) . In addition , the high viral load in lungs of p110γ-KD animals at later points during the infection correlated with an increased proteinosis and cell death in the alveoli , exemplified by higher levels of total protein and higher number of dead cells in the bronchoalveolar lavage ( BAL ) of p110γ-KD mice at day 11 ( Fig 1E and 1F ) . Examining the immune cell infiltrate in the lungs of infected animals at an early time point of infection it was apparent that recruitment of monocyte-derived dendritic cells ( moDC ) natural killer ( NK ) cells and neutrophils was completely intact despite p110γ kinase-deficiency ( Fig 1G and 1H ) . Similarly , numbers of tissue-resident alveolar macrophages ( AM ) were comparable between WT and p110γ-KD animals at day 3 p . i . ( Fig 1H ) . Finally , also levels of hallmark inflammatory cytokines TNFα and IL-1β were similar in the BAL of both mouse strains ( Fig 1I ) . Taken together these results suggested that p110γ plays an important role in host defense against IAV but that the early antiviral response is largely intact . To characterize the adaptive immune response against IAV we again infected p110γ-KD animals with a sub-lethal dose of PR8 IAV and then characterized immune cell infiltration into the BAL and lung at day 7 p . i . , which represents the initiation phase of the adaptive immune response . Both CD4+ and CD8+ T cells were readily detected in BAL and lung at this time point ( Fig 2A ) . Quantifying the total number of T cells in the lungs of infected mice it was evident that p110γ-KD mice exhibited a significantly reduced number of both CD4+ and CD8+ cells present ( Fig 2B ) . Most strikingly , nucleoprotein—34-specific ( Tet+ ) CD8+ virus-specific cells were virtually absent in the lungs of p110γ-KD animals . Similarly , the proportion of IFNγ-producing CD4+ and CD8+ cells in the BAL was lower in mice deficient for p110γ kinase function ( Fig 2C and 2D ) . Furthermore , in the lung draining lymph node ( dLN ) CD4+ , CD8+ and virus specific CD8+ T cells were all considerably reduced in the p110γ- defective condition compared to WT animals ( Fig 2E ) . Conversely , no difference was observable in the fold increase in the number of activated CD4+ and CD8+ T cells in the lungs of infected compared to naïve animals ( Fig 2F ) . Similarly , inflammatory cytokines IL-1β and IL-6 in the BAL of infected mice ( Fig 2G ) were comparable between WT and p110γ-KD mice . In addition , the antiviral B cell response appeared to be completely intact as no difference in antibody titres of different isotypes in the BAL could be observed between WT and p110γ-KD mice ( Fig 2H ) at day 11 p . i . To address the possibility that the deficient T cell response observed in p110γ-KD mice was due to a T cell defect in naïve animals , T cells were examined in blood and lung in the steady-state . This analysis revealed that the number of T cells in the lung as well as the frequency in the blood of naïve mice was comparable between WT and p110γ-KD animals ( S1A and S1B Fig ) . Furthermore , the frequency of CD44+CD62L- activated T cells was similar regardless of p110γ-deficiency ( S1C and S1D Fig ) . To exclude the possibility that p110γ generally regulates development of hematopoietic cells , the composition of blood of p110γ-KD animals was carefully analysed . Granulocytes such as neutrophils are present at normal levels in p110γ-KD mice ( S1E Fig ) and similarly the number of red blood cells is also comparable to WT animals in p110γ-KD mice ( S1F Fig ) . Overall , these results suggested an important and specific role for p110γ in regulating the antiviral T cell response , in particular the CD8+ T cell component but that p110γ is largely dispensable for T cell development and activation in the periphery . To address a potential direct involvement of p110γ in influenza virus propagation in epithelial cells , A549 cells , a lung epithelial cell line , were infected with PR8 IAV and were also treated with inhibitors against p110γ ( i . e . AS605240 ) , p110δ ( i . e . IC-87114 ) or all PI3K subunits ( Wormannin ) . Viability of the cells was not affected by inhibitor treatment ( Fig 3A ) , however the frequency of infected cells as well as the viral titre was significantly reduced in cells treated with Wortmannin , while AS605240 or IC-87114 showed a minor reduction ( Fig 3B and 3C ) . Consistently , p110γ protein was undetectable in A549 cells and mRNA expression of Pik3cg and its regulatory subunit Pik3r5 was barely detectable in sorted lung epithelial cells compared to lung CD103+ DCs ( Fig 3D and 3E ) , while significant expression of another PI3K subunit , Pik3cd , could be detected ( Fig 3F ) . To further address the relative importance of p110γ during IAV infection in vivo in structural and hematopoietic cells , criss-cross bone marrow chimeras were generated . After reconstitution mice were then infected with IAV . Mice which had received WT bone marrow ( BM ) mounted a potent anti-viral T cell response , while animals receiving p110γ BM exhibited a significantly reduced number of CD4+ , CD8+ and virus specific CD8+ T cells at day 7 p . i . ( Fig 3B–3E ) . Overall , these results suggested that p110γ is required in the hematopoietic compartment for mounting an effective T cell response against IAV . To further dissect the underlying mechanism of the impaired antiviral T cell response in the absence p110γ-kinase function , p110γ-KD mice were infected with a sub-lethal dose of IAV and the T cell response was evaluated at the peak of the response at day 10 p . i . At this time-point the number of CD8+ and Tet+CD8+ T cells in the lung was still strongly reduced in p110γ-KD animals compared to WT controls ( Fig 4A ) . However , CD4+ T cells in p110γ-KD mice were now present in similar numbers to WT animals ( Fig 4A ) . Similarly , no significant differences were visible in the number of CD4+ , CD8+ and virus specific Tet+CD8+ T cells in the lung dLN between p110γ-KD and control mice ( Fig 4B ) . In addition , the enhanced morbidity at this time-point post infection of p110γ-KD animals correlated with a more pronounced inflammation in the lung exemplified by a higher number of neutrophils ( Fig 4C ) . To determine whether the reduced number of CD8+ T cells in particular was due to a reduced proliferative capacity of these cells in vivo , WT and p110γ-KD mice were infected with IAV and subsequently injected with EdU to measure the proportion of proliferating cells . At an early time-point of the antiviral T cell response the frequency of EdU+ cells among CD8+ T cells but not CD4+ T cells was reduced in p110γ-KD animals ( Fig 4D–4E ) . To shed further light on the kinetics of the T cell response in p110γ-KD mice the same experimental set up was repeated at a later time-point at of infection . During this peak phase of the infection no deficiency in EdU incorporation could be observed for T cells localized in the lungs or dLN of infected p110γ-KD mice compared to controls ( Fig 4F ) . Overall , these results suggested that there is a defect in the priming of CD8+ T cells lacking a functional p110γ kinase domain during IAV infection in vivo . The T cell response against IAV is strongly dependent on T cell priming by dendritic cells ( DC ) [31] . To address a potential functional role of p110γ kinase activity in DC-mediated priming of T cells we cocultured bone marrow-derived DCs ( BMDCs ) of WT and p110γ-KD origin with OTII CD4+ T cells with different concentrations of the OVA323-339 peptide . Examining the number of CD4+ T cells after 4 days of culture as a readout for T cell proliferation it was apparent that there is no difference between using WT and p110γ-KD BMDCs ( Fig 5A ) . Furthermore , looking at the T cell polarization in terms of Th1 cytokine production , which is the predominant T helper response during IAV infection , it was evident that p110γ-KD BMDCs have no deficiency in inducing IFNγ , GM-CSF or TNFα producing CD4+ T cells ( Fig 5B ) . To address the possibility that p110γ is required for antigen processing by DCs and that it may play a role in the priming of CD8+ T cells , we transferred efluor-670 labeled OT-I T cells into WT and p110γ-KD recipients and injected ovalbumin in alum one day later . 7 days after transfer we then evaluated the efluor-670 staining as well as the number of TCRVα2+ cells in the inguinal lymph-node , which is the TCRα chain used by all OT-I cells . It was clearly evident that in both WT and p110γ-KD recipients OT-I cells had strongly proliferated as most cells had indeed diluted out the dye completely by day 7 ( Fig 5C ) . Similarly , the number of CD8+CD44+CD62L-TCRVα2+ cells was much higher in the animals , which had received transferred OT-I cells , compared to the non-transferred controls ( Fig 5C ) . Furthermore , there was no statistically significant difference between WT and p110γ-KD recipients . p110γ has been classically associated with regulating migration of immune cells towards chemokines [22] but this has thus far not been thoroughly examined in DCs . CCR7 is a critical regulator of migration of DCs towards the lung dLN and has been shown to be critical for the T cell response against IAV[32] . To test a possible role of p110γ in regulating CCR7-mediated migration of DCs we seeded WT and p110γ-KD BMDCs in a trans-well system and quantified migration towards CCL21 , the principal ligand for CCR7 . The frequency of migrating BMDCs was higher in p110γ-KD cells compared to WT although generally only 4–8% of cells migrated at all ( Fig 5C ) . To account for the limitations of using BMDCs as a model for lung-resident DCs , a similar experimental set up was repeated using lung DCs . Due to the strong reduction of lung-resident CD103+ DCs in particular in p110γ-KD animals , lung CD103+ and CD11b+ DC subsets were sorted from the lungs of WT animals and then the same migration assay was done in the presence of p110γ-specific inhibitor AS650240 or DMSO as a control . A significant number of both CD103+ and CD11b+ DCs migrated , regardless of p110γ inhibition ( Fig 5D ) . Overall these results suggested that p110γ is not generally required for DC-mediated priming of T cells in vitro and in vivo as well as for DC migration in vitro . To elucidate the potential role of p110γ in lung DC-mediated antiviral T cell responses in vivo , we characterized the lung DC compartment of p110γ-KD mice in the naïve state and during IAV infection . As described by our group recently[23] p110γ-KD animals have a pronounced deficiency in lung-resident conventional DCs in particular the CD103+ subset ( Fig 6A , 6C and 6D ) , while moDCs were present at comparable levels to WT mice in the steady state ( Fig 6A and 6D ) . During IAV infection this picture changed dramatically . moDCs were recruited to the lung in high numbers regardless of p110γ-kinase deficiency ( Fig 6B ) . Conversely , lung CD103+ DCs decreased strongly in number in both WT and p11t0γ-KD animals until day 7 p . i . , where the numbers start to increase a later time-points of infection ( Fig 6C ) . For CD11b+ DCs the situation mirrors the kinetics of the CD103+ DCs , although by day 7 p110γ-KD CD11b+ DCs manage to reach numbers similar to WT ( Fig 6D ) . p110γ–deficient CD103+ DCs were always present in lower numbers than WT , although the difference to WT mice became somewhat smaller at the peak of IAV infection ( Fig 6C ) . Overall , these results suggested that the conventional lung-resident CD103+ DCs in p110γ-KD mice are severely deficient at the beginning of an IAV infection and remain so for most of its course , although also in the p110γ–deficient situation the number of CD103+ DCs began to increase again at day 7 p . i . IAV To address the question whether potentially the inflammatory environment can partially overcome the pronounced developmental deficiency of lung-resident DCs in the steady-state in p110γ–deficient animals , we instilled either LPS or PolyI:C intratracheally as a single inflammatory stimulus into the lung and examined how the DC compartment changed over time . Intratracheal injection of a single inflammatory stimulus did not lead to an increase in lung CD103+ DCs in p110γ-KD mice ( Fig 6F and 6G ) . By contrast activation of DCs by these mediators induced a reduction in the cell number , possibly due to their migration do the dLN . Thus the lack of functional p110γ cannot be compensated for by a single inflammatory signal . To address the underlying mechanism of how the absence of p110γ kinase activity in DCs would impact on DC mediated immune responses in vivo we administered Cy-5 labeled ovalbumin intratracheally and then observed the migration of DCs to the dLN . After 24 hours cells carrying OVA-Cy5 in the dLN were predominantly DCs in both WT as well as p110γ-KD mice ( Fig 7A ) . However , the proportion of CD103+ DCs carrying OVA in the dLN was considerably reduced in p110γ-KD compared to wild-type mice ( Fig 7B ) . Conversely , the frequency of CD11b+ DCs was somewhat higher in p110γ-KD mice , suggesting a compensatory effect in a situation where pulmonary CD103+ DCs are virtually absent ( Fig 7C ) . To gain a better understanding of the consequences of the strong reduction in pulmonary CD103+ DCs , we sought to evaluate CD103+ DC-specific functions . Lung CD103+ DCs were recently shown to be essential for phagocytosis of apoptotic cells and cross-presentation of antigens in the dLN [33] . To address the efferocytic capacity of pulmonary DCs in p110γ-KD mice we instilled labeled apoptotic thymocytes into the trachea and examined migration of DCs from the lung to the dLN 24h p . i . As previously described , apoptotic cells were almost exclusively transported by CD103+ DCs to the dLN , as opposed to other DC subsets ( Fig 7D ) . Furthermore , while in WT mice a significant amount of CD103+ DCs in the dLN could be found carrying apoptotic thymocytes , these cells were almost completely absent in the dLN of p110γ-KD mice ( Fig 7E ) . Overall , these results suggest that in p110γ-KD animals transport of apoptotic-cell related antigens by cross-presenting DCs is severely deficient . To further investigate the notion that a deficiency in cross-presenting lung CD103+ DCs in p110γ-KD animals is responsible for their enhanced susceptibility to IAV infection , we generated BM chimeras using a mixture of either WT or p110γ-KD with Batf3-/- BM at a ratio of 1:4 . Batf3 is a transcription factor strictly required for development of lung CD103+ DCs [34] . This led to a situation where at least 80% of T cells were WT , however 100% of lung CD103+ DCs were of either WT or p110γ-KD background . This set up allowed us to address the role of p110γ in lung CD103+ DCs specifically in the context of an IAV infection . Upon Infection animals that had p110γ-deficient CD103+ DCs exhibited more pronounced loss of weight and temperature ( Fig 8A and 8B ) compared to animals with WT CD103+ DCs . Furthermore , analyzing the T cell response at day 7 p . i . showed that CD4+ , CD8+ and virus-specific CD8+ T cells were significantly reduced in the lungs of infected animals ( Fig 8C ) . In lung dLN virus-specific CD8+ T cells were also reduced ( Fig 8D ) . Examining the activation state of T cells in the lung it was evident that the frequency of CD44+CD62L- of CD4+ T cells was reduced in animals , which had received p110γ-KD BM ( Fig 8E ) . However this was not the case for CD8+ T cells ( Fig 8F ) . Overall these results suggested that p110γ in lung CD103+ DCs is required for potent antiviral T cell responses against influenza virus .
In this study we describe PI3Kγ as novel factor , which plays a key role in successful host defense against respiratory infection with influenza virus . We show that the highly increased susceptibility of p110γ-kinase dead animals stems from a defective T cell response leading to higher viral titers and more pronounced morbidity . This phenotype is due to a deficiency in the lung-resident DCs , which are essential for the initial priming of the adaptive immune response against influenza [31] . We could show that PI3Kγ is functionally not required in DCs to activate T cells in vitro and in vivo , however , the pronounced reduction of lung DCs in naïve PI3Kγ-deficient animals leads to an impaired transport of antigen to the draining lymph node and thus to a defective antiviral T cell response and consequently a delayed viral clearance . To date the role of PI3Ks in IAV pathogenesis has mainly been analysed from the perspective of the virus , where host PI3Ks were shown to be important for viral replication , in particular of the highly pathogenic PR8 strain [30] . To our knowledge this is the first study reporting an important role of a PI3K family member for the response of the host rather than for viral pathogenesis itself . The well established pro-viral of PI3Ks in general and the novel anti-viral role of PI3Kγ described in this manuscript appear at first conflicting , however , in fact they probably just represent temporally different roles of PI3Ks in the interaction between the virus and its host . PI3Ks have been reported to be important early on during massive viral replication in lung epithelial cells [30] and our results using global inhibition of PI3K signaling support this notion . Furthermore , in this context it appears that PI3Kγ only plays a minor role , which is likely due to its very low expression in lung epithelial cells . The important antiviral role of PI3Kγ only starts to be visible once the PI3Kγ-dependent-DC-mediated T cell response commences with clearance of the virus from the lung . The fact that p110γ-KD mice do not exhibit protection from virus replication earlier on during the infection could reflect that the virus more strongly relies on other PI3K subunits for replication in vivo . The virus could also exploit redundancies between the PI3K subunits in epithelial cells , allowing it to continue replicating , despite the functional lack of PI3Kγ . How other PI3K subunits regulate both viral pathogenesis as well as the host immune response , remains to be addressed in future endeavors . Interestingly , in p110γ-KD animals both CD4+ and CD8+ T cell responses after IAV infection are impaired at an early time-point . The strong defect in CD8+ virus-specific T cells likely stems from the pronounced deficiency in cross-presenting CD103+ DCs in naïve animals , which is similar to that observed in BATF3-/- animals [35] . The minor effect on CD4+ T cells can be explained by the moderate reduction in CD11b+ conventional DCs in the lungs of p110γ-KD animals . This subset has been associated with activating mainly CD4+ T cells [36] , although recently it was shown that these cells too can stimulate CD8+ T cells in the context of influenza infection [37] . The deficiency in CD11b+ DCs however , is then overcome by the massive influx of monocyte-derived DCs as well as a novel CD11b+ DCs that likely rescue the CD4+ T cell response at later time-points during the course of the infection . The virus-specific CD8+ T cell response in the lungs of p110γ-KD animals is still strongly deficient by day 10 post infection , underlining the idea that CD103+ DCs are indeed the key players in generating a potent cytotoxic T cell response [31] , despite the described capacity of CD11b+ DCs to be able to as well [37] . The deficiency in transport of apoptotic cells observable in p110γ-KD mice fits very well into this picture , as it was recently shown that this capacity was indeed crucial for the ability of CD103+ DCs to cross-present antigen derived from dying cells to CD8+ T cells [37] . Nonetheless , when one examines the frequency of CD8+ T cells which have proliferated in the lungs of infected animals the moderate reduction in EdU+ cells in p110γ-KD animals likely does not completely explain the massive defect in the virus-specific CD8+ T cell compartment . Most probably , lung-resident DCs are not only required for initial priming of the CD8+ T cell response in dLN but also for maintenance of CD8+ effector T cells once they are have reached the pulmonary compartment . This notion has been previously suggested for monocyte-derived DCs [38] and is likely also true for conventional lung-resident DCs . Furthermore , CD4+ T cells proliferate more in p110γ-KD than WT mice at late time-points of infection , which can be likely explained by the continuous presence of virus in the p110γ deficient situation . WT animals have cleared the virus by this stage of the infection and thus antigen is no longer present to stimulate T cell proliferation . From a developmental point of view the results presented in this paper also bear some novel insights into how DC development changes from steady-state to inflammation . The pronounced developmental organ-specific deficiency of lung-resident CD103+ DCs in the lungs of p110γ-KD animals , which we recently described [23] , is partially overcome by the prolonged inflammation during a respiratory viral infection . The numbers of CD103+ DCs decrease strongly until day 4 p . i . but then start to recover at later time-points , also in the p110γ- deficient situation . These results suggest the potential presence of an additional developmental pathway overcoming the requirement for Flt3 signaling in lung CD103+ DC development during inflammation . Possibly this mechanism is also regulated by IL-12 and IFNγ , which has been shown to overcome deficiency of BATF3 in chronic infection with Mycobacterium tuberculosis allowing development of lung CD103+ DCs [39] . Unsurprisingly , we could show that for the rerouting of DC development during pulmonary infection , one inflammatory stimulus alone such as LPS or poly I:C in the lung is not sufficient . These observations are also reflected in GM-CSF deficient animals where CD103+ DCs are moderately reduced in adults but this deficiency is then also completely rescued during inflammation [35] . Despite the pre-existing deficiency in lung DCs in p110γ-KD animals , we could also demonstrate that migration of DCs lacking a functional kinase domain of in p110γ is completely normal in vitro and in vivo , thus showing that p110γ is dispensable for chemokine receptor mediated migration to the dLN . This corrects a notion , which has been suggested for DCs by older publications [40] . Overall our results define PI3Kγ as a new key host factor in the defense against influenza virus infection , establishing a more complex picture about the role of PI3Ks in respiratory viral infection and the interplay between the host and its pathogen .
C57BL/6J mice were either bred in-house or purchased from Charles River ( Germany ) . p110γ-KD mice were generated and provided by E . Hirsch , University of Torino and were back-crossed to C57BL/6 for at least 15 generations . BATF3-/- mice were obtained from Jackson ( USA ) . All animals were housed in individually ventilated cages under specific pathogen free conditions at the ETH Phenomics Facility ( Zurich , Switzerland ) and used for experiments at between 6 and 14 weeks of age unless otherwise stated . The number of mice ( n ) indicated in the figure legends always refers to the number of animals per group . Mice were euthanized by an overdose of sodium pentobarbital by intraperitoneal injection . Lungs , spleens or lung draining lymph nodes were removed and then processed as described previously [23] Bronchoalveolar lavage was obtained by inserting a catheter into the trachea and subsequently flushing the lungs with PBS for a total volume of 1ml . BAL cells were then obtained by centrifugation and the supernatant was analysed for inflammatory cytokines using ELISA . Flow cytometry analysis was performed on a FACSCanto II or LSR Fortessa ( BD ) and analyzed with FlowJo software ( Tree Star ) . Fluorochrome-conjugated or biotinylated monoclonal antibodies specific to mouse CD11c ( N418 ) , CD11b ( M1/70 ) , Ly-6C ( HK1 . 4 ) , Siglec-F ( E50-2440 , BD Biosciences ) , CD103 ( 2E7 ) , CD45 ( 30-F11 ) , CD45 . 1 ( A20 ) , CD45 . 2 ( 104 ) , CD4 ( GK1 . 5 ) , CD8α ( 53–6 . 7 ) , MHC class II ( M5/114 . 15 . 2 , eBioscience ) , CD64 ( X54-5/7 . 1 ) , CD19 ( 6D5 ) , CD3e ( 145-2C11 ) , NK1 . 1 ( PK136 , eBioscience ) , Ly-6G ( 1A8 ) , podoplanin ( 8 . 1 . 1 . , eBioscience ) , Ly-6C ( HK1 . 4 ) , CD49b ( DX5 ) , TNF-α ( eBioscience ) IFN-γ ( eBioscience ) and GM-CSF ( BD ) were purchased from Biolegend unless otherwise stated . Dead cells were gated out using the live/dead marker eFluor780 ( eBioscience ) before analysis . PE-conjugated peptide-MHC class I tetramers ( H- 2Db/NP34 ) with the NP34 peptide ( NP366-374; ASNENMETM ) from the nucleoprotein of influenza virus A/PR/8/34 were generated as described [41] . Prior to all flow cytometry stainings , FcγIII/II receptors were blocked by incubating cells with homemade anti-CD16/32 ( 2 . 4G2 ) . For bone marrow chimeras , C57BL/6 CD45 . 2+ mice were lethally irradiated ( 9 . 5 Gy , using a caesium source ) and reconstituted with 5-10x106 BM cells of the background and with the ratio indicated for each experiment . Mice were used for virus experiments weeks post-reconstitution . Influenza virus strain PR8 ( A/Puerto Rico/34 , H1N1 ) was originally provided by J . Pavlovic , University Zurich . At the age of 6 to 14 weeks , mice were infected intratracheally with varying doses of influenza virus , depending on the experiment . The mice were anaesthetized and inoculated with 50μL virus in endotoxin-free PBS . Temperature and weight of animals was monitored daily and animals were euthanized if they fulfilled severity criteria set out by institutional and cantonal guidelines . To determine influenza viral titers in the lungs , samples were collected on various days after infection , homogenized ( Polytron PT 1300 D ) , and serially diluted with MDCK cells as described [42] . The day before mice were sacrificed for analysis , 1 . 5x105 bone marrow-derived dendritic cells ( BMDC ) [43] were incubated overnight with 1 . 6x105 pfu UV-inactivated virus ( PR8 ) in 96-well plates . 12h later , these BMDC were pulsed with 1μg/mL NP34 peptide for 2h before BAL cells from individual mice were added . After 2h of incubation at 37°C , Monensin ( 2μM , Sigma-Aldrich ) was added to retain cytokines in the cytoplasm , and cells were again incubated at 37°C for another 3h . Cells were then harvested and stained for flow cytometry analysis . At the indicated time points , BAL fluid was measured for virus-specific IgA and IgG antibody isotype levels . Ninety-six well plates ( Maxisorp; Nunc ) were coated with UV-inactivated influenza virus ( PR8 ) in PBS overnight at 4°C . Plates were washed and incubated with PBS-1% BSA for 2h at RT for blocking . BAL fluids from individual mice were serially diluted in PBS-0 . 1% BSA starting with a 1:2 dilution for BAL fluids and a 1:50 dilution for sera , followed by incubation at RT for 2h . Plates were washed five times and incubated with alkaline-phosphate-labeled goat anti-mouse antibodies to IgG1 , IgG2c or IgA ( Southern Biotech Technologies , Inc . ) at a 1:1000 dilution in PBS-0 . 1% BSA at RT for 2hrs . Thereafter , plates were washed five times and substrate p-nitrophenyl phosphate ( Sigma-Aldrich ) was added . Optical densities were measured on an enzyme-linked immunosorbent assay reader ( Bucher Biotec ) at 405nm . For these experiments the Cy5 labeling kit ( Axxora ) was used with ovalbumin ( Invitrogen ) to produce OVA-Cy5 according to the manufacturer’s instructions . Mice were anesthetized using isofluorane and injected intratracheally with 40μg OVA-Cy5 and 100ng LPS ( Sigma ) and tissues were analyzed using flow cytometry one-day post injection . Bone marrow cells were harvested from the femurs of donor animals using a syringe and PBS . Cells were subsequently incubated in complete RPMI-1640 medium ( Life Technologies ) with 20ng/ml GM-CSF for 7–9 days depending on the experiment . On the day of use non-adherent cells were harvested by gently pipetting and subsequently used for further assays . DCs were seeded into Costar 5um polycarbonate membrane trans-well plates ( Corning ) and medium containing 100nM CCL21 was added to the bottom of the well . The plates were incubated for 6h at 37°C and subsequently the migrated cells were harvested and analysed using flow cytometry . BMDCs were obtained as described above . T cells were obtained with CD4 MACS-bead ( Miltenyi ) sorting from naïve splenocytes . Cells were then cultured together for 4 days in complete IMDM medium ( Life Technologies ) with the addition of varying concentrations of OVA323-339 peptide ( Mimotopes Australia ) . Before flow cytometric analysis , cells were restimulated with PMA ( Sigma-Aldrich ) ionomycin ( Sigma-Aldrich ) and monensin ( Sigma-Aldrich ) . Mice were anesthetized using isofluorane and injected intra-tracheally with either 100ng LPS ( Sigma-Aldrich ) in PBS or 50μg Poly ( I:C ) ( Invivogen ) in PBS respectively . Control mice were just injected with PBS . At the indicated time-points mice were sacrificed and tissues were analyzed using flow cytometry . Thymocytes were obtained from C57BL/6 mice and subsequently smashed through a 70um cell strainer to get a single cell suspension . Subsequently apoptosis of these cells was induced by a 120mJ exposure to UV-light . Cells were incubated at 37°C for 2h and were then labeled with efluor 670 ( eBioscience ) according to the manufacturer’s instructions . After the labeling procedure 1x 107 cells were injected intra-tracheally into recipient mice and tissues were analyzed one day later . A549 cells were treated with inhibitors for 30 min in infection medium ( DMEM with 50 mM Hepes and 0 . 2% BSA , pH 6 . 8 ) at a final concentration of 5μM . They were then infected with MOI 0 . 5 PR8-NS1-GFP virus [44] . 0 . 2μg/ml TPCK-Trypsin was added and infection was allowed to proceed for 10h . Cells were then analyzed for GFP production using flow cytometry and supernatants were collected for analysis of the virus titre , as described above . The following inhibitors were used: Wortmannin ( Sigma ) , AS605240 ( Sigma ) and IC87114 ( Sigma ) Cells were lysed in laemmli buffer containing 10% β-mercaptoethanol and boiled for 15mins at 95°C . The samples were then separated by SDS-PAGE and transferred to a nitrocellulose membrane . Thereafter the membranes were blocked in 10% milk powder in TBST . Membranes were then incubated in the primary and secondary antibodies at RT for 1h each respectively , with washing in TBST in between . After washing the Supersignal West Pico Chemiluminescent substrate was added and the membranes were imaged on Chemidoc MP imaging system ( Biorad ) . The following antibodies were used: goat anti-mouse p110γ ( Santa Cruz Biotechnology ) , bovine anti-goat HRP ( Santa Cruz Biotechnology ) , goat anti-mouse actin ( Santa Cruz Biotechnology ) . For analysis of PI3K subunit expression RNA was isolated from cells with TRIzol reagent ( Invitrogen ) and was reverse-transcribed with GoScript reverse transcriptase according to the manufacturer’s instructions ( Promega ) . Quantitative real-time RT-PCR was performed with KAPA SYBR FAST . The expression of PI3K subunits was normalized to that of Tbp . Mean values , SD , SEM , and Student’s t test ( unpaired ) and One-way ANOVA with CI 95% were calculated using Prism ( GraphPad Software , Inc ) . p < 0 . 05 ( * ) , p < 0 . 01 ( ** ) , p < 0 . 001 ( *** ) , p < 0 . 0001 ( **** ) . All animal experiments were approved by the local animal ethics committee ( Kantonales Veterinärsamt Zürich , licenses ZH270/2014 and 113/2012 ) , and performed according to local guidelines ( TschV , Zurich ) and the Swiss animal protection law ( TschG ) .
|
Acute respiratory viral infections like influenza virus can cause life-threatening disease in infected individuals . Phosphoinositide-3-kinases have been suggested to be important factors used by the virus to infect and replicate in host cells , and thereby cause viral pneumonia . However , to date the role of these signaling molecules has not been thoroughly addressed in the context of an infection in whole animals , rather than just cell culture systems . Here we show that one of the PI3K subunits , PI3Kγ , is in fact critically required for the clearance of the infection . This is because PI3Kγ regulates the immune response against the virus through the generation and maintenance of antiviral CD8+ T cell responses . We show that in the absence of PI3Kγ a specialized dendritic cell subset in the lung is deficient and this leads to a strongly impaired immune response against influenza virus . We thus identify PI3Kγ as a novel host molecule that is important for the immune defense against influenza virus infection
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
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] |
2016
|
PI3Kγ Is Critical for Dendritic Cell-Mediated CD8+ T Cell Priming and Viral Clearance during Influenza Virus Infection
|
Hospital networks , formed by patients visiting multiple hospitals , affect the spread of hospital-associated infections , resulting in differences in risks for hospitals depending on their network position . These networks are increasingly used to inform strategies to prevent and control the spread of hospital-associated pathogens . However , many studies only consider patients that are received directly from the initial hospital , without considering the effect of indirect trajectories through the network . We determine the optimal way to measure the distance between hospitals within the network , by reconstructing the English hospital network based on shared patients in 2014–2015 , and simulating the spread of a hospital-associated pathogen between hospitals , taking into consideration that each intermediate hospital conveys a delay in the further spread of the pathogen . While the risk of transferring a hospital-associated pathogen between directly neighbouring hospitals is a direct reflection of the number of shared patients , the distance between two hospitals far-away in the network is determined largely by the number of intermediate hospitals in the network . Because the network is dense , most long distance transmission chains in fact involve only few intermediate steps , spreading along the many weak links . The dense connectivity of hospital networks , together with a strong regional structure , causes hospital-associated pathogens to spread from the initial outbreak in a two-step process: first , the directly surrounding hospitals are affected through the strong connections , second all other hospitals receive introductions through the multitude of weaker links . Although the strong connections matter for local spread , weak links in the network can offer ideal routes for hospital-associated pathogens to travel further faster . This hold important implications for infection prevention and control efforts: if a local outbreak is not controlled in time , colonised patients will appear in other regions , irrespective of the distance to the initial outbreak , making import screening ever more difficult .
A growing body of literature shows the influence of contact networks on the spread of infectious diseases . This extends from individual hosts [1–5] to entire countries [6–10] or organisations , such a healthcare institutions for pathogens that are predominantly transmitted through hospital contacts [11–14] . The focus on contact networks , between various entities , reflects the importance of social structure on the successful spread of a pathogen , and the fact that the risk of contracting an infectious disease depends not only on the properties of the host or the pathogen [15] . Each contact between an entity may potentially serve as a transmission route , aiding pathogen spread . Traditionally , individuals in a person-based network were considered to be connected simply when there was any contact between them over the study period , resulting in an unweighted ( binary ) network; the characteristics of a contact , such as its length or intensity [16–18] , were not taken into account . However , advances in techniques to determine the level of contact between individuals , such as wearable sensors that measure face-to-face contact [4 , 19–25] , have resulted in more weighted networks becoming available . Like these recent person-to-person networks , hospital networks comprise weighted connections between institutions , as the number of shared patients ( sometimes compensated for their length of stay ) can be used to estimate the extent of contact . However , some differences between these networks should be considered . Person-to-person contact is almost by definition symmetrical: if person A was in close contact with person B , the reverse should also be true [22 , 23] . In contrast , hospital networks are nearly always asymmetrical , because the number of patients moving from hospital A to B is not necessarily the same as the number of patients moving from B to A . Furthermore , the geographical position of the hospital nodes is fixed and usually known , making it straightforward to compare network and geographical distances . Previous studies have shown how infection prevention and control ( IPC ) efforts against hospital-associated pathogens in general , and bacteria showing antimicrobial resistance ( AMR ) in particular , should be coordinated according to the structure of the hospital network [26] . Correct estimates of the network distance between hospitals are therefore important for risk assessments during outbreaks of hospital-associated pathogens . However , this is complicated by the properties of these hospital networks . While the distance between two individuals in an unweighted network is simply the minimum number of steps needed to reach one from the other [27] , the distance through a dense weighted network is less straightforward , because of the interplay between the number of intermediate steps and the weight of the contact [28] . A direct weak link may , for instance , constitute a longer distance than a route through many intermediate nodes , but with stronger links . We here estimate the distances between hospitals in the English hospital network , while considering the spread of AMR as an example . AMR poses a significant risk to hospitalised patients , and effective IPC is paramount [29] . We simulate the spread of hospital-associated antibiotic-resistant pathogens with various transmission characteristics , to determine the optimal balance between the number of intermediate steps and the weight of the connections to measure the connectivity between hospitals in England . We compare the network distance to geographical distances to determine the potential differences between them . The resulting distance metric can inform IPC strategies after observed outbreaks .
We used data from the English National Health Service ( NHS ) Hospital Episode Statistics ( HES ) for financial years 2013/14 and 2014/15 to construct a network of hospital organisations ( trusts ) in England based on patient movements . The NHS-HES includes all inpatient admissions to hospital trusts in England , based on provider episodes . We included all acute care hospital trusts , including specialist hospitals ( such as oncological ) , and joined all consultant episodes per admission ( called a ‘spell’ in NHS-HES , the time between admission and discharge in the same hospital ) . We sorted the spells per patient by admission date , and for each admission during 2014/15 determined if the previous discharge ( if any ) happened within the 365 days prior to the admission . For each pair of discharge and subsequent admission event we tallied the two hospitals involved in this single patient movement ( mij ) . Information from all spells during 2014/15 taken together thus form a contact matrix , Mij , of patient movements between hospitals i and j . This contact matrix therefore included both direct ( i . e . patient transfers between hospitals ) and indirect ( i . e . with a stay at home in between ) patient movements . Each node in the network represented a single hospital trust and the edges represented the number of patients who moved from one hospital trust to the other . The network was weighted ( number of patients moving ) and directional ( in any pair of hospital trusts , the numbers of patients moving from and to each hospital could differ ) . Based on the community structure of the measured hospital network , we divided hospitals into referral regions . The community assignment with maximum modularity was determined using a standard community structure detection algorithm [30] . We simulated the spread of a hospital-associated pathogen through the hospital network using a stochastic , discrete-time model with time steps ( ΔT ) of 7 day , much in the same way as used by Ciccolini et al . [31] and Van Bunnik et al [32] . In the simplest version of the model , we assume that each hospital is either “susceptible” ( pathogen not present ) or “infected” ( pathogen present ) , and infected hospitals have a fixed prevalence , which determines the proportion of discharged patients that are colonised with a hospital-associated pathogen . The probability of a hospital getting infected depends on the state of the other hospitals , the assumed prevalence , and the patient movements between them . The probability of getting infected in the initial model can be described as: P i S → I = 1 - ∏ j : H i ∈ I N H ( 1 - γ ) C I M i j Δ T ( 1 ) where Hj denotes each hospital , whose introductions to hospital j are summed if they are part of the set of infected hospitals , I . γ is the between-hospital transmission parameter equal , to the probability that an imported colonised individual successfully spreads the pathogen . CI is the attained prevalence after infection of the hospital . Mij denotes the patient movements between hospitals i and j , as measured in the NHS-HES data . We chose parameter values such that about half of the hospitals were affected after one year , using an equilibrium prevalence ( CI ) of 20% . Table 1 lists the parameters used for the main analysis , as well the parameters values used to test the sensitivity of parameter choices . Because the assumed binary infection state of the hospitals is a fairly crude approximation of the within-hospital dynamics of hospital-associated pathogens ( since the infectivity of a hospital is likely to increase after the initial successful introduction ) , we expanded the model to include the increasing prevalence within the hospital . We modelled this using a standard SIS model; with transmission rate β and removal rate δ , the infected class is described as d I d t = β S I - δ I . ( 2 ) As the susceptible class is simply the rest of the total population , using S = 1 − I , the within-hospital epidemic curve can be derived as follows: I ( t ) = I ( ∞ ) ( 1 - ( 1 - I ( ∞ ) I ( 0 ) ) ) e - ( β - δ ) t ( 3 ) where I ( ∞ ) denotes the equilibrium prevalence given by I ( ∞ ) = 1 - 1 R 0 , with R 0 = β δ , and I ( 0 ) the starting prevalence at introduction , set at 0 . 001 ( i . e . one patient among a thousand admitted ) . This function can then be used to calculate the within-hospital prevalence at each time point , P i S → I ( 0 ) ( t ) = 1 - ∏ j : H i ∈ I N H ( 1 - γ ) I ( t ) i M i j Δ T ( 4 ) The parameter values were chosen again such that approximately half the hospitals became affected after one year , together with a within-hospital R0 = 1 . 25 , reflecting R0 values often used for studying the spread of AMR in hospital settings [33] , resulting in an equilibrium prevalence of 20% . We used each hospital in turn as the initial starting point of the outbreak , and repeated the simulation 100 times per starting hospital , resulting in 15200 iterations of the model , which delivers stable estimates ( see S1 Fig ) . We tracked the time of infection per hospital , and estimated the mean time to infection for each pair of starting and receiving hospitals . In order to determine the shortest paths between hospitals , we applied a metric proposed by Opsahl et al [28] , that allows weighting between the number of steps and edge weight ( Fig 1 ) . The shortest path through the weighted network , dwα between node i and j is d w α = min ( 1 M i h α + ⋯ + 1 M h j α ) ( 5 ) If α is set to 0 , the shortest path is completely determined by the number of steps , as each existing connection has value 1 , irrespective of the weight of the connection . Conversely , if α is set to 1 , the shortest path is fully determined by the weight of the connections ( i . e . the number of shared patients ) . Technically , α can be set to above one , where the shortest path is skewed towards the strongest links , but we considered only values within the range of 0 to 1 . To determine the risk posed by an outbreak in any of the other hospitals , we calculated the centrality of each hospital . We considered three centrality metrics: degree , closeness , and betweenness centrality . Closeness centrality is given by the inverse of the distances to all hospitals , as defined by the shortest paths C C w α ( i ) = ∑ j N H 1 d w α ( i , j ) ( 6 ) while the betweenness centrality is defined as the proportion of all of these shortest paths that pass through node i , C B w α ( i ) = g j k w α ( i ) g j k w α ( 7 ) , where g j k w α ( i ) is the number of shortest paths passing through hospital i , and g j k w α is the total number of shortest paths between all hospitals ( excluding hospital i: ( N − 1 ) ( N − 2 ) ) . We used the degree centrality defined as the sum over all connection weights , adjusted for the importance of steps by α C D - i n w α ( i ) = ∑ j N H M j i α ( 8 ) To determine the optimal balance between the number of intermediate hospitals and the strength of the connections to accurately reflect the spread of infectious pathogens through the hospital network , we calculated the shortest paths and centralities for various values of α and determined their correlation with the simulation results . We used Kendall’s tau ( τ ) to measure the correlation between the mean simulated time to infection and the distances and centrality metrics , as it gives the most intuitive interpretation compared to other measures of rank correlation ( τ gives the proportion of randomly chosen pairs that are concordant ) [34] . To investigate the influence of weak links ( i . e . connections between hospitals exchanging few patients ) that could theoretically be removed most easily , we repeated the analysis with altered hospital networks . For each alteration , we removed the links with a weight under a certain threshold , calculated the shortest paths between hospitals for various values of α , and repeated the simulations for the scenario using a binary hospital infection state ( Eq 1 ) and the scenario using the SIS within-hospital model ( Eqs 2 and 4 ) .
The hospital network consisted of 152 acute hospital trusts ( Fig 2A ) that admitted 4 , 091 , 977 patients with a total of 16 , 643 , 460 admissions in financial year 2014/2015 . On average each hospital exchanged 7 , 481 ( IQR: 4583–9244 ) patients with 124 ( IQR: 114–137 ) other hospitals , resulting in 18 , 847 unique hospital to hospital connections made by individual patients , comprising 82% of all possible links . The weight of the connections was highly skewed ( Fig 2B ) , with 90% of the connections based on movements of 50 patients or fewer , comprising 10% of the total number of patient exchanges . Although highly connected hospitals ( i . e . those sharing patients with many other hospitals ) shared more patients ( Fig 2C ) , the mean number of patients exchanged per connection was not higher for the well-connected hospitals . The simulated spread of a pathogen through the hospital network showed a fast increase in the proportion of hospitals within the referral region of the initially affected hospital where the pathogen was present , followed by the other regions after some time ( Fig 3 ) . The order in which the pathogen reached hospitals in other regions depended on the position ( region ) of the initially infected hospital . For some , the hospitals in one of the neighbouring regions were clearly the next ones to be affected ( e . g . Liverpool after Manchester and vice versa ) , while for others ( e . g . Newcastle ) there was no discernible order of regions , and they were all affected at more or less the same time . The difference in time for the pathogen to reach the other hospitals in the starting region and hospitals in the other regions was shortest when London hospitals were the first affected . When including a delay between the time a hospital became infected and the time it starts to spread the pathogen to its neighbouring hospitals , the difference in time to infection between regions became slightly bigger . In particular , the time from the initial region becoming infected to the other regions becoming infected increased , indicating that spread within the regions depended mostly on the direct connections with the initial hospital , while other regions were more often reached by indirect routes . The shortest path distance based purely on the connections weights ( α = 1 ) correlated with the simulated time it took the infection to spread between any two hospitals ( Fig 4A ) . However , incorporating the number of intermediate hospitals in the distance metric , by scaling factor α , increased the association with time to infection ( Fig 4A ) . This association was maximal around α = 0 . 25 , in particular for simulations with higher within-hospital R0 values . The shortest route between two hospitals therefore tended to have fewer steps ( intermediate hospitals ) instead of more exchanged patients . This implies that the route of transmission between two hospitals does not necessarily follow the connections between hospitals sharing many patients . The optimal value for α did not depend on the parameter choices in our model ( S2–S4 Figs ) , and although some parameter combinations did show weaker or stronger correlations , the position of the maximal correlation only slightly altered . If we consider the mean time to infection from all other hospitals , which translates to the general risk these hospitals pose to the focal hospital , this correlated well with closeness centrality ( Fig 4B ) at values of α around 0 . 2–0 . 3 , again preferentially incorporating the direct weak connections over indirect strong routes . The –local– degree centrality ( Fig 4C ) shows similar results , with a peak around α = 0 . 4 − 0 . 5; although marginally weaker , the peak correlation for simulations including within-hospital delays was still over Kendall τ = 0 . 7 . This indicates that a hospital’s direct connections to its neighbouring hospitals are a good enough predictor of its risk of admitting patients colonised with antibiotic-resistant bacteria from anywhere in the country . However , betweenness centrality ( Fig 4D ) showed far weaker correlation with the mean time to infection . This is probably a result of the dense structure of the hospital network: there exist many alternative routes next to the shortest paths through the hospitals with high betweenness that are just marginally longer and therefore – epidemiologically speaking – just as important . None of the tested centrality metrics showed clear geographical correlation ( Fig 5A , 5B & 5C ) , while hospitals with high degree or closeness centrality were the same ( mainly the large tertiary care ) institutions ( Fig 5D & 5E ) . Using α = 0 . 25 , we observed that for hospitals in close proximity , network and geographical distance were strongly related , with hospitals sharing many patients also being geographically close to one another . However , hospitals that were geographically further away than about 200km all showed more or less the same network distance ( Fig 6A ) . The network distance to these distant hospitals depended fairly strongly on the initial hospital they were measured from ( Fig 6B , 6C and 6D ) . As all network metrics favoured the number of connections or steps instead of the weight of the connections , to test their influence we removed the all weak links below thresholds of 10 , 50 , 100 , 200 , or 300 patients from the network . Using the hospital in Oxford as the initial outbreak hospital , the time to infection of other regions increased as the threshold used to remove links increased ( Fig 7A & 7C ) , in particular for a subset of regions , where hospitals became completely disconnected from the rest of the network after removing all connections with fewer than 308 exchanged patients . Removing weak links had no effect on the dynamics within the initial region , in contrast to the spread between regions . Furthermore , the optimal estimate of α increased as the threshold increased , focussing the metric more on connection weight than on number of steps ( Fig 7B & 7D ) .
We considered three exemplar network metrics , capturing different structures , while more are available . It would also possible to evaluate a larger number of metrics in a meta-modelling approach to determine the full effect of the network structure on the risk of a hospital receiving hospital-associated pathogens; in particular , as effects that are not captured by one network metric may be captured by another . This could result in a combined metric , with different metrics adding to the hospital’s risk , which we deem beyond the scope of this single paper . It should be noted that the epidemiological distance used in our simulation is purely based on the simulations using the observed hospital network . The simulation model is an approximation , and results may be influenced by specific assumptions . For example , we assume that all patients within a single hospital trust have an equal opportunity to transmit the pathogen to each other . However , the physical and organisational structure of a hospital affects these probabilities , resulting in preferential transmission between patients admitted to the same ward , often with similar conditions . Apart from clustering in the network , medical conditions may also alter the susceptibility of patients , causing different rates and different connectivity depending on the particular condition . Differences in rates may be exacerbated in wards with larger proportions of frail patients , resulting higher mortality . This could imply that the condition- or ward-specific sub-networks might even have to be weighted differently depending on the question at hand . Sub-networks may also exist on the level of the institutions , as the network is based on hospital organisations that could comprise multiple hospital sites . However , these hospital sites within one organisation can be expected to share even more patients than hospital organisations within a single region , thus creating an extra layer within the network . Although these assumptions may alter the absolute distance between hospitals in the network , the relative difference is unlikely to change dramatically as we can expect the within-trust networks to be much more tightly connected , thus forming a single entity , compared to the between-trust network . The network distance between hospitals is not merely a reflection of the number of shared patients between them , but also the number of intermediate hospitals that need to be passed en-route from one to the other . Because the network of hospitals consists of many weak links , formed by hospitals sharing very few patients , the distance between far away hospitals may therefore easily be underestimated . This particularly applies when a pathogen has spread to other hospitals in the initial region and many more weak links become available for its onward spread to different regions . Our observations hold important implications for IPC efforts: if a local outbreak is not controlled in time , colonised patients will appear in other regions from unexpected hospitals , making import screening ever more difficult .
|
Shared patients can spread hospital-associated pathogens between hospitals , together forming a large network in which all hospitals are connected . We set out to measure the distance between hospitals in such a network , best reflecting the risk of a hospital-associated pathogen spreading from one to the other . The central problem is that this risk may not be a directly reflected by the weight of the direct connections between hospitals , because the pathogen could arrive through a longer indirect route , first causing a problem in an intermediate hospital . We determined the optimal balance between connection weights and path length , by testing different weighting factors between them against simulated spread of a pathogen . We found that while strong connections are important risk factor for a hospital’s direct neighbours , weak connections offer ideal indirect routes for hospital-associated pathogens to travel further faster . These routes should not be underestimated when designing control strategies .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"methods",
"Results",
"Discussion"
] |
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] |
2017
|
Measuring distance through dense weighted networks: The case of hospital-associated pathogens
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Kenya has experienced outbreaks of chikungunya in the past years with the most recent outbreak occurring in Mandera in the northern region in May 2016 and in Mombasa in the coastal region from November 2017 to February 2018 . Despite the outbreaks in Kenya , studies on vector competence have only been conducted on Aedes aegypti . However , the role played by other mosquito species in transmission and maintenance of the virus in endemic areas remains unclear . This study sought to determine the possible role of rural Aedes bromeliae and Aedes vittatus in the transmission of chikungunya virus , focusing on Kilifi and West Pokot regions of Kenya . Four day old female mosquitoes were orally fed on chikungunya virus-infected blood at a dilution of 1:1 of the viral isolate and blood ( 106 . 4 plaque-forming units [PFU]/ml ) using artificial membrane feeder ( Hemotek system ) for 45 minutes . The engorged mosquitoes were picked and incubated at 29–30°C ambient temperature and 70–80% humidity in the insectary . At days 5 , 7 and 10 post-infection , the mosquitoes were carefully dissected to separate the legs and wings from the body and their proboscis individually inserted in the capillary tube containing minimum essential media ( MEM ) to collect salivary expectorate . The resultant homogenates and the salivary expectorates were tested by plaque assay to determine virus infection , dissemination and transmission potential of the mosquitoes . A total of 515 female mosquitoes ( 311 Ae . bromeliae and 204 Ae . vittatus ) were exposed to the East/Central/South Africa ( ECSA ) lineage of chikungunya virus . Aedes vittatus showed high susceptibility to the virus ranging between 75–90% and moderate dissemination and transmission rates ranging from 35–50% . Aedes bromeliae had moderate susceptibility ranging between 26–40% with moderate dissemination and transmission rates ranging from 27–55% . This study demonstrates that both Ae . vittatus and Ae . bromeliae populations from West Pokot and Kilifi counties in Kenya are competent vectors of chikungunya virus . Based on these results , the two areas are at risk of virus transmission in the event of an outbreak . This study underscores the need to institute vector competence studies for populations of potential vector species as a means of evaluating risk of transmission of the emerging and re-emerging arboviruses in diverse regions of Kenya .
Chikungunya virus ( CHIKV ) is vector-borne virus of genus Alphavirus and family Togaviridae that is principally transmitted from human to humans by Ae . aegypti and Ae . albopictus . The first CHIKV outbreak was documented in Makonde village in Tanzania in 1956 [1 , 2] and since then , various outbreaks have been experienced in more than 60 countries in Africa , Asia , Europe and America [3 , 4] . In Africa high infection was reported in union of Comoros island in the2004- 2005 outbreak [5] , Congo in the 1998–2000 outbreaks [6] and Mauritius and Madagascar in 2005 and 2006 respectively [7] . CHIKV is re-emerging in Kenya , after the 2004–2005 outbreaks in Lamu Island . It has caused several outbreaks the northeastern and coastal Kenya from May 2016 and late 2017to early 2018 respectively [8] . In addition , previous studies have reported high seroprevalence rates ( 59% ) of CHIKV infection in Busia District and 24% in Malindi Kenya [9] . Chikungunya virus strains are classified into three distinct genotypes; Asian , West African , and East/Central/South African ( ECSA ) . This virus causes chikungunya fever , an acute febrile illness characterized by severe arthralgia , fever , skin rash , and arthritis-like pain in small peripheral joints that lasts for weeks or months , joint swelling and conjunctivitis [10–12] . Both Ae . aegypti and Ae . albopictus have been implicated in the CHIKV transmission cycle in the African region and other parts of the world , based on vector competence studies [13 , 14] and virus isolation from infected field collected mosquitoes [15–17] . International travels and global expansion leading to the spread of the two main CHIKV urban mosquito vectors , Ae . aegypti and Ae . albopictus , have enhanced the ability of the virus to spread to new regions where environmental conditions are permissive for viral transmission [18–20] . Extrinsic incubation period ( EIP ) in mosquitoes infected with CHIKV ranges from 2 to 9 days , with an average of 3 days in the tropics such as East Africa [21] Aedes simpsoni consists of a complex of mosquito species including vectors of important arbovirus diseases such as yellow fever . In Kenya , Ae . bromeliae is the dominant species of the Ae . simpsoni complex found in the peridomestic areas , Ae simponi simpsoni has never been documented in the country [22] . Studies involving the ecology and vector competence of Ae . vittatus and Ae . bromeliae on chikungunya have been conducted in Senegal [23] , and on dengue , and yellow fever virus in Kenya [24] . In Rabai , Kenya , Ae . bromeliae breeds in the domestic and peridomestic areas while Ae . lilii breeds in the forest [25 , 26] . Aedes bromeliae preferably feed on human hosts for their blood meal , maintaining the virus in the rural cycle [24] and breed not only on water reservoirs held by plants , including trees holes and plant leaf axils [27 , 28] , but also in artificial water containers [29 , 30] . Aedes vittatus is a savannah species that is abundant in rocky areas , prevalent in African forest galleries and is also common in villages near forests . Female Ae . vittatus have daily and nocturnal activities with a significant crepuscular peak [23 , 31] . They bite a wide range of vertebrate hosts , with a strong anthropophilic trend in specific locations [32] , and breed mostly on natural habitats mainly in rock pools/holes and tree holes during the rain seasons . In absence of these breeding sites the vector breeds in domestic areas especially in household water-holding containers [23] . The vector has a high susceptibility to infection and dissemination , and most importantly is able to transmit the West Africa lineage of CHIKV [23 , 33] . Aedes vittatus and Ae . bromeliae have the potential to expand their distribution and abundance due to their ability to adapt to human dwellings using available breeding habitats , such as domestic containers , in absence of their preferred breeding sites [26 , 33 , 34] . Determination of the vector competence of mosquito populations is a key parameter in evaluating the risk of CHIKV transmission and spread in Kenya . Despite several outbreaks of CHIKV in Kenya , focus is usually on Ae . aegypti and no vector competence studies have been conducted to determine the role played by other mosquito species in its transmission and maintenance . We evaluated the competence of Ae . bromeliae populations from Rabai sub-county in Kilifi County and Ae . vittatus populations from Kacheliba sub-county in West Pokot County of Kenya as an important factor in assessing the risk of transmission of ECSA lineage of CHIKV in these regions . This would provide the necessary baseline data to inform the public health sector on best vector control practice , and effective preventive and control interventions in case of increased risk of virus transmission .
This study was conducted in Rabai sub-county , Kilifi County in the coastal region of Kenya and Kacheliba sub-county in West Pokot County ( Fig 1 ) . Kilifi County ( latitude 3 . 63°S , longitude 39 . 85°E ) has a mean annual temperature of 30°C , relative humidity of 82% and receives approximately 88 . 25 mm of rainfall annually . The county has a bimodal pattern of rainfall with the long rains occurring between April and July , with the highest rainfall occurring in the month of May and short rains in November and December . In Kilifi , the rainfall patterns towards the hinterlands are unreliable due to the influence of the Indian Ocean . The main topographical features include the coastal plains , island plains and Dodori River Plain . The presence of forest areas around the town inhabited by primates and other wildlife species poses a risk of zoonotic disease transmission . Minimum temperatures are always above 20°C , the maximum temperatures reach 30°C to 34°C . The natural vegetation consists of coconut trees , banana plantations and a variety of agricultural crops . Characteristic soil types consist of sandy soil with patches of high loam soil . West Pokot County lies between latitudes 1 . 13°N to 2 . 70°N and longitudes 34 . 77°E to 35 . 79°E in the Rift Valley region of Kenya , bordering the Republic of Uganda to the west , Trans-Nzoia County to the south , Elgeyo-Marakwet and Baringo Countiesto the southeast and Turkana County to the north and northeast . It covers an area of 9 , 169 . 39 km2 . West Pokot County has a bimodal rainfall pattern . The long rain season occurs between May and June with mean daily temperature of 32°C , rainfall of approximately 60 . 25 mm and 82% relative humidity . Aedes bromeliae eggs , larvae and pupae were collected from peridomestic areas in four villages in Rabai sub-county: Mbarakani , Bengo , Changombe and Kibarani ( Fig 1 ) . The eggs were collected using ovitraps that consisted of black ovicups lined with oviposition paper and half-filled with water . After obtaining consent from the home/residence owner to sample in their private land , the ovitraps were placed in the peridomestic areas for four days to allow the mosquitoes to lay eggs . Larvae and pupae were collected from natural habitats , mainly rock pools/holes and tree holes , plant axils , especially bananas , and flower axils using larval sampling tools . Aedes vittatus larvae and pupae were collected from rock pools/holes and tree holes in peridomestic and forest areas of Kacheliba sub-county . Field collected eggs were briefly dried on a damp cotton wool to induce diapause , and transported to a level 2 ( BSL2 ) insectary at Kenya Medical Research Institute ( KEMRI ) for colonization . To avoid an oviposition from a single female mosquito , several larval collections from the same area were mixed . All collected larvae and pupae were reared to adults in the field laboratory and then transported to the KEMRI insectary for identification . In the insectary , the oviposition papers with eggs were dispensed in water to allow hatching and the emerging larvae were fed on fish fingerlet meal ( Tetramin baby ) until pupation . The pupae were transferred in small cups containing water to within 4 liter plastic cages with netting material for eventual development to adults . The adults were knocked down at -20°C for 45 seconds and morphologically identified using an identification key [35–38] under a dissecting microscope to select Ae . bromeliae and Ae . vittatus for use in the study . The adult mosquitoes were provided with 10% glucose solution on cotton wool and maintained at temperature between 28–32°C , 70–80% relative humidity and 12:12 hour light:dark ( L:D ) photoperiod . In order to stimulate egg production the mosquitoes were fed on anaesthetized clean laboratory mice placed on top of the cage for 45 minutes . The eggs collected were hatched into F1 ( first filial generation ) and adult mosquitoes were maintained as described . The Lamu001 strain of ECSA lineage CHIKV , isolated from human during the 2004–2005 outbreak in Lamu Island [6] , was used for all the infection assays performed in this study . The virus was passaged in confluent monolayers of Vero cells in T-25 cell culture flasks , grown in Minimum Essential Medium ( MEM ) , ( Sigma-Aldrich , St . Louis , MO ) with Earle’s salts and reduced NaHCO3 , supplemented with 10% heat inactivated fetal bovine serum Fetal bovine serum ( or foetal bovine serum ) is serum taken from the fetuses of cows . Fetal Bovine Serum ( or FBS ) is the most widely used serum in the culturing of cells . In some papers the expression foetal calf serum is used . ( FBS FBS abbr . fasting blood sugar FBS Fasting blood sugar . See Fasting glucose . ) , ( Sigma-Aldrich ) , 2% L-glutamine ( Sigma-Aldrich ) glutamine ( gl`təmēn ) , organic compound , one of the 20 amino acids commonly found in animal proteins . and 2% antibiotic antimycotic solution containing 10 , 000 units penicillin , 10 mg streptomycin and 25μg amphotericin B per ml ( Sigma-Aldrich , St . Louis , MO ) . The inoculated monolayer was incubated at 37°C for 1 hour , to allow for virus adsorption and then maintenance medium ( MEM , with 2% Fetal Bovine Serum , 2% glutamine , 2% antibiotic/antimycotic ) was added and incubated at 37°C . 80% cytopathic effect ( CPE ) was observed after two days . The CPE—Customer Premises Equvirus was harvested , aliquoted in cryovials and stored at -80°C until use [39] . Quantification of CHIKV was performed by plaque assay . 10-fold serial dilutions of the amplified CHIKV was carried out and inoculated in 6-well plates containing confluent Vero monolayers as described by Gargan [40] . This was grown in minimum essential medium ( MEM ) , with Earle’s salts and reduced Sodium bicarbonate ( NaHCO3 ) , supplemented with 10% heat-inactivated Fetal Bovine Serum ( FBS ) , 2% L-glutamine , and 2% antibiotic/antimycotic solution with 10 , 000 units penicillin , 10 mg streptomycin and 25 μg amphotericin B per ml and incubated at 37°C in 5% CO2 overnight . Each well was inoculated with 100 μl of the respective virus dilution , incubated for 1 hour with frequent rocking to allow for adsorption . The infected cells were maintained using 2 . 5% methylcellulose mixed with 2X maintenance medium ( MEM , GIBCO Invitrogen corporation , Carlsbad , California ) and incubated at 37°C with 5% CO2 for 4 days; then fixed for 1 hour with 10% formalin , stained for 2 hours with 0 . 5% crystal violet , washed and the plaques counted and calculated to quantify the virus using the following formula [39]: Numberofplaquesd×V=PFU/ml where d is the dilution factor and V is the volume of diluted virus added to the wells . The wild filial generation ( F0 ) and first generation ( F1 ) of female Ae . bromeliae and Ae . vittatus , respectively , were deprived of glucose for 24 hours before exposure to the infectious blood meal , using an artificial membrane feeding system ( Hemotek ) . The virus/blood mixture was put in membrane feeders covered with freshly prepared mouse skin , and maintained using the hemotek system which employs an electric heating element that maintains the temperature of the blood meal at 37°C . Batches of 50–100 female mosquitoes aged 4–5 days were fed on the virus-blood mixture at a ratio of 1:1 ( CHIKV isolate and defibrinated sheep blood ) using a Hemotek feeding system for 60 minutes . Only fully engorged mosquitoes were transferred to 4-litre plastic cages ( 15–30 mosquitoes/cage ) with a net on top and maintained with 10% glucose at 28–30°C , relative humidity of 70–80% , and 12:12 hour L: D photoperiod . The non-engorged mosquitoes were destroyed . Mosquito mortality was monitored in the cages by removing and counting dead mosquitoes daily . The experiment was done in three replicates to obtain the sufficient sample size . On 5 , 7 and 10 days post-infection ( dpi ) , a representative sample ( at least 30% ) of the orally exposed mosquitoes were picked , cold anesthetized and carefully decapitated with the legs/wings and bodies placed into separate 1 . 5 mL microfuge tubes ( Eppendorf ) . Each mosquito body was placed separately in a well labelled 1 . 5ml tubes containing 1000 μl of homogenization media ( HM ) , made of MEM , supplemented with 15% FBS , 2% L-glutamine , and 2% antibiotic/antimycotic . Mosquito bodies were homogenized using a mini bead beater ( BioSpec Products Inc , Bartlesville , OK 74005 USA ) with the aid of a copper bead ( BB-caliber airgun shot ) and clarified by centrifugation at 12 , 000 rpm ( Eppendorf centrifuge 5417R ) for 10 minutes at 4°C . The supernatants were inoculated in Vero cells in 12 well plates , grown in MEM , supplemented with 10% FBS , 2% L-glutamine and 2% antibiotic/antimycotic . One hundred microliters of the appropriate dilutions of the abdominal homogenates was added to each of ten wells of the 12-well plate to infect the cells and the remaining two wells were used for controls , negative control was comprised of male mosquitoes from the study vectors comprising of a pool of 25 mosquitoes . The plates were incubated at 37°C in a 5% CO2 incubator with frequent agitation after every 15 minutes for 1 hour to allow for virus adsorption . The infected cell monolayers were then overlaid with 2 . 5% methylcellulose supplemented with 2% FBS , 2% L-Glutamine and 2% antibiotic/antimycotic and incubated at 37°C in 5% CO2 . On day 4 , plates were fixed for 1 hour with 10% formalin , and stained for 2 hours with 0 . 5% crystal violet , washed on running tap water , dried overnight and the plaques observed on a light box . The CHIKV positive bodies were used to determine the infection rates . For each positive abdomen , corresponding legs were homogenized and their infection status determined as described above for the abdomens . Plaques were counted and calculated to determine the viral titer . If the virus was detected in the mosquito’s body but not in the legs , the mosquito was considered to have a non-disseminated infection , limited to the midgut . Detection of virus in the body and legs was considered evidence of successful infection and dissemination , respectively [41] . After exposing the mosquitoes to the infectious blood meal , engorged mosquitoes were picked , placed into new cages , reared under the insectary conditions and maintained with 10% sucrose . On 5 , 7 and 10 days dpi , mosquitoes were sucrose-starved and deprived of water for 16 hours , then cold anesthetized for about 40 seconds before the legs and wings from each of them were carefully removed and placed on sticky tape . Individual mosquito proboscises were inserted into a capillary tube containing 10–20 ul HM . Mosquitoes were allowed to expectorate saliva for 30 minutes . Media containing saliva was then expelled into a cryovial containing 200 ul of MEM and stored at -80°C until tested . A volume of 80 μl of the saliva sample was inoculated into each well of a 24-well plate containing confluent Vero cell monolayers . Plates were incubated for 1 hour to allow for adsorption , with frequent agitation . The infected cells were maintained using maintenance media ( 1 ml per well ) and incubated at 37°C with 5% CO2 . Plates were observed for 7 days and the supernatant of wells showing CPE were harvested and virus quantified by plaque assay as discribed above . Plaques were counted and calculated to quantify the virus . Scientific and ethical approval to carry out this study was obtained from the KEMRI Scientific Ethical Review Unit ( SERU ) ( KEMRI/SERU/CVR/002/3449 ) . The animal use component was reviewed and approved by KEMRI Animal Care and Use Committee ( ACUC ) ( KEMRI/ACUC/01 . 05 . 17 ) . The KEMRI ACUC adheres to national guidelines on the care and use of animals in research and education in Kenya enforced by National Commission for Science , Technology and Innovation ( NACOSTI ) . The Institute has a foreign assurance identification number F16-00211 ( A5879-01 ) from the Office of Laboratory Animal Welfare ( OLAW ) under the Public Health Service and commits to the International Guiding Principles for Biomedical Research Involving Animals . Three parameters describing vector competence were determined: infection ( number of infected mosquito bodies per 100 mosquitoes orally exposed and tested ) , dissemination ( number of mosquitoes with positive legs per 100 mosquitoes infected ) and transmission rates ( number of mosquitoes with positive saliva per 100 mosquitoes with disseminated infection ) . Test of proportions were used to get the infection , transmission and dissemination rates with their 95% confidence interval ( CI ) . Chi-square test with or without Yates’ correction or Fisher’s exact test were used to assess the differences between the two species at each time point and between the three time points for each species . Test of difference between means was done for the titers to determine if there was significant difference in incubation days for each species for the infection and dissemination . Statistical significance was considered for p < 0 . 05 .
The feeding success rate of the two mosquito species on infected blood meal was high , ranging from 70–80% for Ae . bromeliae and 40–50% for Ae . vittatus . The blood meal titres were determined before and immediately after mosquito exposure . The infection rate for Kilifi and West Pokot mosquitoes were measured from a total of 311 Ae . bromeliae ( 110 on 5 dpi , 101 on 7 dpi and 100 on 10 dpi ) and 204 Ae . vittatus ( 69 on 5 dpi , 69 on 7 dpi and 66 on 10 dpi ) . Both species were susceptible to chikungunya virus infection with average infection rates of 37% and 79% for Ae . bromeliae and Ae . vittatus , respectively . Aedes vittatus had high midgut infection rate , with no significant difference between the extrinsic incubation periods . The overall dissemination rate was high for Ae . vittatus with more than 46% of the mosquitoes with midgut infection having a disseminated infection . Aedes bromeliae had moderate midgut infection rate on 5 and 7 dpi , but low infection rate on 10 dpi . Overall Ae . bromeliae showed relatively low dissemination with 34% of those with midgut infection having disseminated infection ( Table 2 ) . Aedes vittatus was highly susceptible to CHIKV with infection rates of 81% , 78% , and 79% on 5 , 7 and 10 dpi respectively compared to Ae . bromeliae which was moderately susceptible with infection rates of 44% , 41% and 26% on 5 , 7 and 10 dpi respectively ( Fig 2A ) . Infection rates for Ae . vittatus were higher relative to that of Ae . bromeliae . Statistically significant differences were observed for infection rates 5 dpi between Ae . bromeliae ( 40 . 9% , 95% CI [31 . 6–50 . 7%] ) and Ae . vittatus ( 81 . 2% , 95% CI [69 . 9–89 . 6%] ) p < 0 . 001; 7 dpi between Ae . bromeliae ( 43 . 6% , 95% CI [33 . 7–53 . 8%] ) and Ae . vittatus ( 78 . 3% , 95% CI [66 . 7–87 . 3%] ) p < 0 . 001; and 10 dpi between Ae . bromeliae ( 26 . 0% , 95% CI [17 . 7–35 . 7%] ) and Ae . vittatus ( 78 . 8% , 95% CI [67 . 0–87 . 9%] ) p < 0 . 001 ( Table 2 ) . Dissemination rates for Ae . vittatus were higher relative to those of Ae . bromeliae . However , statistical significant difference was only observed 5 dpi , Ae . bromeliae ( 26 . 7% , 95% CI [14 . 6–41 . 9%] ) and Ae . vittatus ( 46 . 4% , 95% CI [33 . 0–60 . 3%] ) p< 0 . 042 . Viral dissemination was observed as early as 5–7 dpi for both species . The proportion of disseminated infection for Ae . bromeliae increased significantly with increase in the number of days post infection with higher rate on day 10 ( 43% ) . Aedes bromeliae had dissemination rate of 26% , 36% and 43% at 5 , 7 and 10 dpi ( Fig 2B ) . Aedes vittatus had disseminated infection rates of 46% , 43% and 50% at 5 , 7 and 10 dpi , respectively , but these differences were not statistically significant ( chi-square test , p>0 . 05 ) ( Fig 2B ) . The overall data shows that 114 out of 277 mosquitos with midgut infection disseminated the virus to the legs , the Ae . vittatus population from West Pokot County had higher dissemination rate ( 46% ) , than the Ae . bromeliae ( 34% ) population from Kilifi county . Both species were able to transmit the virus as early as 5 dpi . The transmission rate for Ae . bromeliae was higher on day 10 ( 55% ) compared to other days post infection . Aedes vittatus had higher transmission rate on day 7 ( 48% ) which significantly declined on day 10 ( 35% ) post infection ( Fig 2C ) . The overall data for both the Kilifi and West Pokot mosquito population shows that 46 out of 114 ( 40% ) were able to transmit the virus . Although Ae . vittatus had higher infection and dissemination , there was no significant difference on overall transmission in both vectors ( Ae . vittatus 41% and Ae . bromeliae 41% ) . Aedes bromeliae dissemination efficiencies increased with increase in the number of days post infection , Ae . vittatus had high dissemination efficiencies on 7 dpi ( Table 2 ) . Overall transmission rates for Ae . vittatus was higher ( Fig 2 ) relative to that of Ae . bromeliae though no statistical significance was observed ( chi-square test , p>0 . 05 ) . Aedes bromeliae and Ae . vittatus were analysed to assess viral titers in bodies and legs plus wings by titration in Vero cells . Aedes bromeliae bodies showed mean viral titers of 5 . 0 ± 0 . 33 log10 PFU/mL , 5 . 3 ± 0 . 34 log10 PFU/mL , 5 . 3 ± 0 . 45 log10 PFU/mL at 5 , 7 , and 10 days post infection , respectivelyn . The mean CHIKV titers in the bodies increased progressively , reaching a value of 5 . 3 ± 0 . 45 log10 PFU/mL 10 dpi ( Fig 3 ) . The viral presence in the legs was detected as early as 5 dpi with a titer of 4 . 0 ± 0 . 58 log10 PFU/mL , and titers of 4 . 3 ± 0 . 52 log10 PFU/mL and 4 . 3 ± 0 . 62 log10 PFU/mL on day 7 and 10 dpi , respectively . Aedes vittatus bodies showed mean viral titers of 5 . 7 ± 0 . 32 log10 PFU/mL , 5 . 8 ± 0 . 32 log10 PFU/mL , 4 . 9 ± 0 . 31 log10 PFU/mL at 5 , 7 , and 10 dpi , respectively ( Fig 3 ) . The viral presence in the legs was detected as early as 5 dpi with a titer of 3 . 6 ± 0 . 37 log10 PFU/mL , and titers of 4 . 4 ± 0 . 44 log10 PFU/mL and 4 . 2 ± 0 . 40 log10 PFU/mL 7 and 10 dpi respectively . Our results highlighted that among our Ae . bromeliae and Ae . vittatus populations , CHIKV was able to infect mosquitoes and replicate over time , disseminating to the wings and legs and reaching the salivary glands . There was no significant difference in infection and dissemination mean titers between the vectors . In general , viral dissemination only occurred when body titers were ≥ 105 for both strains . Ae . bromeliae had a midgut infection barrier that was stronger than that of Ae . vittatus . No difference in leg titers was observed between mosquitoes that did and did not transmit the virus ( Table 3 ) . No statistical difference for mean titers for the Ae . bromeliae and Ae . vittatus observed for all timepoints ( chi-square test , p>0 . 05 ) .
This is the first study to determine the ability of Ae . bromeliae and Ae . vittatus mosquito populations from Kenya to transmit the ECSA lineage of CHIKV . This study has demonstrated that the two are laboratory competent vectors for ECSA lineage of CHIKV . The recent outbreak of chikungunya in Africa , America , Asia and Europe [18 , 42 , 43] , clearly demonstrates the potential of the disease to spread to new areas and cause massive epidemics . The risk of importation of CHIKV to new areas is due to international and local travels from epidemic areas and exporting infected vectors to new areas where there are susceptible people and competent vectors [14 , 44] . The full competence of a vector is not only determined by the ability of the vector to get infected , but also by its ability to transmit the pathogen [45] . In this study we determined the capacity of the vectors to get infected , disseminate and transmit the virus . The CHIKV titers ( 106 . 4 PFU/ml ) used to infect mosquitoes in this study , are similar to published viremia levels associated with human infections ( often >105 PFU/mL blood ) in nature [46] . It has also been shown that a titer of 104 PFU/ml in monkeys was sufficient to infect mosquitoes [41] . Our results show that these two mosquito species are susceptible to infection and have ability to transmit CHIKV ( Table 2 ) . Although all mosquito species tested had ingested infectious blood meals , not all mosquitoes were infected and not all that were infected had the virus disseminated . This shows that other factors , such as the midgut escape barrier , affect the replication and dissemination of the virus in a mosquito [47] . The Ae . bromeliae population had moderate midgut infection which ranged from 26–44% across the different days post infection . Virus infection in the midgut was detected as early as 5 dpi . This is similar to previous studies which showed that the mosquito bodies infection with CHIKV in East Africa ranges from 2–9 days [21] . Aedes bromeliae had the highest transmission rate 10 dpi , compared to Ae . vittatus , which had its highest transmission rate 7 dpi , suggesting Ae . bromeliae requires more days for the virus to infect the salivary glands and eventually transmit to a susceptible host . Aedes vittatus breeds mostly on rock pools/holes and tree holes as demonstrated by their representing over 70% of the total collected in these habitats . Breeding of Ae . vittatus in rock pools and tree holes has been previously documented [23 , 33 , 48] . This study showed that the West Pokot population of Ae . vittatus has the potential to transmit CHIKV as has been demonstrated in other studies [23] . Our data showed that Ae . vittatus midgut infection and dissemination rates 5 dpi were relatively high suggesting the presence of weak midgut infection and escape barriers . Our data suggest the West Pokot Ae . vittatus population is efficient in transmitting CHIKV and indicates a potential risk if the virus is introduced in the area . Our study demonstrated that not all Ae . bromeliae and Ae . vitattus are capable of transmitting the CHIKV via capillary feeding; showing that dissemination is dependent on the midgut infection [49] . However , such in vitro experiments may not represent the actual amount of virus inoculated in a host during feeding . Despite the two species being exposed to the same virus titers , Ae . vittatus showed high infection and dissemination rates compared to Ae . bromeliae . This may be due to other intrinsic factors such as varying strength of midgut infection barrier and midgut escape barrier that individually affect the susceptibility of different mosquito species to infections [50] . It was observed that Ae . vittatus had a higher midgut infection than Ae . bromeliae , but there was no significant difference in transmission between the two species regardless of the incubation period . Since this is determined by the ability of the virus to penetrate into the saliva glands and be secreted into the saliva , the data support the notion that the salivary gland barrier is independent of the midgut infection and [51] . For both species , a higher viremia in their infected legs correlated with the ability to transmit the virus by the capillary method . Although this method is not a fully accurate representation of transmission , it does confirm the presence of virus in the salivay and can be used as a model to test for transmission of viruses which have no documented animal models for such experiments . Mosquitoes usually secrete less virus into a capillary tube than when feeding on an animal [52] and transmission rates are often lower when they are determined by collection of saliva as compared to allowing the mosquito to feed naturally on a susceptible animal [53] . Therefore , failure to detect CHIKV in the saliva collected in a capillary tube does not necessarily mean that the mosquito would not have transmitted the virus by bite if it fed on a susceptible human . In this case , our transmission rates should be considered as minimum transmission rates . Additionally , although Ae . vittatus and Ae . bromeliae from Kenya are efficient laboratory vectors , their potential role in CHIKV transmission depends on other factors in relation to mosquito ecology such as densities , survival , longevity , anthropophily and duration of gonotrophic cycles , which have been shown to interfere with transmission and maintenance of CHIKV .
This study demonstrated that Ae . vittatus and Ae . bromeliae populations in Kenya are laboratory competent vectors of ECSA lineage CHIKV and it indicates the potential for CHIKV transmission to occur in these locations should the virus find its way there through travel or introduction via a sylvatic host . It is therefore recommended that the public health authorities should continually monitor and carry out surveillance of the CHIKV and virus genotypes circulating within particular regions as well as identify vectors mediating these transmissions to prevent their adverse effects before an outbreak .
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Kenya experienced its first chikungunya outbreak in 2004/2005 along the coastal area , followed by sporadic outbreaks in Mandera in 2016 , and subsequently in Mombasa city in late 2017 and early 2018 . Despite the rising risk of transmission of the virus in the country based on evidence of outbreaks in Kenya , vector competence studies have only been limited to Ae . aegypti , while the role played by other Aedes species largely remain unknown . This study demonstrated the ability of Ae . bromeliae and Ae . vittatus to transmit chikungunya virus under controlled laboratory conditions . Vector competence remains the most important approach in disease risk assessment that provides knowledge to the public health sector in developing vector control guideline .
|
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2018
|
Vector competence of Aedes bromeliae and Aedes vitattus mosquito populations from Kenya for chikungunya virus
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Interferon-induced transmembrane proteins ( IFITMs ) inhibit infection of diverse enveloped viruses , including the influenza A virus ( IAV ) which is thought to enter from late endosomes . Recent evidence suggests that IFITMs block virus hemifusion ( lipid mixing in the absence of viral content release ) by altering the properties of cell membranes . Consistent with this mechanism , excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry . Here , we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells . IFITM3 over-expression did not inhibit lipid mixing , but abrogated the release of viral content into the cytoplasm . Although late endosomes of IFITM3-expressing cells accumulated cholesterol , other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion . These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion . The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet . We propose that IFITM3 interferes with pore formation either directly , through partitioning into the cytoplasmic leaflet of a hemifusion intermediate , or indirectly , by modulating the lipid/protein composition of this leaflet . Alternatively , IFITM3 may redirect IAV fusion to a non-productive pathway , perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes .
The recently identified interferon-induced transmembrane proteins ( IFITMs ) inhibit infection of diverse enveloped viruses [1]–[3] . Ectopic expression of IFITM1 , -2 and -3 restricts a growing number of unrelated viruses , including IAV [1] , [2] , [4]–[7] . IFITM3 has been shown to potently restrict infection by IAV and the Respiratory Syncytial Virus in vivo [8]–[10] . In contrast , arenaviruses and some retroviruses , such as murine leukemia virus ( MLV ) , are resistant to IFITM restriction [2] , [6] . The IFITMs have been reported to inhibit HIV-1 entry , albeit less potently than IAV and apparently in a cell type-dependent manner [11]–[13] . The mechanism by which IFITMs inhibit infection of diverse viruses is not fully understood . IFITM2 and -3 are predominantly found in late endosomes ( LE ) and lysosomes [13] , [14] , whereas IFITM1 is also found at the cell periphery [4] , [15] . Different membrane topologies of IFITMs have been proposed [16] , but recent data suggests that IFITM3 is a type II transmembrane protein [17] . Accumulating evidence implies that IFITMs may interfere with virus-endosome fusion [1] , [2] , [5] , [13] , [14] . The fact that IFITMs seem to expand acidic intracellular compartments [13] indicates that the fusion block is downstream of the low pH trigger . Effective restriction of viruses that enter from the LE , such as IAV , Ebola virus ( EBOV ) and SARS coronavirus seems consistent with the cellular localization of IFITM2 and -3 proteins . However , these proteins also restrict Vesicular Stomatitis Virus ( VSV ) that appears to fuse with early endosomes [18] . IFITMs have been reported to curtail viral infection by modifying properties of cellular membranes , such as fluidity and spontaneous curvature [3] , [5] , [14] . These effects could be related , in part , to the accumulation of cholesterol in LE as a result of IFITM-mediated disruption of the interaction between the vesicle-membrane-protein-associated protein A ( VAPA ) and oxysterol-binding protein ( OSBP ) [14] . Since lipids play an important role in membrane fusion , these findings offer an attractive paradigm for a broad antiviral defense mechanism that involves altering the lipid composition of cellular membranes . The recent finding that amphotericin B , which forms complexes with sterols [19] , rescues IAV infection in IFITM2- and IFITM3-expressing cells [20] is in line with the notion that cholesterol may be directly or indirectly involved in IAV restriction . However , lipid composition-based models do not readily explain the lack of restriction of amphotropic MLV and arenaviruses , which enter cells via distinct endocytic routes [21] , [22] . These findings indicate that IFITMs may restrict virus entry from a subset of intracellular compartments . In order to define the mechanism of IFITM restriction , it is important to identify the viral entry step ( s ) targeted by these proteins , define compartments in which restriction occurs , and elucidate potential changes in intracellular membranes that may be responsible for this phenotype . Here , we examined the mechanism of IFITM3 restriction of IAV using single particle imaging and a direct virus-cell fusion assay . Our results show that IFITM3 does not inhibit the lipid mixing stage of IAV fusion but blocks the release of viral contents into the cytosol , and that this phenotype does not correlate with cholesterol accumulation in intracellular compartments . Specifically , IFITM3 inhibits the conversion of hemifusion to fusion through a mechanism that does not rely on cholesterol accumulation . Together these findings reveal a previously unappreciated view of IFITM-mediated restriction and suggest new avenues of investigation to delineate the mechanism by which these proteins block infection .
We chose to focus on IFITM3 to study the mechanism of IAV restriction because this protein potently inhibits infection in vitro and in vivo [8]–[10] . Since published data suggest that IFITM3 likely inhibits the viral fusion step , a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [23] . HIV-1 particles carrying the β-lactamase-Vpr ( BlaM-Vpr ) chimera and pseudotyped with the influenza HA and NA proteins from the H1N1 A/WSN/33 strain ( referred to as IAVpp ) were allowed to fuse with cells transduced with an empty vector or with an IFITM3-expressing vector . The resulting cytosolic BlaM activity was measured as previously described [24] . Out of several cell lines tested , A549 and MDCK cells over-expressing IFITM3 were least permissive to IAVpp fusion ( Fig . 1A ) . In agreement with the previous reports [2] , [13] , we found that IFITM3 over-expression partially inhibited VSV G glycoprotein-mediated fusion of pseudoviruses ( VSVpp ) carrying the BlaM-Vpr chimera ( Fig . 1A ) . Similar to inhibition of IAVpp fusion , the IFITM3-mediated restriction of VSVpp was most potent in A549 and MDCK cells . As expected , fusion of particles pseudotyped with the Lassa fever virus glycoprotein ( LASVpp ) , which directs virus entry through an IFITM3-resistant pathway [2] , [6] , was not considerably affected by IFITM3 over-expression . We next checked if the strong suppression of virus fusion in A549 and MDCK cells was related to the level of IFITM3 expression . Immunostaining for IFITM3 in these and CHO cells which exhibited modest restriction of viral fusion ( Fig . 1A ) did not reveal a clear correlation between IFITM3 expression and inhibition of IAVpp or VSVpp fusion ( Fig . 1B ) . Of note , potent IAV restriction in A549 and MDCK cells was not related to the usage of HIV-1 core-based pseudoviruses . Influenza virus-like particles containing the IAV BlaM-M1 chimera [25] also failed to efficiently fuse with A549-IFITM3 and MDCK-IFITM3 cells while fusing well with vector-transduced cells ( Fig . 1C ) . We also found that both vector-transduced A549 and MDCK cells were highly susceptible to IAV infection , as determined by virus titration ( see Materials and Methods ) . These two cell lines were therefore chosen for studies of IFITM3-mediated restriction described below . IFITM-based restriction has been studied using a cell-cell fusion model , as well as by forcing viral fusion with the plasma membrane by lowering the pH [5] , [20] . Since fusion with the plasma membrane is more amenable to mechanistic studies than endocytic entry , we asked whether IFITM3 can restrict forced IAV fusion . Exposure to acidic buffer induced IAVpp fusion with A549-Vector cells pretreated with Bafilomycin A1 ( BafA1 ) , which blocked low pH-dependent entry from endosomes ( Fig . 1D ) . The extent of forced fusion was lower compared to the conventional entry route . By contrast , forced IAVpp fusion with A549-IFITM3 cells was ∼3-fold more efficient than endocytic fusion with cells not treated with low pH or BafA1 , showing that IFITM3 does not restrict IAVpp fusion at the cell surface . Interestingly , IFITM1 suppressed IAVpp-plasma membrane fusion at low pH ( Fig . 1D ) , in agreement with the Jaagsiekte sheep retrovirus ( JSRV ) and IAV fusion data [5] , [20] . The inability of IFITM3 to block IAV fusion with the plasma membrane is consistent with its lower abundance at the cell surface [13] , [14] , [20] and shows that the mechanism of restriction must be studied in intracellular compartments . Preponderance of evidence implies that hemifusion is a universal intermediate ( reviewed in [26] , [27] ) that precedes the formation of a fusion pore . Having shown that IFITM3 over-expression inhibits viral fusion ( Fig . 1A , C ) , we asked whether this protein also blocks the upstream hemifusion step . This was accomplished by labeling the A/PR/8/34 virus membrane with a self-quenching concentration of vybrant DiD ( vDiD ) , using a modification of the previously published protocol [28] . Incorporation of self-quenching quantities of a lipophilic dye enables the visualization of single lipid mixing events based on the marked increase in fluorescence upon dye redistribution to an endosomal membrane ( see for example [28] , [29] ) . Significantly , to control for fluctuations in the vDiD fluorescence caused by deviation from a focal plane , the viral surface proteins were labeled with the amine-reactive AlexaFluor-488 ( AF488 ) dye . The relatively steady AF488 signal before and after hemifusion is allowed correcting for the vDiD intensity fluctuations due to moving in and out of focus . The vDiD/AF488 co-labeling protocol only modestly ( <2-fold ) reduced IAV infectivity compared to the mock-labeled viruses ( Fig . S1A ) . Immunofluorescence staining of AF488-labeled virions with anti-HA antibodies revealed an excellent co-localization of the two signals ( Fig . S1B , C ) , thus supporting the notion that AF488/vDiD-labeled particles are bona fide virions . Labeled viruses were allowed to enter A549-Vector cells , and the resulting lipid mixing activity was examined by single particle tracking . A fraction of virions exhibited a marked increase in the vDiD signal ( Fig . 2A , B ) . Redistribution of vDiD was mediated by low pH-dependent conformational changes in the IAV HA glycoprotein , as evidenced by potent inhibition of lipid mixing by anti-HA antibodies ( Fig . 2C ) and by NH4Cl ( Fig . 3A ) . Without simultaneous monitoring of the viral content release into the cytoplasm , vDiD dequenching does not discriminate between hemifusion ( operationally defined as lipid mixing without content transfer [30] ) and full fusion . To avoid over-interpreting dequenching events , we will refer to these events as lipid mixing or hemifusion . A similar vDiD dequenching pattern was observed in MDCK cells transduced with an empty vector ( data not shown ) . Analysis of lipid mixing showed that 2 . 2±0 . 4% and 5 . 6±0 . 6% of cell-bound particles released vDiD in A549 and MDCK cells , respectively ( Fig . 3A ) . By comparison , a much greater fraction of virions ( 38 . 3±0 . 6% ) hemifused with CHO cells ( data not shown ) , in agreement with the previously reported data [28] . Importantly , IAV lipid mixing was readily detected in IFITM3+ A549 and MDCK cells ( Figs . 2D–G and 3A ) . Not only was lipid mixing not inhibited in A549-IFITM3 cells , but a >3-fold greater fraction of particles released vDiD in these cells compared to control cells ( Fig . 3A , P<0 . 001 ) . By comparison , IFITM3 over-expression in MDCK cells did not significantly promote vDiD dequenching ( Fig . 3A ) . Thus , contrary to the cell-cell fusion results [5] , IFITM3 does not inhibit and can even promote IAV lipid mixing , consistent with the block of virus entry at a post-hemifusion stage . Accordingly , the addition of oleic acid , which augments hemifusion by altering spontaneous membrane curvature , did not rescue IAVpp or VSVpp fusion with A549-IFITM3 cells ( Fig . S2 ) . This is in agreement with the recent infectivity results [20] , but in contrast with the rescue of fusion between JSRV Env- and IFITM-expressing cells by this fatty acid [5] . The higher frequency of vDiD dequenching in A549-IFITM3 cells could be caused by the increased endosome acidity compared to control cells [13] . However , the distribution of waiting times to the onset of lipid mixing was independent of IFITM3 expression or the type of target cells ( A549 vs . MDCK , Fig . 3B , P = 0 . 37 ) . The fact that the kinetic curves do not reach plateau indicates that IAV entry into A549 and MDCK cells is not completed within the first hour . Our results thus demonstrate that IFITM3 restricts the IAV fusion at a post-hemifusion step , most likely at the point of fusion pore opening , as evidenced by the dramatic decrease of the BlaM signal in A549 and MDCK cells expressing this protein ( Fig . 1A ) . Under our conditions , vDiD dequenching was typically completed within a few minutes for both control and IFITM3+ cells ( Fig . 2 ) . This dequenching rate is much slower than sudden increases in fluorescence of the IAV membrane markers described previously [28] , [31] . While a portion of vDiD dequenching could be completed within seconds ( Fig . S3 ) , these fast events were not common . Slow dequenching was also typical with the vDiD/AF488-labeled X31 virus , as well as with the X31 virus labeled with a 15-fold excess of DiD , using the published protocol for single virus imaging [28] ( data not shown ) . Slow vDiD dequenching during the first hour of virus-cell co-incubation did not appear to result from IAV degradation in LE/lysosomes , since the surface-exposed AF488 label persisted long after vDiD dequenching was completed and because anti-HA antibodies blocked vDiD dequenching ( Fig . 2 ) . In addition , we did not detect any correlation between the lag before the onset of lipid mixing and the vDiD dequenching slope ( Fig . S4A ) . This result reinforces the notion that late lipid mixing events are mediated by HA and not by virus degradation . Control experiments , in which samples were not exposed to laser light during the first 30 min at 37°C , did not reveal fast dequenching events reaching completion in less than 1 min ( data not shown ) . This control argues against phototoxicity-related attenuation of virus fusogenicity as the cause for sluggish lipid redistribution . Since free vDiD diffusion between a virus and a small endosome should be completed in less than a second [32] , [33] , an initial membrane connection between IAV and an endosome must severely impair lipid movement . To assess whether early fusion intermediates in control and IFITM3+ cells restrict vDiD diffusion to the same extent , we examined the rate of vDiD dequenching . Single particle analysis revealed that , in A549 cells , the average vDiD dequenching profile ( Fig . 3C ) was independent of IFITM3 expression , as were the initial slopes of vDiD dequenching ( Fig . S4B , P>0 . 5 ) . These results indicate that IFITM3 over-expression does not affect the properties of fusion intermediates responsible for vDiD redistribution , such as the size and/or architecture of a hemifusion site ( e . g . , [34] , [35] ) . We then asked whether the rate of vDiD dequenching varied depending on the cell type . The average rate of vDiD fluorescence increase in MDCK cells was ∼2-fold greater than in A549 cells ( Figs . 3C and S4B , P<0 . 02 ) . This demonstrates our ability to detect changes in the rate of vDiD transfer and shows that lipid transfer lasts several minutes irrespective of the cell type . We also examined the final extent of vDiD dequenching , which is proportional to the surface area of a target membrane over which it redistributes . This parameter was not significantly affected by IFITM3 expression in A549 cells or by the cell type ( MDCK vs . A549 cells , Fig . 3D ) . Together , similar kinetics and extents of viral lipid dilution in control and IFITM3+ cells suggest that neither the size/architecture of early fusion intermediates nor the surface area of target endosomes is considerably affected by IFITM3 expression . To investigate the relationship between lipid mixing and productive IAV infection , we compared the fraction of cells “receiving” at least one vDiD dequenching event in live cell imaging experiments to the fraction of cells that got infected under the same conditions . The only difference was that virus imaging was not continued beyond 1 h after initiation of fusion , whereas infection proceeded overnight . We found that one or more vDiD dequenching events occurred in 15% of A549 cells while 44% of cells got infected ( Fig . S5 ) . Under the same conditions , 20% of MDCK cells “hosted” one or more dequenching events and 36% were infected . The greater fraction of infected cells compared to those permissive to hemifusion is likely due to the shorter time widow for single virus imaging , which is likely to miss late vDiD dequencing events ( Fig . 3B ) . The lower apparent fraction of cells supporting vDiD dequenching could also be caused by the presence of viruses that did not incorporate self-quenching amounts of vDiD . Importantly , the comparable efficiencies of lipid mixing and infection , indicate that the former events likely culminate in productive infection . To determine whether IFITM3 impairs the IAV's ability to form small fusion pores , we attempted to load the virus with a content marker by soaking in a concentrated solution of sulforhodamine B , as described in [36] . However , only a small fraction of AF488-labeled particles stained with sulforhodamine , and the retained dye was lost in live cell experiments under conditions that blocked IAV fusion ( data not shown ) . We therefore resorted to using HIV pseudoviruses bearing A/WSN/33 HA and NA glycoproteins and co-labeled with the capsid marker , YFP-Vpr , and the content marker , Gag-iCherry [24] , [37] . Upon virus maturation , the “internal” mCherry is proteolytically cleaved off the HIV-1 Gag-iCherry and released through a fusion pore , as manifested by the loss of the red signal ( Fig . 4 and [37] ) . The YFP-Vpr signal , which remained associated with the viral core after fusion , provided a reference signal for single particle tracking . Under our conditions ∼1% of double-labeled pseudoviruses entering A549-Vector cells lost their content marker , while approximately 2% fused with MDCK-Vector cells . In sharp contrast , the mCherry release in IFITM3+ A549 and MDCK cells or in vector-transduced cells in the presence of NH4Cl could not be detected ( Fig . 4E , P<0 . 001 ) . Thus , IFITM3 does not adversely affect IAV hemifusion but severely inhibits viral content release into the cytoplasm . Together these findings suggest that the mechanism of IFITM3-mediated restriction arises from the entrapment of viruses at a hemifusion intermediate prior to fusion pore formation . A recent study has shown that , through disrupting the interaction between VAPA and OSBP , IFITM3 causes cholesterol accumulation in LE [14] . Based on this finding , the authors proposed that high levels of endosomal cholesterol may inhibit IAV fusion and/or the release of nucleocapsid . Staining with filipin revealed that IFITM3+ A549 cells exhibited increased levels of intracellular cholesterol ( Fig . 5A ) . However , the filipin signal was still primarily associated with the plasma membrane and the total cellular cholesterol was not elevated in IFITM3+ cells ( Fig . S6 ) . In addition , the overall intensity of intracellular cholesterol poorly correlated with the level of IFITM3 expression ( Fig . 5C ) . By comparison , pretreatment of A549-Vector cells with U18666A , which inhibits transport of LDL-derived cholesterol from LE/lysosomes ( reviewed in [38] ) , resulted in a dramatic shift in the filipin staining pattern from the plasma membrane to endosomes ( Fig . 5B ) . Aberrant accumulation of cholesterol in LE is also known to occur in cells lacking the functional NPC1 cholesterol transporter [39] . We therefore knocked down NPC1 expression in A549 cells using shRNA ( shNPC1 , Fig . 5D ) and examined the resulting cholesterol distribution ( Fig . 5B ) . Reduced NPC1 expression correlated with excess cholesterol in intracellular compartments , which was also much more pronounced than endosomal filipin staining in A549-IFITM3 cells . We next asked whether the cholesterol accumulation induced by U18666A pretreatment or by down regulation of NPC1 can phenocopy the IFITM3-mediated restriction of viral fusion . Neither IAV lipid mixing ( vDiD dequenching ) nor fusion ( BlaM signal ) was inhibited by silencing NPC1 in A549 cells ( Fig . 5E , F ) . VSVpp also fused with shNPC1-transduced cells as efficiently as with control cells ( Fig . 5E ) . These results show that excess cholesterol does not inhibit viral fusion or hemifusion . In control experiments , silencing the NPC1 expression potently suppressed fusion of Ebola GP-pseudotyped particles ( EBOVpp , Fig . 5E ) , which use NPC1 as a receptor [40] , [41] . Similar to the NPC1 knockdown phenotype , pretreatment of A549 cells with 10 µM U18666A , which caused cholesterol buildup in endosomes ( Fig . 5B ) , did not inhibit fusion of IAVpp or VSVpp ( Fig . 5G ) . As will be shown below for MDCK cells , higher doses of U18666A can inhibit viral fusion ( Fig . 5G ) , but this effect is due to elevation of endosomal pH as opposed to cholesterol accumulation in endosomes . To generalize the effects of excess cholesterol in A549 cells , we tested whether endosomal cholesterol can inhibit viral fusion in MDCK cells . As in A549 cells , IFITM3 over-expression in MDCK cells caused moderate accumulation of cholesterol in endosomes ( Fig . 6A ) , while pre-treatment with U18666A caused a much more dramatic buildup of intracellular cholesterol ( Fig . 6B ) . However , unlike A549 cells , IAVpp and VSVpp fusion was significantly inhibited in U18666A-treated MDCK cells ( Fig . 6C ) . Since prolonged exposure to U18666A has been reported to raise endosomal pH [42] , we sought to determine if insufficiently acidic pH could prevent IAV hemifusion/fusion with pretreated MDCK cells . The pH in IAV-carrying endosomes was measured using virions co-labeled with the pH-insensitive AF488 ( green ) and CypHer5E ( red ) , which fluoresces brighter at acidic pH [28] ( Fig . S7A ) . Cells were incubated with viruses for 45 min , and the red/green signal ratio from individual particles was measured ( Fig . S7B ) . The average pH in virus-containing endosomes of MDCK-IFITM3 cells was slightly less acidic than in control cells: 5 . 38±0 . 03 ( n = 498 ) vs . 4 . 98±0 . 04 ( n = 242 ) , respectively ( Fig . 6D and F , P<0 . 001 ) . Interestingly , as shown in Figure 6E , endosomal pH in U18666A-treated MDCK cells was markedly shifted to neutral values ( 6 . 44±0 . 05 , n = 160 , P<0 . 001 ) . Since the pH threshold for triggering A/PR/8/34 fusion is reported to be around 5 . 6 [43] , elevation of endosomal pH in U18666A-treated MDCK cells is the likely cause of inhibition of viral fusion . Together our results imply that U18666A most likely attenuates IAV fusion with MDCK cells by raising endosomal pH and not through inducing cholesterol accumulation . We also took advantage of the available CHO cell line that does not express NPC1 [44] to further ascertain the role of endosomal cholesterol in IAV fusion . These cells ( designated CHO-NPC1− ) exhibited exaggerated endosomal cholesterol staining , in sharp contrast to a peripheral staining pattern in parental CHO cells ( Fig . 7A ) . In spite of the high endosomal cholesterol content in CHO-NPC1− cells and of the elevated level of total cholesterol ( Fig . S6 ) , IAVpp fused with these cells as efficiently as with parental cells ( Fig . 7C ) . The NPC1-null cells also supported IAV lipid mixing , albeit at somewhat reduced level compared to control ( Figs . 7D and S8 ) . Pretreatment of CHO cells with U18666A also trapped cholesterol in endosomes and raised the total cholesterol content ( Figs . 7B and S6 ) , but only modestly diminished the extent of IAVpp or VSVpp fusion ( Fig . 7E ) . Interestingly , in contrast to the decreased endosome acidity in MDCK cells , endosomes in U18666A-treated CHO cells were more acidic than in control cells ( Fig . S9 ) . In control experiments , both the lack of NPC1 expression and U18666A pretreatment blocked EBOVpp fusion ( Fig . 7C , E ) , consistent with its reliance on NPC1 receptor and high sensitivity to disruptions of cholesterol transport [45] . Together , our results show that the cholesterol accumulation achieved through two different interventions – U18666A pretreatment and NPC1 silencing – does not phenocopy IFITM3-mediated restriction of viral fusion . This implies that ( i ) elevated levels of endosomal cholesterol do not generally confer resistance to viral fusion , and ( ii ) the mechanism by which IFITM3 blocks transition from hemifusion to full fusion is not through the mislocalization of cholesterol .
The IFITMs restrict the cellular entry of multiple pathogenic enveloped viruses . Recent studies lead to a model that IFITMs inhibit virus-host hemifusion [5] and that the membrane-rigidifying properties of cholesterol may contribute to antiviral actions [14] . In contrast to these studies , our results now demonstrate that IFITM3 prevents the release of viral genomes into the cytosol by inhibiting viral entry after hemifusion but prior to fusion pore formation ( Fig . 8 ) . Moreover , we found that IFITM3 can promote hemifusion in some cells , perhaps secondary to its acidifying the endosomal pathway . IFITM3 therefore does not negatively regulate the properties of contacting leaflets involved in hemifusion , but stabilizes the cytoplasmic leaflet of the endosomal membrane , thereby disfavoring the formation of fusion pores [35] . In one potential scenario IFITM3 is located directly at the site of arrested hemifusion , perhaps “toughening” the endosomal membrane to create a barrier to viral entry ( Pathway 1 ) . A considerable colocalization of IFITM3 with internalized IAV ( [3] and Fig . S10 ) is consistent with Pathway 1's direct mechanism of inhibition . Alternatively , IFITM3 might arrest hemifusion through an indirect mechanism , perhaps involving modulation of lipid and/or protein composition of the cytoplasmic leaflet ( Pathway 2 ) . Recent findings that changes in global membrane properties interfere with productive entry would appear to support an indirect mechanism [5] , [14] . Lipids , such as unsaturated fatty acids and cholesterol that confer negative spontaneous curvature to membranes can promote hemifusion ( a net negative curvature structure ) and disfavor a fusion pore ( a net positive curvature intermediate ) , as has been previously shown for oleic acid [35] . Although this prediction is consistent with efficient lipid mixing in endosomes of IFITM3+ cells observed in our imaging experiments , several studies [20] , [46]–[48] and our own results do not support cholesterol accumulation as playing a role in fusion inhibition . We found that cholesterol-laden endosomes in cells pretreated with U18666A or expressing undetectable/low levels of NPC1 supported efficient viral fusion . It is thus possible that IFITM3 interferes with cellular functions of VAPA other than the interaction with OSBP , such as regulation of SNAREs and modulation of lateral mobility of membrane proteins ( reviewed in [49] ) . IFITM3 appears to induce the formation multivesicular bodies and increase the number of ILVs [13] , [14] . One can therefore envision that IFITM3 may inhibit infection by redirecting viruses to a non-productive pathway , perhaps involving fusion with ILVs instead of the limiting membrane of LE ( Fig . 8 , Pathway 3 ) . If , as suggested in [14] , IFITM3 disallows back fusion of ILVs with the limiting membrane , then virus-ILV fusion products will likely be degraded . Indeed , back fusion has been implicated in the VSV core release into the cytosol following the virus-ILV fusion [50] . It should be stressed that this “fusion decoy” model does not explain the ability of IFITM1 to interfere with fusion at the cell surface ( [5] and Fig . 1D ) . It is also not clear why the Old World arenaviruses , which have been reported to enter from MVBs [51] , are not restricted by IFITMs . The indistinguishable extents of vDiD dequenching in control and IFITM3+ cells ( Fig . 3D ) indicate that target endosomes have similar sizes . While this appears to argue against redirection of IAV fusion to small ILVs , the lack of a post-hemifusion decay of vDiD fluorescence in A549 and MDCK cells ( Figs . 2 and S3 ) is consistent with IAV fusion with abundant ILVs in endosomes of IFITM3+ cells . This is because a lipophilic dye in the limiting membrane of an endosome should be quickly removed through membrane trafficking [24] , [31] , [52] . Because post-dequenching decay was not observed irrespective of the level of IFITM3 expression , it is possible that IAV may infect several cell lines by fusing with small intralumenal vesicles followed by the nucleocapsid release through back fusion ( Fig . 8 , dashed black arrows ) . This pathway could explain the similar extents and rates of vDiD dequenching in control and IFITM3-expressing cells , which are indicative of similar lipid intermediates and of the size of a target membrane , respectively . As discussed above , slow vDiD dequenching observed by single IAV imaging can be rationalized in the context of fusion with the limiting membrane of endosomes ( Pathways 1 and 2 ) , as well as in the context of fusion with ILVs ( Pathway 3 ) . Slow dilution of this dye in Pathway 3 could occur through multiple rounds of IAV fusion with small ILVs , whereas Pathways 1 and 2 would predict restricted lipid diffusion through early fusion intermediates formed at the limiting membrane . Although the latter notion is in agreement with the reported restriction of lipid movement through hemifusion sites and small fusion pores [34] , [35] , [53] , [54] , these intermediates are usually short-lived under physiological conditions and tend to resolve into larger structures that do not impair lipid movement [28] , [32] , [35] . Clearly , more detailed studies of virus-endosome hemifusion and fusion are needed to understand the nature of slow lipid redistribution between IAV and endosomes . The IFITMs may now arguably be one of the most broadly acting and clinically relevant restriction factor families [1] , [3] . While both IFITM3's membrane-associated topology and its localization to the site of viral attenuation suggest it acts to restrict viral entry via a direct mechanism , additional work remains to be done to fully elucidate its actions . Nonetheless , as the primary effector of IFN's anti-IAV actions , IFITM3 represents a previously unappreciated class of restriction factor that prevents viral entry by stabilizing a hemifusion intermediate , likely comprised of an invading virus fatally tethered to the interior of the endosome's limiting membrane . Future single virus experiments combining the detection of both viral lipid and content release events ( see for example [52] ) should provide further insights into IAV entry pathways and the mechanism of IFITM3-mediated restriction . Indeed , such efforts may also bring to light unknown viral countermeasures , which are perhaps employed by the IFITM-resistant New and Old World arenaviruses .
HEK 293T/17 cells and human lung epithelial A549 cells were obtained from ATCC ( Manassas , VA ) and grown as previously described [55] . Wild-type CHO cells and CHO-NPC1− cells , a gift from Dr . L . Liscum ( Tufts University ) [44] , were grown in Alpha-MEM ( Quality Biological Inc , Gaithersburg , MD ) supplemented with 10% FBS and penicillin-streptomycin . The A549 , MDCK , HeLaH1 and CHO cells stably expressing IFITM3 or IFITM1 were obtained by transducing with VSV-G-pseudotyped viruses encoding wild-type IFITM3 and IFITM1 or with the vector pQCXIP ( Clontech ) and selecting with puromycin , as described previously [2] . The pR8ΔEnv , BlaM-Vpr , pcRev , HIV-1 Gag-iCherryΔEnv and pMDG VSV G expression vectors were described previously [37] , [55] . The YFP-Vpr was a gift from Dr . T . Hope ( Northwestern University ) . The pCAGGS vectors encoding influenza H1N1 WSN HA and NA were provided by Donna Tscerne and Peter Palese , and the pCAGGS BlaM1 ( WSN ) plasmid was a gift from Dr . A . Garcia-Sastre ( Mount Sinai ) . Vectors expressing phCMV-GPc Lassa and pcDNA3 . 1-Ebola GP ( Zaire ) were gifts from Dr . F . -L . Cosset ( Université de Lyon , France ) [56] and Dr . L . Rong ( University of Illinois ) [57] , respectively . U18666A was from Tocris Bioscience ( Bristol , UK ) . Poly-L-lysine , filipin , sulphorhodamine B Bafilomycin A1 and the Cholesterol Kit were from Sigma-Aldrich . AlexaFluor-488 amine-reactive carboxylic acid , vybrant-DiD ( vDiD , 1 , 1′-dioctadecyl-3 , 3 , 3′ , 3′-tetramethylindodicarbocyanine , 4-chlorobenzenesulfonate salt ) , Hoechst-33342 and Live Cell Imaging buffer were purchased from Life Technologies ( Grand Island , NY ) . CypHer5E Mono NHS Ester was from GE Healthcare ( Pittsburgh , PA ) . Antibodies used were rabbit anti-IFITM3 ( to N-terminus ) from Abgent ( San Diego , CA ) , mouse anti-IAV-NP and goat anti-IAV-polyclonal antibodies from Millipore ( Billerica , MA ) , rat anti-mouse-IgG-FITC from eBioscience ( San Diego , CA ) , and goat anti-rabbit-Cy5 from Jackson Immunoresearch ( West Grove , PA ) . Pseudovirus production and titration were described previously [58] . Pseudoviruses were produced by transfecting HEK293T/17 cells using JetPRIME transfection reagent ( Polyplus-transfection SA , NY ) . For LASV and EBOV pseudoviruses , 5 µg of the phCMV-GPc Lassa or 5 µg of the pcDNA3 . 1-Ebola GP was included in the transfection mixture . Fluorescently labeled influenza pseudoviruses were produced using 1 µg of pR8ΔEnv , 2 µg of HIV-1 Gag-iCherryΔEnv [37] , 2 µg of YFP-Vpr , 1 µg of pcRev , and 2 µg of each WSN HA- and NA-expressing vectors . Ebola GP pseudoviruses were concentrated 10× , using Lenti-X™ Concentrator ( Clontech , Mountain View , CA ) . To generate influenza BlaM1 VLPs , HEK293T cells were transfected with pCAGGS-BlaM1 ( 5 µg ) and 2 . 5 µg of each pCAGGS-WSN HA and pCAGGS-WSN NA . After 12 h , the transfection reagent was removed , and cells were further cultivated in phenol red-free growth medium . The influenza virus surface proteins and the lipid membrane were labeled with AF488 and vDiD , respectively . A hundred µg of influenza virus from the purified H1N1 A/PR/8/34 stock ( 2 mg/ml , Charles River , CT ) was diluted in 95 µl of sodium bicarbonate buffer ( pH 9 . 0 ) supplemented with 50 µM AF488 . The mixture was incubated for 30 min at room temperature , after which time , 5 µl of vDiD ( from 1 mM stock in DMSO ) was added followed by an additional incubation for 90 min in the dark at room temperature with mild agitation . The labeled viruses were purified through a NAP-5 gel filtration column ( GE Healthcare ) in 145 mM NaCl solution buffered with 50 mM HEPES , pH 7 . 4 . Approximately 50% of AF488-labeled particles incorporated detectable amounts of vDiD with minimal contamination by free dye aggregates . The infectious IAV titer was determined in MDCK or A549 cells after incubation with serially diluted inoculum for 15 h at 37°C . Cells were fixed , permeabilized , blocked and incubated with rabbit R2376 anti-WSN HA antibody ( a gift from Dr . D . Steinhauer , Emory University ) for 2 h at room temperature . Cells were then washed and incubated with secondary Cy5-conjugated goat anti-rabbit antibodies ( Jackson ImmunoResearch , PA ) in 10% FBS-containing buffer supplemented with 10 µg/ml Hoechst-33342 for 1 h . The number of infected cells per image field was determined by fluorescence microscopy and normalized to the total number of cells ( stained nuclei ) . The infectious titer ( IU/ml ) was calculated by taking into account the ratio of the area of well and the image area and correcting for dilution and volume of viral inoculum . The β-lactamase ( BlaM ) assay for virus-cell fusion was carried out as described previously ( [24] and Methods S1 ) . Briefly , pseudoviruses bearing β-lactamase-Vpr chimera ( BlaM-Vpr ) were bound to target cells by centrifugation at 4°C for 30 min at 1550×g . Unbound viruses were removed by washing , and fusion was initiated by shifting to 37°C for 90 min , after which time cells were placed on ice and loaded with the CCF4-AM substrate ( Life Technologies ) . The cytoplasmic BlaM activity ( ratio of blue to green fluorescence ) was measured after an overnight incubation at 12°C , using the Synergy HT fluorescence microplate reader ( Bio-Tek , Germany ) . IAV was pre-bound to A549-IFITM3 cells in the cold , followed by incubation at 37°C for 90 min and immunostaining with mouse anti-IAV-NP ( Millipore , Billerica , MA ) ( when applicable ) and rabbit anti-IFITM3 antibody ( N-terminus , Abgent , San Diego , CA ) , as described in [13] . Rat anti-mouse-IgG-FITC ( eBioscience , San Diego , CA ) and goat anti-rabbit-Cy5 antibodies were used for secondary staining . Cellular distribution of cholesterol was examined by incubation with 0 . 25 mg/ml filipin added during the incubation with secondary antibodies . Images were collected on a LSM 780 laser scanning microscope ( Carl Zeiss , Germany ) using a 63× oil immersion objective . All staining methods involved fixation with 2% paraformaldehyde , permeabilization with 0 . 25% Triton-X100 , blocking in with 10% FBS and dilution in phosphate buffered saline ( with calcium and magnesium ) , and sequential incubation with primary and secondary antibodies for 2 h and 1 h , respectively . To silence the NPC1 gene , A549 cells were transduced with five shRNAs encoded by pLK0 . 1 lentiviral vector ( Sigma ) and selected with puromycin . The samples for Western blotting were processed as described in [24] . The NPC1 protein band was detected with rabbit anti-NPC1 ( Abcam , Cambridge , MA ) and horseradish peroxidase-conjugated Protein G ( Bio-Rad , Hercules , CA ) , using a chemiluminescence reagent from GE Healthcare . Cells grown on glass-bottom Petri dishes ( MatTek , MA ) were chilled on ice and washed with cold Hank's balanced salt solution ( HBSS ) . Predetermined amount of viral suspension ( MOI∼0 . 01 ) was added to the cells and spinoculated at 4°C for 20 min . The cells were then washed twice with cold HBSS and placed on the stage of an LSM 780 confocal microscope . Virus entry was initiated by adding 2 . 5 ml of pre-warmed imaging buffer and imaged at 37°C using a C-Apo 40×/1 . 2NA water-immersion objective . Three Z-stacks separated by ∼2 µm were acquired every 7–8 s through the MultiTime macro ( Carl Zeiss ) . To block IAV hemifusion and fusion , experiments where performed in HBSS supplemented with 50 mM HEPES/70 mM NH4Cl ( pH 7 . 6 ) or containing 200 nM of BafA1 . The time lapse images were first visually inspected to identify vDiD dequenching or loss of mCherry events . The number of relevant events in each experiment was independently determined by two trained individuals . Particle trajectories and their mean/total fluorescence intensities were obtained using Volocity ( PerkinElmer , MA ) . The onset of lipid mixing and the initial slope of vDiD dequenching were determined by fitting to a pair of straight lines ( Fig . S11 ) . IAV particles were co-labeled with the AF488 dye ( pH-insensitive ) and CypHer5E , which fluoresces brighter at acidic pH . The ratios of the CypHer5E and AF488 signals were converted to pH values using a calibration curve obtained by exposing coverslip-immobilized viruses to citrate-phosphate buffers of different acidity ( Fig . S7 ) . Images were collected from 3 different fields , and sum of single-particle fluorescence was calculated . The mean ratios of CypHer5E to AF488 signals as a function of pH were used for the calibration curve . Cells were inoculated with labeled viruses for 45 min at 37°C , as described above . Images were collected from at least 10 different fields , and single particle-based ratio of fluorescence signals was calculated . Outliers with a near-background CypHer5E signal were rejected to reduce the uncertainty in pH measurements . Statistical significance was assessed using the pairwise t-test or rank sum test . Single-particle fusion events in control and IFITM3 expressing cells were compared by the z-test .
|
Interferon-induced transmembrane proteins ( IFITMs ) block infection of many enveloped viruses , including the influenza A virus ( IAV ) that enters from late endosomes . IFITMs are thought to prevent virus hemifusion ( merger of contacting leaflets without formation of a fusion pore ) by altering the properties of cell membranes . Here we performed single IAV imaging and found that IFITM3 did not interfere with hemifusion , but prevented complete fusion . Also , contrary to a current view that excess cholesterol in late endosomes of IFITM3-expressing cells inhibits IAV entry , we show that cholesterol-laden endosomes are permissive for virus fusion . The ability of IFITM3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes , either directly or indirectly , through altering its physical properties . IFITM3 may also redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"cell",
"biology",
"biology",
"and",
"life",
"sciences",
"immunology",
"microbiology",
"molecular",
"cell",
"biology",
"biophysics"
] |
2014
|
IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion
|
Linking synaptic connectivity to dynamics is key to understanding information processing in neocortex . Circuit dynamics emerge from complex interactions of interconnected neurons , necessitating that links between connectivity and dynamics be evaluated at the network level . Here we map propagating activity in large neuronal ensembles from mouse neocortex and compare it to a recurrent network model , where connectivity can be precisely measured and manipulated . We find that a dynamical feature dominates statistical descriptions of propagating activity for both neocortex and the model: convergent clusters comprised of fan-in triangle motifs , where two input neurons are themselves connected . Fan-in triangles coordinate the timing of presynaptic inputs during ongoing activity to effectively generate postsynaptic spiking . As a result , paradoxically , fan-in triangles dominate the statistics of spike propagation even in randomly connected recurrent networks . Interplay between higher-order synaptic connectivity and the integrative properties of neurons constrains the structure of network dynamics and shapes the routing of information in neocortex .
Understanding any complex system requires a mechanistic account of how dynamics arise from underlying architecture . Patterns of connections shape dynamics in diverse settings ranging from electric power grids to gene transcription networks[1–5] . It is critical to establish how synaptic connectivity orchestrates the dynamics of propagating activity in neocortical circuitry , since dynamics are closely tied to cortical computation . For example , trial-to-trial differences in network dynamics[6–9] can be used to decode sensory inputs and behavioral choice[10 , 11] . It is particularly important to understand the transformation from connectivity to activity within local populations of neurons since this is the scale at which the majority of connections arise . Locally , neocortical neurons are highly interconnected , and their connectivity schemes are characterized by the prevalence of specific motifs[12] . At the level of local populations , functional coordination has been demonstrated in diverse ways , e . g . on the basis of active neurons[13 , 14] and their correlation patterns[15] . Yet predicting population responses on the basis of pairwise connections alone has proven to be difficult . Establishing a mechanistic link between connectivity and dynamics in neocortical networks is intricate and non-trivial because individual neurons themselves are complex computational units[16–20] . Fundamentally , neurons are state dependent non-linear integrators of synaptic input[21–23] . When neurons in neocortex process information , they are generally subjected to numerous synaptic inputs which activate diverse receptors , and concomitant gating of voltage-dependent channels[24–26] . In consequence , neocortical neurons tend to operate in a high-conductance state , which lessens the impact of any one synaptic input[21 , 27] . Because inputs are weak individually , collective synaptic bombardments are necessary to depolarize a neuron to threshold for action potential generation . As a result , it is difficult to predict the flow of activity through a synaptic network based solely on knowledge of single connections , without the context of ongoing activity in the entirety of the system . Network models are an important tool for linking synaptic connectivity to dynamics in neocortex because they enable precise measurement and manipulation of simulated connectivity . In this work , we generate networks comprised of leaky integrate-and-fire model neurons with naturalistic dynamics that mimic recordings from superficial neocortical layers . Despite random synaptic topology in the model network , we find that small-world topological organization emerges in maps of propagating activity . This paradoxical divergence of dynamics from synaptic connectivity is not explained by coactivity alone . Rather , recruitment preferentially occurs in a selective subset of active connected pairs . In the model , activity is preferentially routed through clustered fan-in triangles , despite their statistical scarcity . Because they result in coordinated presynaptic timing , fan-in triangle motifs are particularly effective for spike generation . By comparison , among neurons converging on a common target but lacking presynaptic interconnectivity , presynaptic timing is less synchronous on average , and postsynaptic recruitment is less likely . Moreover , when we decrease the need for cooperative presynaptic action , by doubling synaptic weights in network models , the fan-in triangle motif becomes significantly less prevalent . We evaluate the prediction of our model using high speed two-photon imaging of emergent network activity ex vivo , in somatosensory cortex . We verify that propagating activity in real neuronal networks has small-world characteristics and elevated clustering , Decomposing this clustering , we discover that neocortical circuitry also manifests propagating activity that is dominated by the fan-in triangle motif . These results suggest a mechanistic account for the widespread findings of clustered activity in neuronal populations [14 , 28–31] . We suggest that clustered fan-in triangles are a canonical building block for reliable cortical dynamics .
Multineuronal dynamics are the computational substrate for sensation and behavior , implemented by synaptic architectures . Propagating multineuronal activity arises from three main sources: the underlying connectivity itself , recent network history , and the non-linear integrative properties of individual neurons . Here , multineuronal activity was modeled using conductance-based leaky integrate-and-fire neurons , stimulated with brief periods of Poisson input and recorded during self-sustained firing ( Fig 1A ) . Model neurons were connected with heterogeneous synaptic weights drawn from a heavy-tailed distribution , in a random arrangement ( Erdős-Rényi; pee = 0 . 2 ) . Simulated dynamics were asynchronous , irregular , and sparse , with critical branching ( see Methods ) . A synaptic network was constructed for each simulation , consisting of excitatory model neurons and their synaptic connectivity . For each structural iteration of the model we generated three distinct maps of activity ( and in two of the cases , multiplex connectivity and activity ) : a functional network , the active subnetwork , and a recruitment network ( Fig 2 ) . Edges in the functional network summarized network dynamics and represented frequency of lagged firing between every pair of nodes ( with maximum interspike interval T = 25 ms; see Methods ) . The active subnetwork was a subgraph of the synaptic network and consisted of model neurons active at least once and all their interconnections ( regardless of lagged firing relationships ) . Finally , the recruitment network was a subgraph of the functional network defined by its intersection with the synaptic network , to map the routing of activity through synaptic interactions . In this way , non-zero edges in the recruitment network linked synaptically connected nodes that also spiked sequentially in the interval T at least once . For T = 25 ms , 10 . 9 ± 3 . 52 excitatory presynaptic input spikes immediately preceded each postsynaptic spike ( mean±std ) . Surprisingly , although underlying synaptic connectivity was Erdős-Rényi ( i . e . random ) , functional activity networks were small world ( Fig 1B ) [32] . To judge the small world character of these networks , global clustering coefficient and characteristic path were normalized by their respective abundances in density-matched Erdős-Rényi networks and combined as a quotient[33] . Comparison with density-matches was important given that sparseness itself results in enhanced smallworldness[34] . Functional networks were marked by significantly increased small world scores ( functional network: 2 . 8±0 . 23; synaptic network: 1 . 0±0 . 035; n = 5 , p = 0 . 0079 , Wilcoxon rank-sum ) resulting from increased clustering ( function: 2 . 8±0 . 23; synaptic network: 1 . 0±0 . 035 , n = 5 , p = 0 . 0079 ) , with characteristic path lengths similar to random-matches ( function: 1 . 0±6 . 4x10-4; synaptic network: 0 . 99±0 . 033; n = 5 , p = 0 . 69 ) . The lag interval T was chosen to encompass important network timescales for synaptic plasticity and integration[35 , 36] . We also generated functional networks using intervals of 10 and 50 ms , which showed that the emergence of non-random features does not depend strongly on choice of T ( functional network for T = 10ms: small world ratio 3 . 2±0 . 24 , n = 5 , p = 0 . 0079; functional network for T = 50ms: small word ratio 2 . 6±0 . 22 , n = 5 , p = 0 . 0079 ) . Given modest sampling conditions ( e . g . binning near timescales of synaptic integration ) , functional relationships can indicate locations of probable synaptic recruitment[35] . However , a subset of edges in functional networks are 'false positives'—they reflect polysynaptic relationships and other combined statistical dependencies rather than monosynaptic connectivity and recruitment[35 , 37] . To determine whether these measurement artifacts were responsible for the statistical differences between functional and synaptic networks , we turned to recruitment networks . Pruned of false positives , recruitment networks were significantly more small world than functional networks constructed from the same activity ( 4 . 6±0 . 87; n = 5 , p = 0 . 0079 ) , with even shorter characteristic paths ( recruitment: 0 . 65±0 . 072 , n = 5 , p = 0 . 0079 compared to function , Wilcoxon rank-sum ) and a similar elevation in clustering ( recruitment: 3 . 0±0 . 26; n = 5 , p = 0 . 22 ) . Thus , emergent statistical structure in the functional networks reflected coordinated timing among multiple synaptically connected neurons . As demonstrated by non-random recruitment , i . e . clustering in the recruitment network , activity did not propagate homogeneously through the random topology . However , it remained a possibility that the seemingly non-random routing of activity was simply the byproduct of shared activity , without being selective on the basis of connectivity . As a control , the active subnetwork establishes the role of interactions among neurons with elevated firing rates ( including pairs of neurons which never recruited one another within the interval T ) . Compared to functional networks , the corresponding active subnetwork exhibited reduced small world ratio ( active network: 2 . 2±0 . 26 , n = 5 , p = 0 . 0159 ) and reduced clustering ( 1 . 3±0 . 041 , p = 0 . 0079 ) , despite somewhat shorter characteristic paths ( 0 . 60±0 . 055 , n = 5 , p = 0 . 0079 ) . If directed connections that never fired sequentially were pruned from the active subnetwork , it would attain the same binary topology as the recruitment network . Comparing the active network with the recruitment network , global clustering ratio was significantly increased ( from 1 . 3±0 . 041 to 3 . 0±0 . 26 , n = 5 , p = 0 . 0079 , Wilcoxon rank-sum ) . Thus , the select connections which were directly involved in propagation of spiking activity were more clustered than activated connections as a whole ( Fig 1C ) . We next evaluated whether neuronal pairs that never fired sequentially differed from those that did . Comparisons were performed between in-degree matched samples . Connected neurons that never fired in succession shared significantly fewer neighbors than those that did fire sequentially at least once ( n = 500 pairs , p = 3 . 1 x 10−17 , Wilcoxon rank-sum ) . In the model , activity was selectively routed through interconnected neighborhoods . Connectivity within a triplet is the simplest way two nodes can share a common neighbor and be clustered . However , this measure fails to account for the direction of connection . Since direction is crucial in synaptic communication , we turned to a formulation which differentiates directed triangle motifs[38] . From the perspective of a reference postsynaptic neuron , clustered neighbors can be arranged into four kinds of three-edge triangle motifs: fan-in , fan-out , middleman , and cycle arrangements ( Fig 3A ) . Taken in isolation , fan-in , middle-node , and cycle triangles are isomorphic to one another through rotation , i . e . dependent on labeling the reference node ( which is necessary to compute local clustering ) . Measures of undirected clustering can be decomposed fractionally into these four components . Because the underlying model synaptic connectivity was random , none of the four triangle motifs were more prevalent than the others , and each contributed equally to synaptic clustering ( Fig 3B ) . By contrast , in recruitment networks , fan-in triangle motifs were highly overrepresented ( Fig 3C ) . The overrepresentation of fan-in triangle motif was also present in the functional network: for example , iterative Bayesian inference[35] was sensitive to asymmetric directed clustering in model activity ( fan-in: 0 . 38±0 . 052 , fan-out: 0 . 29±0 . 032 , middleman: 0 . 19±0 . 016 , cycle: 0 . 15±0 . 0076; mean±std , threshold at the 95th percentile ) . To understand whether these higher order asymmetric features emerge from chance correlations tied to firing rates , we generated Poisson populations that were rate-matched on a neuron-by-neuron and trial-by-trial basis . This resulted in an inhomogeneous distribution of firing rates across all trails . Our Poisson null populations had identical expected spike counts as model activity in each 100ms bin but no synaptic interactions and no causal propagation of activity . Undirected clustering was significantly lower in iterative Bayesian maps of uncoupled Poisson rate-matched activity compared to connected network models ( Poisson rate-match: 0 . 0052±3 . 6x10-4; simulated activity: 0 . 024±0 . 013; Wilcoxon rank-sum p = 0 . 036; n = 3 ) , and the fan-in triangle motif was not elevated relative to other clustering patterns ( Fig 3D ) . The Poisson populations demonstrated that elevated fan-in triangle motifs do not result trivially from the analysis procedure but instead are the result of synaptic interactions between neurons . Interestingly , we found that model neurons with high fan-out clustering were characterized by elevated firing rates ( Fig 4A and 4B ) , but model neurons which comprised the fan-in triangle motif actually contracted towards low firing rates ( Fig 4C and 4D ) . Fan-in triangles were more abundant in propagating activity than would be expected from their frequency in the synaptic network or component firing rates alone . Like undirected clustering , the emergence of fan-in clustering in maps of propagating activity was robust to choice of T . Fan-in clustering was highly elevated in recruitment maps for T = 10 ms ( undirected: 0 . 0068±0 . 0007; fan-in 0 . 011±0 . 0017; fan-out: 0 . 0028±0 . 0001; middle-node: 0 . 0068±0 . 0007; cycle: 0 . 0052±0 . 0004; mean±std for 5 simulations ) and T = 50 ms ( undirected: 0 . 019±0 . 0015; fan-in 0 . 027±0 . 0027; fan-out: 0 . 0077±0 . 0003; middle-node: 0 . 019±0 . 0013; cycle: 0 . 015±0 . 0007; mean±std for 5 simulations ) . Because of the different levels of sparseness in the numbers of connections these values should not be compared across values of T . Instead these analyses demonstrate that the over-representation of fan-in triangles is robust across a number of timescales . To investigate the mechanism for overrepresentation of fan-in triangles in recruitment networks , we measured spike timing at their locations . The signature of fan-in triangle motifs is convergence from interconnected presynaptic neurons , a motif that could potentially facilitate cooperative summation of synaptic inputs . Consistent with this postulate , presynaptic neurons in fan-in triangle motifs were marked by increased probability of firing in the 10 ms prior to postsynaptic spiking ( Fig 5A and 5B ) . We next compared differences in presynaptic timing relationships at loci of fan-in triangle motifs compared to loci of simple convergence , to assess the role of presynaptic interconnectivity . For this analysis , random samples were obtained from epochs of coincident firing: 50 ms windows where every neuron in a triplet was active , centered on a spike in the postsynaptic reference neuron . To avoid confounds from juxtaposing multiple motifs , neuron triplets with any additional connections , including recurrent loops , were excluded for this specific analysis alone . As a result only fan-in triangles with exactly three interconnections were analyzed in this case . We found fan-in presynaptic neurons were stereotypically ordered in a manner consistent with the direction of their interconnection , resulting in an asymmetric distribution of intervals between their firing ( Fig 5C ) . In addition to the temporal structure imposed by this asymmetry , mean absolute timing difference between presynaptic neurons in clustered fan-in motifs was modestly but significantly more temporally precise than were neurons in simple convergence motifs ( 13 . 5±10 . 2 ms compared to 14 . 9±10 . 7 ms; Wilcoxon rank-sum on mean-absolute timing difference , p = 0 . 0035 , n = 1000 samples ) . Moreover , we found that coincidence in fan-in triangle motifs occurred nearly twice as frequently as in motifs of simple convergence ( 1 . 9 ± 0 . 17 times more frequent , mean ± std; Wilcoxon rank-sum , p = 0 . 0079 , n = 5 model datasets ) . Accounting for expected frequency of the two connection patterns in the underlying synaptic network , coincident activity is far more common at sites of fan-in triangles than at sites of simple convergence ( linear regression: slope 3 . 0 , y-intercept 0 . 00075 , n = 5 simulations , r2 = 0 . 91 , p = 0 . 011 ) ( Fig 5D ) . We postulated that clustering is efficacious for synaptic integration and examined whether the prevalence of clustering was predictive of postsynaptic membrane potentials . Pooling over all neurons and time bins , we binned the distribution of membrane voltages into segments that contained equal numbers of samples ( Fig 6A ) . On average , because the model was active in the analyzed simulations , membrane voltages were depolarized from the resting equilibrium potential of -65 mV ( median: -60 . 2 mV; lower quartile: -63 . 6 mV; upper quartile: -56 . 8 mV ) . To test our hypothesis , we generated functional networks that related recent presynaptic activity ( within a 25 ms interval ) to postsynaptic voltage ( Fig 6B; see Methods ) , yielding one network for each division of the voltage distribution ( Fig 6C ) . These networks can be viewed as reverse correlograms conditioned on postsynaptic voltage , and differed in the statistics of their topologies across different voltage regimes . At more negative membrane potentials , the active neurons which connected to the postsynaptic reference neuron ( and accounted for its recent excitatory synaptic drive ) were only modestly more clustered than random sparseness-matched controls . As the postsynaptic neuron depolarized , the presynaptic nodes driving that depolarization became increasingly clustered , peaking at the threshold for firing ( Fig 6D ) . Characteristic paths were similar to random graphs at all subthreshold voltages . As a result of elevated clustering during membrane depolarization , small world ratios peaked at the most depolarized voltages corresponding to threshold for action potential generation . These data support the hypothesis that activity among clustered presynaptic neurons is particularly effective for recruiting the postsynaptic neuron to spike . The statistical incongruence of function and synaptic connectivity indicates that spiking activity does not flow in an egalitarian fashion through the synaptic network . Instead , patterns of local clustering influence and direct where propagating activity occurs most frequently . That is , patterns of activity are shaped by higher-order patterns in synaptic connectivity and not just pairwise couplings . To further explore the dependence of activity flow on higher order synaptic connections we evaluated postsynaptic recruitment in a network model with a modest increase in mean synaptic strength . Synaptic connections were twice as strong on average compared to the network models used throughout the remainder of this study but remained too weak to drive spiking alone ( Fig 7A ) . The two network designs did not differ in connection density . After synaptic weights were doubled , functional networks became more similar in topology to synaptic networks ( small world ratio decreased; Wilcoxon rank-sum , p = 0 . 0079 , n = 5 ) ( Fig 7B ) . The double-strength models were less clustered ( Fig 7C ) ( Wilcoxon rank-sum , p = 0 . 0079 , n = 5 ) , and exhibited longer average path lengths ( Wilcoxon rank-sum , p = 0 . 0079 , n = 5 ) . Directed clustering was compared across the two families of models . Recruitment networks were analyzed with binary edges to control for their distinct mean synaptic weights . In addition to their decreased overall clustering , the fan-in triangle motif was significantly rarer in double-strength recruitment networks ( Fig 7D ) ( from 0 . 030±0 . 0051 to 0 . 022±0 . 0025 , p = 0 . 030 , n = 6 ) , while the fan-out triangle motif showed a small but significant increase in abundance ( from 0 . 0040±2 . 0x10-4 to 0 . 0046±3 . 2x10-4 , p = 0 . 0043 , n = 6 ) . Stronger presynaptic inputs reduced the need for extensive postsynaptic integration , allowing individual presynaptic cells to have a more independent impact on their postsynaptic partners . As a result , statistics of propagating activity were more faithful to underlying pairwise connections in the models with increased synaptic strength . In model simulations , fan-in triangle motifs were abundant in maps of function and recruitment . We next evaluated whether the preponderance of fan-in triangle motifs would be robust to additional complexity in single-neurons and their connections . Unlike the simple model neurons that we used for simulation , real neurons are complex elements[16] and the connections between them are structured[12 , 39] . If clustered fan-in triangle motifs are a general feature of high-conductance nodes in a complex system , where coordinated inputs drive integration , the fan-in triangle will be overabundant in experimental dynamics . This postulate would be falsified if all directed clustering motifs were equally common in functional networks . To investigate , we analyzed high speed imaging data ( 20 Hz ) of spontaneous circuit activity collected ex vivo in mouse somatosensory cortex ( Fig 8A ) ( following [40] ) . We generated functional networks from the imaged experimental data using an iterative Bayesian approach which is robust to relatively small numbers of observations [33] . We then measured the prevalence of fan-in motifs in the functional topology ( Fig 8B ) . Importantly , iterative Bayesian inference was not biased toward detection of fan-in triangle motifs , as demonstrated with rate-matched Poisson spiking ( see Fig 3D ) . Though imperfect indicators , functional weights probabilistically identify the likelihood of true monosynaptic excitatory connectivity[35] . As a result , expected error rate for inferred connections can be adjusted with a sliding threshold on functional weight . Stricter thresholds yield a more accurate approximation of the underlying recruitment network at the cost of restricted sampling . Using inferred recruitment networks , beginning at the top quartile of inferred weights , directed clustering was computed in five-percentile increments . Confidence intervals were obtained using bootstrap resampling under the assumption of a 30% false-positive rate . As confidence of synaptic connectivity increased , the fan-in triangle motif became increasingly abundant and fan-out triangles less so ( Fig 8C ) . Differences between the two motifs were significant ( threshold at 95th percentile , p = 4 . 8x10-34 , n = 100 bootstrap resampled functional networks , Wilcoxon ranksum ) . We next measured whether strong functionally coupled neurons were more spatially proximal than random pairs . We defined strong functional connections as those exceeding a 95% threshold on non-zero weights since previous work has indicated that these particular functional connections are more likely to reflect a causal synaptic connection[35] . We found that the median pairwise distance separating strong functionally connected cells was 249 μm , whereas randomly chosen pairs of neurons were separated by a median 263 μm ( Wilcoxon-ranksum p = 0 . 0336 , nfunctional = 638 , nrandom = 10000 ) . We then measured triplets of neurons with functional connections that form triangles to determine whether these neurons were more spatially proximal to one another than randomly chosen triplets of neurons . To investigate , proximity was quantified as the perimeter around the triangle formed by vertices at the spatial location of each neuron . Neurons in functional triangles with mutual connectivity and at least three functional connections were inscribed by perimeters of median length 807 μm , compared to median perimeter of 823 μm for randomly selected triplets that were unconstrained by direction and number of edges ( Wilcoxon rank-sum p = 0 . 0097 , ntriangles = 2556 , nrandom = 10 , 000 ) . Interestingly , triplets of neurons connected into arrangements of either simple divergence or simple convergence ( i . e . neurons in wedges , lacking interconnectedness between the common neighbors ) , were even more distant , inscribed by a perimeter of median 839 μm ( Wilcoxon rank-sum , ntriangles = 2556 , nwedges = 14 , 882 ) . Thus , clustered triplets ( triangles ) tended to be arranged significantly more locally than simple convergent or simple divergent triplets ( wedges ) . We then compared measures of clustering between the model , which was comprised of random connections , and the experimental data which almost certainly contained structured connectivity [12 , 39] to evaluate how the measure of fan in and fan-out triangles depend on the underlying structural topology . To do so we used a measure of clustering propensity[41] which allowed us to make comparisons of networks which have very different connection densities . Clustering propensity ( 1-ΔCfan-in and 1-ΔCfan-out ) results in a normalized value where 1 is extreme clustering as seen in lattices , and 0 indicates no clustering above that expected in Erdős-Rényi random networks . For the model , fan-in clustering was scored at 0 . 18 ± 0 . 019; and for the experimental data , fan-in clustering was scored at 0 . 20 ± 2 . 0x10-4 ( Wilcoxon ranksum p = 1 . 74x10-4 , nmodel = 5 simulations; ndata = 100 bootstrap samples ) . Thus , fan-in clustering was modestly but significantly more abundant in maps of propagating activity based on experimental recordings . We note that we compared thresholded graphs at the 80%-level ( i . e . top 20% of non-zero edges ) for this measure because the experimentally derived functional networks were not well-matched by regular lattices below this density . Finally , we measured timing relationships among imaged active neurons . Reliable timing relationships were measured independent of other functional analyses , using cross-correlations on the normalized fluorescence traces ( Methods ) . Presynaptic coactivity was assessed as the product of the two z-scored presynaptic traces and compared to postsynaptic fluorescence as a straightforward cross correlation . The resulting average cross-correlogram for fan-in triangles was stronger and more asymmetric than those measured from simple-convergence motifs ( Fig 8D ) . Thus , presynaptic activity in fan-in triangles was more predictive of postsynaptic firing than presynaptic activity in motifs of simple convergence . These results are consistent with fan-in triangles supporting coincident input and favoring reliable propagation of activity . Results from the model indicated that the fan-in triangle motif temporally coordinates presynaptic inputs , rendering them more capable of driving recipient neurons to threshold . Supporting our prediction of its fundamental importance for reliable recruitment , in acutely dissected neocortical tissue with more complex patterns of connectivity and intrinsic neuronal properties , we find a robust elevation of the same directed motif .
Using a model composed of random connections among leaky integrate-and-fire neurons with conductance-based synapses , we found that maps of propagating activity were structured and non-random . Small-world patterning in the dynamics emerged because a specific higher-order connection pattern was particularly effective for postsynaptic integration: convergence of synaptic input from connected neighbors . Synaptic connections between neighbors favored coincident timing of inputs onto their targets . This coincident activation led to efficient postsynaptic integration . As a consequence , clustering among active presynaptic cells tracked depolarization of model postsynaptic neurons . Thus , activity was preferentially routed through fan-in triangle motifs . In experimental recordings of emergent activity in hundreds of neurons ex vivo , after mapping inferred recruitment patterns [33] , we found that fan-in triangles were even more dramatically overrepresented than in the model . These results are contextualized by increasing recognition of non-random functional structure in networks of neurons: Rich club structure has been reported ex vivo and in vivo[31] . Clustered[30] , small world functional networks[28] , and nucleation of dynamics[29] have also been observed in neuronal cultures . Since cultured populations differ from neocortex in the details of their topological makeup , these findings across model systems further suggest that clustering in general and the fan-in triangle motif in particular may be a canonical feature of propagating activity among interconnected neurons . Despite differences in details of connectivity and neuronal intrinsic properties , dynamics are constrained by the requirement for coincident summation of individually weak inputs . Constraining dynamics with beyond-pairwise relationships can be helpful for cortical computation . Theoretical work has shown that non-uniform features of connection topology impact information transfer[42] , and higher-order correlations were particularly impactful in low spike-rate regimes[43] . These complementary results from complex networks , statistical physics and network biology suggest that , by shaping feasible dynamics , the fan-in triangle motif could enhance information transfer from inputs to outputs . We hypothesize that local circuits are organized around fan-in triangle motifs , promoting cooperative patterns of firing and stabilizing[44] the propagation of activity despite individually unreliable neurons . This canonical mechanism provides the coordination necessary to propagate signal despite weak synaptic connections . Indeed , reliable sequential firing was associated with number of fan-in triangles even after controlling for overall in-degree . Although clustering among fan-in triangles has not been tested directly until now , paired patch clamp recordings have shown that local neocortical circuitry is characterized structurally by abundant triplet motifs[12 , 39] . Our data and modeling suggest a functional consequence for a subset of these synaptic motifs: connected presynaptic neurons help establish coordinated timing among convergent inputs , leading to cooperative summation at the postsynaptic membrane . Such cooperativity has been shown to be one potential mechanism capable of generating spike trains that are consistent with experimental observations in vivo[45] . While there are certainly explicit developmental rules that govern neuron to neuron connectivity , our results suggest that higher-order connectivity need not require specification a priori . It could emerge autonomously if fan-in triangle motifs within a random network were stabilized and magnified during network development , e . g . by pruning non-recruiting connections through activity-dependent plasticity . Thus , higher-order synaptic motifs that are particularly effective for postsynaptic recruitment could potentially self-organize[46] . These results do not indicate a complete schism between synaptic connectivity and dynamics—one clearly depends on the other . However , their relationship is complicated by the integrative properties of single neurons . Synaptic integration constrains feasible dynamics , and distributed synaptic motifs route the propagation of activity . These interactions are a source of higher-order dynamical structure . The routing of information is coordinated by higher-order synaptic patterns and the context of ongoing activity because the routing of spikes is determined by relative timing and collective interactions .
Simulations were implemented using the Brian Brain Simulator[47] . Model populations consisted of 1000 excitatory neurons , 200 inhibitory neurons and 50 Poisson input units . Connection probabilities depended on source and target identity . For example , inhibitory-excitatory connections occurred with probability 0 . 25 ( Pee = 0 . 2 , Pei = 0 . 35 , Pie = 0 . 25 , Pii = 0 . 3 ) . Conductance based synaptic weights were drawn from a heavy-tailed distribution and assigned randomly[48 , 49] . Weights were drawn randomly from a lognormal distribution with mu = -0 . 64 and sigma = 0 . 51 . These parameters are the mean and standard deviation of the corresponding normal curve . The resulting lognormal ensemble has expected mean of 0 . 60 and variance of 0 . 11 , in multiples of the leak conductance . Connections from inhibitory to excitatory cells were scaled by a further 50% to simulate efficacious somatic contacts . A small tonic excitatory drive gt was supplied to all units to help stabilize sparse spiking . Synaptic bombardments induced exponentially shaped membrane conductances with leaky-integrate-and-fire summation . Conductance-based synapses are important for recapitulating synaptic integration in the high-conductance state[21 , 50] . We used sparse and randomly connected networks in which we did not impose any synaptic organization beyond cell-type dependent connection probabilities . Trials began with 50 ms of activity in the input pool at 15 Hz , exciting the network via random input projections . After input units were silenced , the recording period began , and activity flowed through the network for 100 ms . Input units projecting to excitatory cells randomly and independently with probability 0 . 1 . Every 100 trials ( an epoch ) , new random projections were drawn from the input pool to the excitatory population , simulating a diversity of activity . Participation during a single input epoch totaled 64±0 . 98% of neurons ( mean ± std ) , growing to encompass 85 . 5% of neurons when all sets of input projections were considered ( i . e . over all epochs ) . Excitatory reversal potential Ee was 0 mV , as was Et . Inhibitory reversal potential Ei was -90 mV . Reversal potential for leak current Eleak was -65 mV . Firing threshold was -48 mV , and post-spike reset was -70 mV . In addition to after spike hyperpolarization induced by the reset potential , a 1 ms absolute refractory period was imposed on model neurons . Leak conductance gleak was fixed at 0 . 20 mS . Tonic depolarizing conductance gt was equal in magnitude to the leak conductance . Membrane time constant τm was 20 ms; excitatory synaptic time constant τe was 10 ms; and inhibitory synaptic time constant τi was 5 ms . Additional description can be found in[35] . Spiking dynamics were compared to in vivo activity according to the following criteria: asynchrony[51] was measured with spike-rate correlations , by convolving spike times with a Guassian kernel of width σ = 3 ms . Among excitatory neurons in the recording period , mean correlation coefficient was 0 . 0019[50] . This asynchrony emerged in the presence of heterogeneous connection strengths , raising the possibility of combining stable propagation with rich internal dynamics[49 , 52] . Irregularity was measured with interspike-intervals , which were observed to have mean squared-coefficient of variation of 0 . 81 , consistent with other reports of irregular activity[53] . To measure inter-spike intervals , model activity was stimulated with Poisson firing for 50 ms , then allowed to evolve for 950 ms in isolation . This procedure was repeated 100 times . Excitatory spiking activity was characterized by a median branching coefficient of 1 . 00 ( for 10 ms bins ) , indicating near-critical dynamics[54–57] . Firing rates in the excitatory population during the recording period were 1 . 33 ± 3 . 15 Hz ( mean ± std ) consistent with findings in awake behaving mice[58] . Collective spiking generated spike-driven conductances that dwarfed the leak conductance , in keeping with definitions of high-conductance state[21] . Call the directed network of synaptic connections among excitatory neurons Esyn and the population of excitatory cells Ve . Construct the directed graph of synaptic connections: Gstructural≡ ( Ve , Esyn ) To map functional relationships using lagged firing , define recent activity for neuron i at time t as firing at least once in the 25 ms preceding t . More formally , we can define random variable Si representing the activity of neuron i such that sit≡{2ifneuroniis firing at timet1ifneuroniis not firing but was active within the last25ms0otherwise} In that case , Eijlag≡P ( sj=2|si>0 ) The recruitment network encompassed synaptically connected neurons manifesting lagged patterns: Eijomniscient≡{EijlagifEijsyn>00otherwise} Grecruitment≡ ( Ve , Eomniscient ) Iterative Bayesian networks were measured with a heuristic optimization procedure , described further below and in [35] , following [28] . Since shortest path measurements assume a cost matrix , edge weights were first inverted so strong connections were cheap and zero-weighted connections were infinitely costly . Shortest paths between all pairs were computed using Dijkstra’s algorithm . Mean path length was compared to sparseness-matched Erdős-Rényi graphs analyzed in the same way . Local clustering coefficients were computed using the neighbors of neighbors formulation[32] and aggregated as the mean over all neurons . Sparseness-matched Erdős-Rényi graphs were analyzed in the same fashion . Clustering score was the ratio of the actual mean to sparseness-matched null mean . Small-world topologies can be quantified as a ratio of ratios , clustering elevation divided by mean path length reduction[33] . Clustering was also investigated using a related definition , the number of connected undirected triangles as a fraction of all possible undirected triangles ( transitivity formulation ) . Directed clustering was computed in the same way , using directed triangles instead of undirected[38] . To compare clustering between data and model networks , across connection densities that were very different , we followed the small-world propensity approach[41] . In that work , clustering levels ΔC are normalized as the fractional distance between density-matched lattice and random graphs . We termed this measure clustering propensity , expressing it as 1 – ΔC so that 1 signified extreme clustering and 0 signified no clustering beyond that expected at random . We made a straightforward extension to this approach to account for directed clustering , simply substituting directed triangle counts for undirected triangle counts , with appropriate normalizations[38] . Quantifications based on clustering propensity recapitulated our findings quantifying clustering as fractional abundance over random expectation . For the set of voltage bins with lower bounds a and upper bounds b , construct one network for each bin k , where edge ( i , j ) k is quantifying the probability model neuron j will have postsynaptic potential Mj between ak and bk conditioned on presynaptic model neuron i being recently active . Recently active was defined as firing within 25 ms relative to postsynaptic voltage measurement . A final condition was imposed: that connected pairs also share a synaptic connection , a convenience of measurement unique to simulated networks . Functional topologies were measured for simulations having typical synaptic weight distributions ( n = 5 ) and for simulations where random draws from the synaptic weight distributions were scaled to double strength ( n = 6 ) . Ratios for global clustering , characteristic path , and smallworldness were quantified following[33] , as above , on the two sets of weighted , symmetrized topologies . Directed clustering was measured following[38] . The directed clustering measurements were conducted on binary topologies to control for potential differences stemming from their different underlying mean synaptic weights . All procedures were performed in accordance with and approved by the Institutional Animal Care and Use Committee at the University of Chicago . One juvenile mouse ( postnatal day 14 , of strain C57BL/6 ) was anesthetized by intraperitoneal injection of ketamine-xylazine and rapidly decapitated . The brain was dissected and placed in oxygenated , ice-cold artificial cerebrospinal fluid ( Cut-ACSF; contents contain the following in mM: 3 KCl , 26 NaHCO3 , 1 NaH2PO4 , 0 . 5 CaCl2 , 3 . 5 MgSO4 25 dextrose , and 123 sucrose ) . The brain was then sliced coronally using a vibratome ( VT1000S; Leica ) into 450 μm thick slices . These slices encompassed the mouse whisker somatosensory cortex . Slices were then transferred into 35°C oxygenated incubation fluid ( Incu-ACSF; contents contain the following , in mM: 123 NaCl , 3 KCl , 26 NaHCO3 , 1 NaH2PO4 , 2 CaCl2 , 6 MgSO4 , 25 dextrose ) for 30 min . Bulk loading of Ca2+ dye was then performed , via transfer of slices into a Petri dish containing ∼2 ml of Incu-ACSF and an aliquot of 50 μg Fura-2AM ( Product code , Invitrogen , location ) dissolved in 13 μl DMSO and 2 μl of Pluronic F-127 ( Code , Invitrogen , location ) ( following [9] ) . Throughout the duration of imaging , slices were continuously perfused with a standard ACSF solution ( contents contain the following , in mM: 123 NaCl , 3 KCl , 26 NaHCO3 , 1 NaH2PO4 , 2 CaCl2 , 2 MgSO4 , and 25 dextrose , which was continuously aerated with 95% O2 , 5% CO2 ) . Visualization of Fura-2AM loaded neurons was performed via serial 5 min recordings , collected using the HOPS scanning technique ( a suite of software and custom microscopy setup developed in-house , see [40] ) . This method allowed us to monitor action potential generation within individual neurons , by detecting contours of loaded cells from a raster image , then computing an efficient traveling salesman tour over those cell bodies . Our dwell time parameter was fixed at a value of 16 samples/cell/frame . Population framerate was 20 Hz , resulting in ~450 neurons sampled once every ~50 ms . Changes in emitted fluorescence were analyzed with a threshold-crossing approach . First , a signal-to-noise cutoff was implemented by measuring the ratio of the 99th percentile divided by the mean for the fluorescence trace of each cell . Cells exceeding 1 . 55 by this metric were retained for further analysis . Of the 444 sampled neurons , 189 exceeded our strict criterion on signal-to-noise ( see Methods ) . Among these cells with clean fluorescent signals , instances of elevated firing were identified from excursions in the signal exceeding two-sigma , with inflection points more precisely identified by following these excursions backwards to the bin of their most recent median-crossing . The resulting binary vector identified high-probability periods of spiking activity across the imaged population[14 , 59] . Recurring timing relationships can be used to identify likely synaptic connections between individual pairs , particularly lagged firing near the timescale of synaptic integration . We used an iterative Bayesian inference algorithm to parse these lagged firing patterns[28 , 35] . The inference algorithm was initialized five times , and final weights were pooled as an average . The combined network was thresholded to isolate its strongest relationships . With increasing threshold , functional relationships became more precise in indicating true monosynaptic connectivity , and also more confidently overabundant in the fan-in triangle motif . To understand the impact of mistaken inferences from a different perspective , independent of relationships between functional weight and true connectivity , bootstrap resampling was used to estimate how errors in inferred connectivity affected estimates of directed clustering measures . For an error rate of 30% estimated from simulated experimental constraints[35] , differences in directed clustering were significant even after redacting possible false positives ( 100 bootstrap-resampled topologies; Fig 8C and 8E ) . In a typical simulated network , the density of the recruitment network was 0 . 049 , meaning only about one quarter of synaptic connections were a site of propagating activity . Since only those pairs are visible in patterns of lagged firing , the density of recruiting connections was shown for an additional definition of optimal performance ( one potentially more appropriate for models with sparse firing ) . Average cross-correlations were computed over a two-second sliding window using z-scored fluorescence traces . The first signal was computed as the product of two putative presynaptic fluorescence traces , as a simple score of their activity and/or coactivity . The second signal was the postsynaptic fluorescence trace . Their raw cross-correlation measures the timing offsets between putative presynaptic activity and postsynaptic firing . The functional relationships used to define fan-in triangle motifs versus simple convergence motifs inferred using iterative Bayesian inference , on the basis of single-frame lagged activity , measured in 50 ms bins .
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Active networks of neurons exhibit beyond-pairwise dynamical features . In this work , we identify a canonical higher-order correlation in network dynamics and trace its emergence to synaptic integration . We find that temporally coordinated firing preferentially occurs at sites of fan-in triangles—a synaptic motif which coordinates presynaptic timing , leading to greater likelihood of postsynaptic spiking . The influence of fan-in clustering leads to the surprising emergence of non-random routing of spiking in random synaptic networks . When synaptic weights are made artificially stronger in simulation , so that cooperative input is less crucial , dynamics are no longer dominated by fan-in triangles but instead more closely reflect the random synaptic network . Thus , the emergence of fan-in clustering in maps of synaptic recruitment is a collective property of individually weak connections in neuronal networks . Because higher-order interactions are necessary to shape the timing of presynaptic inputs , activity does not propagate uniformly through the synaptic network . Like water finding the deepest channels as it flows downhill , spiking activity follows the path of least resistance and is routed through triplet motifs of connectivity . These results argue that clustered fan-in triangles are a canonical network motif and mechanism for spike routing in local neocortical circuitry .
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2016
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Higher-Order Synaptic Interactions Coordinate Dynamics in Recurrent Networks
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Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp . The different host innate immune responses are partially related to the different severities of leptospirosis . In this study , we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages ( MPMs ) and human peripheral blood monocytes ( HBMs ) to leptospiral infection . We uncovered a series of different expression profiles of these two immune cells . The percentages of regulated genes in several biological processes of MPMs , such as antigen processing and presentation , membrane potential regulation , and the innate immune response , etc . , were much greater than those of HBMs ( >2-fold ) . In MPMs and HBMs , the caspase-8 and Fas-associated protein with death domain ( FADD ) -like apoptosis regulator genes were significantly up-regulated , which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection . In addition , the key component of the complement pathway , C3 , was only up-regulated in MPMs . Furthermore , several cytokines , e . g . interleukin 10 ( IL-10 ) and tumor necrosis factor alpha ( TNF-alpha ) , were differentially expressed at both mRNA and protein levels in MPMs and HBMs . Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level . Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection , this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis . Taken together , these findings will facilitate further molecular studies on the innate immune response to leptospiral infection .
Leptospirosis is an important tropical infectious disease around the world , particularly in humid tropical and subtropical countries [1] , [2] . The causal agents include several pathogenic Leptospira spp . , of which the highly virulent strains ( e . g . Leptospira interrogans ) chronically infect reservoir hosts ( e . g . wild rodents ) without causing severe symptoms; however , L . interrogans acutely infects humans and causes severe organ failure and mortality in some individuals . The urine released from a chronically infected reservoir host contains a high concentration of leptospiral cells , which can survive and replicate in moist soil and water for a long time before infecting the next subject . The pathogen can infect humans through mucous membranes or abrasions in the skin , penetrate into the blood stream , and rapidly diffuse into the liver , lung , kidney , and other organs [1] . The clinical symptoms are complex , including hemorrhage , diarrhea , jaundice , severe renal impairment , aseptic meningitis , etc . [2] . Multiple components of the pathogen , such as lipopolysaccharide ( LPS ) [3] , peptidoglycans [4] , glycolipoproteins [5] , lipoproteins [6] , and transmembrane or outer membrane proteins ( OMPs ) [6] , are involved in induction of the host immune response and cytokine secretion . Although previous research has shown that humoral immunity is important in leptospirosis [7] , [8] , the role of innate immunity in controlling leptospiral infection has recently been uncovered in cell infection models and animal infection models . Phagocytosis is key to the early defenses of hosts to bacterial infection , while pathogenic Leptospira can escape complement attack and phagocytosis upon infection [9] , [10] . In in vitro cell infection models , unlike nonpathogenic L . biflexa , pathogenic L . interrogans can rapidly attach and invade macrophages [11] , [12] and induce apoptosis [13] . Pathogenic Leptospira have also been found to survive and replicate in human macrophages but are killed in murine macrophages [14] . The LPSs of pathogenic Leptospira activate human macrophages only through Toll-like receptor 2 ( TLR2 ) [3] , while they activate murine macrophages through both TLR2 and TLR4 [15] . In addition , the cytokine expression differs between mouse and human macrophages as revealed by in vitro cell infection models [16] . These previous studies suggest that the different innate immune responses of murine and human macrophages correlate with the differences of murine resistance and human susceptibility to leptospirosis . The expression patterns of cytokines and chemokines in different animal infection models have also been comparatively analyzed to reveal the mechanisms of anti-Leptospira immunity and identify predictors of leptospirosis [17] , [18] , [19] . Though hamsters [19] , [20] and the TLR4-deficient murine models [10] , [21] that mimic human acute leptospirosis , were appropriately used , the immune responses of acute infections in animal models may not fully resemble those in humans . The immune responses demonstrated in human primary cells infected by pathogenic Leptospira may improve our understanding of human leptospirosis . In addition , the approaches used to study immune responses have been limited to specific genes and pathways , and the kinetic signaling transduction and molecular activation process of host immunity remain largely unknown [17] . In this study , we applied gene expression microarrays and cytokine arrays to comparatively analyze responses of murine and human macrophages to leptospiral infection and identify more activated inflammatory genes and signaling pathways in this in vitro cell infection model .
Dunkin-Hartley ICO:DH ( Poc ) guinea pigs and BALB/c mice were bred and maintained under specific pathogen-free conditions in the animal facilities of Capital Medical University . All animal experiments complied with the Regulations for the Administration of Affairs Concerning Experimental Animals in China , the Chinese Standards on Experimental Animals , and the Manual for Bacterial Inspection ( GB/T 14926 . 42-2001 ) . Animal protocols were approved by the Animal Ethics Review Committee of Capital Medical University ( Approval number: CCMU-AE20110129 ) . Written informed consents were signed by the participants , and the protocols were approved by the Ethics Review Committee of Capital Medical University ( Approval number: CCMU-PE20110212 ) . The standard cultures of pathogenic L . interrogans serovar Lai strain Lai ( 56601 ) and saprophytic Leptospira biflexa serovar Patoc strain Patoc I ( Paris , 651505 ) were obtained from the Division of Parasitic Vaccines , Institute for Biological Product Control , National Institute for Food and Drug Control ( NIFDC ) , which is also the National Center for Medical Culture Collections of China . The strains were cultivated at 28°C in Ellinghausen-McCullough-Johnson-Harris ( EMJH ) liquid medium for passage . The virulence of L . interrogans was preserved by iteratively infecting specific pathogen-free Dunkin-Hartley ICO: DH ( Poc ) guinea pigs ( 10 to 12 days old; each weighing less than 150 g ) . The L . interrogans was recovered from the kidneys of the infected guinea pigs , washed three times using autoclaved phosphate-buffered saline ( PBS ) , and cultured for three generations in EMJH medium to exclude possible sources of animal components . L . interrogans and L . biflexa were respectively proliferated in 50 ml of EMJH mediums for use in the experimental infections . The close relative of L . interrogans [22] , nonpathogenic L . biflexa , was used as a control to verify the pathogenic Leptospira-specific gene regulations and cytokine expressions in this study . Murine peritoneal macrophages ( MPMs ) were isolated from male BALB/c mice ( 6 to 8 weeks old ) by washing the peritoneal cavities with cold RPMI 1640 medium . Macrophages were seeded in 75-cm2 tissue culture flasks ( Corning , NY , USA ) , and the cell numbers were counted in a hemocytometer . The cells were cultured in RPMI 1640 medium ( supplemented with 10% heat-inactivated fetal bovine serum , 100 µg/ml penicillin , and 100 µg/ml streptomycin ) for 2 h at 37°C under a humidified atmosphere containing 5% CO2 . After preincubation , non-adherent cells were removed gently by washing with 0 . 01 M PBS ( pH 7 . 4 ) , and the purity of the remaining macrophages was tested by Wright's staining . Human peripheral blood monocytes ( HBMs ) were isolated from twelve healthy donors using standard Ficoll-Hypaque gradient centrifugation as previously described [23] . The monocytes were seeded and counted according to the above-mentioned protocol for MPMs and then preincubated overnight at 37°C under a humidified atmosphere containing 5% CO2 . After preincubation , the monocytes were thoroughly washed with autoclaved PBS to remove non-adherent cells and then continuously incubated for 5 days in medium containing 500 U/ml granulocyte-macrophage colony stimulating factor ( GM-CSF; eBioscience , CA , USA ) to differentiate the cells into macrophages . The MPM and HBM cultures were washed three times with autoclaved PBS , renewed with new medium without antibiotics , and further cultured for 12 h before infection . Leptospiral cells ( L . interrogans or L . biflexa ) were harvested by centrifugation at 8 , 000 g for 10 min at 20°C , and then washed twice with PBS . The bacterial pellets were resuspended in RPMI 1640 medium ( supplemented with 10% heat-inactivated FBS ) at 37°C to a final concentration of 108 bacteria/ml . Ten ml of bacterial suspension ( 109 bacteria ) was added to 107 cells ( bacteria∶cell = 100∶1 , the total culture medium volume was 20 ml ) , and the tissue culture flasks were centrifuged at 2500 g for 15 min to synchronize the infection . Then , the infection models were incubated at 37°C under a humidified atmosphere containing 5% CO2 . Host cell samples were collected at three intervals ( 1 , 2 , and 4 h ) , respectively , in the cell infection models; and three biological replicates were performed for each sample . The high efficiency of leptospiral infection were revealed by indirect immunofluorescence and examined by confocal microscopy . Briefly , the lysosomes of the infected MPMs and HBMs ( for 1-h ) were labeled with 1∶1000 diluted lysosome marker Lamp1 Abs ( Invitrogen , CA , USA ) , then labeled with Alexa Fluor 488-conjugated F ( ab′ ) 2 anti-rabbit Ab ( Invitrogen , CA , USA ) . While , the leptospiral cells were labeled with 1∶200 diluted rat antiserum against L . interrogans strain Lai or L . biflexa strain Patoc I ( NIFDC , Beijing , China ) , and then labeled with Texas Red-conjugated F ( ab′ ) 2 anti-rat IgG Ab ( Invitrogen , CA , USA ) . The fluorescence signals were captured by an Olympus FV1000 laser scanning confocal fluorescence microscope . Previous cytokine mRNA kinetic expression studies have shown that most cytokine transcripts can be detected as early as 1 h after infection in an animal infection model [17] , indicating that rapid gene regulation of innate immune response can be detected within a 4-h period in this study . The MPM and HBM cultures not infected by L . interrogans or L . biflexa were used as negative controls . At sample harvesting , MPM and HBM cultures were washed three times using autoclaved PBS to remove leptospiral cells . The total RNA was extracted using TRIzol reagent ( Invitrogen , CA , USA ) , then purified using the RNeasy Mini Kit ( QIAGEN , Hilden , Germany ) with on-column DNase digestion ( QIAGEN , Hilden , Germany ) according to the RNeasy Mini handbook . RNA quantity and integrity were determined using an RNA 6000 Nano Laboratory-on-a-Chip kit and a Bioanalyzer 2100 ( Agilent Technologies , CA , USA ) . For each sample , approximately 10 µg of total RNA was mixed with 600 ng of oligo d ( T ) primers ( TaKaRa , Otsu , Japan ) and denatured at 65°C for 5 min . Then , the first strand cDNA was synthesized using 2 µl ( 400 U ) of SuperScript III reverse transcriptase ( Invitrogen , CA , USA ) , according to the protocol recommended by the manufacturer . The double-stranded cDNA ( ds cDNA ) sample was then synthesized using the second Strand Synthesis section of the M-MLV RTasecDNA Synthesis Kit ( TaKaRa , Otsu , Japan ) , according to the manufacturer's instructions . Following RNase H ( Invitrogen , CA , USA ) and RNase A ( Ambion , TX , USA ) digestion for 1 h , the ds cDNA sample was purified using a QIAquick PCR Purification Kit ( QIAGEN , Hilden , Germany ) , according to theQIAquick Spin handbook . The Phalanx OneArray mouse whole genome microarray ( Phalanx , Hsinchu , Taiwan ) containing 31 , 802 highly sensitive 70-mer sense-strand polynucleotide probes , including 29 , 922 mouse gene probes and 1880 experimental control probes , was used . In addition , the Phalanx OneArray human whole genome microarray containing 32 , 050 highly sensitive 60-mer sense-strand polynucleotide probes , including 30 , 968 human gene probes and 1082 experimental control probes , was used . The microarray experiments were performed by SinoGene Scientific Co . , Ltd . , Beijing , China , according to the microarray manufacturer's instructions . Briefly , the ds cDNA templates were labeled using Amersham Mono-functional Cy5 CyDye and hybridized to the OneArray whole genome microarray with Phalanx hybridization buffer using cover slides . After overnight hybridization at 50°C , nonspecific binding targets were washed away using three different washing steps . The slides were dried using centrifugation and scanned using a GenePix 4000B microarray scanner ( Molecular Devices , CA , USA ) . The Cy5 fluorescence intensities of each spot were analyzed using GenePix Pro 6 . 0 software ( Molecular Devices , CA , USA ) . The signal intensity of each spot was corrected by subtracting background signals in the immediate surroundings . Spots with flag <0 and a signal-to-noise ratio <3 , and the control probes were filtered out . Spots that passed the above-mentioned criteria were normalized using quantile normalization , according to the manufacturer's recommendation , and tested for differential expression . The differentially expressed genes were further analyzed using Agilent GeneSpring GX software ( version 10 . 0 ) and CapitalBio MAS ( Molecule Annotation System , version 3 . 0 , CapitalBio , Beijing , China ) . Considering that primary cells are a mixture of several related cell types with limited purity [24] , regulated genes with more than 3-fold ( P<0 . 05 ) up-regulation or down-regulation were defined as significantly regulated genes in this report . The regulations induced during the 4-h leptospiral infection ( same regulation trends at 1 h , 2 h , and 4 h ) were counted in the statistical analysis . Meanwhile , some instantaneous regulations at the 2-h time points were also included if needed . To validate the microarray data , the highly regulated genes , differentially expressed genes between MPMs and HBMs , six randomly selected genes , and sixteen highly regulated and differentially expressed genes from a new batch of RNA samples were quantitatively analyzed using qRT-PCR . The cell infection model , RNA extraction , and cDNA synthesis were performed according to the microarray cDNA synthesis protocol . In addition , MPMs and HBMs infected by L . biflexa ( bacteria∶cell = 100∶1 ) were designed as controls to verify that the differentially expressed genes in leptospiral infection were pathogenic Leptospira-specific regulations . The primers were designed using Premier software version 5 ( Premier Biosoft International , CA , USA ) ( Tables S1 , S2 , and S3 ) . RT reaction mixtures contained 0 . 5 µg of total RNA , 100 ng of random hexamer primers ( TaKaRa , Otsu , Japan ) , 0 . 5 µl ( 100 U ) of Superscript III reverse transcriptase ( Invitrogen , CA , USA ) , and 500 µM concentrations each of dATP , dCTP , dGTP , and dTTP . After denaturation at 65°C for 5 min , the samples were incubated at 50°C for 1 h , followed by 10 min at 70°C to synthesize the first strand cDNA . Samples of 30 ng of cDNA were mixed with 25 µl of 2×SYBR Green PCR Master Mix ( TaKaRa , Otsu , Japan ) . Assays were performed in triplicate with the ABI PRISM model 7500 sequence detection instrument . A relative quantification method was used to calculate the regulation folds in different infection samples . For each target gene , an amplicon was obtained using the qRT-PCR primers and standard RT-PCR , then the concentration of the purified amplicon was determined using Qubit 2 . 0 fluorometer ( Invitrogen , CA , USA ) . The gradient dilutions of the amplicon were used as control templets in qRT-PCR to construct a gene-specific standard curve . The templet quantifications in different infection samples were finally obtained by comparison with the standard curve . The melting curves were used to evaluate whether the accumulation of SYBR Green-bound DNA was gene specific . Semi-quantitative protein membrane arrays ( RayBiotech , GA , USA , Cat . No . AAM-CYT-3-8 for MPMs , and AAH-CYT-6-8 for HBMs ) containing 62 mouse or 60 human cytokine antibodies were used to verify cytokine and chemokine expression of macrophages at the protein level in this study . The membrane arrays were spotted with capture antibodies to various cytokines and chemokines including tumor necrosis factor alpha ( TNF-alpha ) , TNF-beta , interleukin ( IL ) -1beta , IL-10 , IFN-gamma , MIP-1alpha , MCP-1 , MIP-1beta , IP-10 , et al . Briefly , 5×105 MPMs and HBMs were infected with 5×107 L . interrogans or 5×107 L . biflexa cells ( bacteria∶cell = 100∶1 ) for 4 h , respectively . The final culture medium volume of each sample was 1 ml in a block of a 24-well plate ( Corning , NY , USA ) , which corresponded to the culture medium volume used in the previous infection model for the microarray assay . MPMs and HBMs that were not infected were used as negative controls . The host cells were washed three times using autoclaved PBS , and the proteins were extracted using cell lysis buffer ( RayBiotech , GA , USA , with protease inhibitor ) . The membrane arrays were incubated with 2 ml of cell lysates for the immunoblot assay . Captured cytokines and chemokines were visualized using immunohistochemistry following the manufacturer's instructions . Three biological replicates were performed .
Unlike the heat-killed Leptospira used in prior infection models [16] , a live and strong virulent L . interrogans strain was used to infect primary macrophages to establish the cell infection model in this study . The infection times were strictly limited to a short period ( 4-h ) , and only a few live leptospiral cells ( less than 1% ) survived in macrophages . Most of the extracellular bacteria , along with apoptotic and dead host cells , were removed by repeated PBS washes . The infection efficiencies were checked by indirect immunofluorescence of the Leptospira-containing phagosomes . The high infection ratio ( Leptospira∶cell = 100∶1 ) guaranteed that almost all the macrophages were infected by Leptospira ( Figure S1 ) . To verify the integrity and purity of total RNA , RNA samples were determined using a Bioanalyzer 2100 high performance capillary electrophoresis ( HPCE ) instrument . Bacterial 23S and 16S RNA bands were not detected by HPCE ( Figure S2 ) . The microarray data were validated using qRT-PCR . The transcriptional levels of the six randomly selected genes were determined using qRT-PCR performed on a new batch of RNA samples . No PCR amplification was detected in the negative controls . The qRT-PCR values of the infected MPMs and HBMs at 1- , 2- , and 4-h time points were plotted against the corresponding microarray data values , respectively . The high correlation coefficient values ( R2≥0 . 85 ) indicated that the microarray signal represented by multiple oligonucleotide probes was valid for transcriptomics research ( Figures S3 and S4 ) . The gene regulation folds of sixteen highly regulated genes and differentially expressed genes between MPMs and HBMs , such as TLR1 , LPS-binding protein , NF-kappa B inhibitor alpha , IL-10 , TNF-alpha , IL-6 , CCL5 , CXCL9 , MIP-1 alpha , MIP-1 beta , MIP-2 , C3 , CASP8 and FADD-like apoptosis regulator , IL-1-alpha , IL-1-beta , and BCL-2 genes , were further verified by qRT-PCR . As a control , the regulation folds of these genes in MPMs and HBMs infected by L . biflexa were also calculated to confirm that the different regulations of eight genes ( i . e . TLR1 , LPS-binding protein , IL-10 , MIP-1 alpha , MIP-1 beta , MIP-2 , C3 , and BCL-2 ) between MPMs and HBMs were pathogenic Leptospira-specific regulations ( Figure S5 ) . The protein expression levels of the 62 murine and 60 human cytokines as well as the chemokines of the macrophages infected by L . interrogans were verified by using immunoblot assays ( i . e . RayBiotech semi-quantitative protein membrane arrays ) . The quantitative data of the microarrays and the cytokine arrays were comparatively analyzed using hierarchical cluster analysis of Cluster 3 . 0 software and visualized by TreeView software ( Figure 1 ) . At 4-h intervals , most of the cytokine and chemokine protein expression levels corresponded to their mRNA expression levels . However , the protein expression fold changes of several of the most highly regulated cytokines , such as GCSF ( CSF3 ) ( 19 . 31-fold at mRNA level , and 5 . 78-fold at protein level ) and TNF alpha ( 5 . 62-fold at mRNA level , and 1 . 46-fold at protein level ) in MPMs , and IL-10 ( 26 . 14-fold at mRNA level , and 7 . 21-fold at protein level ) , IL-1alpha ( 11 . 03-fold at mRNA level , and 5 . 74-fold at protein level ) , and TNF-beta ( 15 . 89-fold at mRNA level , and 9 . 06-fold at protein level ) in HBMs , were less than the mRNA fold changes . These results may be due to the feedback or post-transcriptional regulations of these cytokines . As a control , the cytokine expression of MPMs and HBMs infected by L . biflexa were also calculated to verify that the different regulations between MPMs and HBMs were pathogenic Leptospira-specific regulations ( Figure 1 ) . Nine cytokines and chemokines ( i . e . CXCL13 , CCL11 , IL-13 , IL-17 , IL-3Rb , CCL19 , CCL1 , CCL25 , and VCAM-1 ) were differentially regulated in MPMs infected by L . interrogans and L . biflexa . And , ten cytokines and chemokines ( i . e . CXCL6 , CCL1 , IL-10 , IL-16 , IL-5 , IL-7 , CCL22 , CCl18 , TGF-b3 , and TNF-b ) were differentially regulated in HBMs infected by L . interrogans and L . biflexa . The microarray project was deposited in the DDBJ as BioProject ID PRJDB733 , and the microarray data was deposited in the NCBI-GEO as ID GSE45170 . In total , 1891 up-regulated genes and 431 down-regulated genes in MPMs , and 1932 up-regulated genes and 629 down-regulated genes in HBMs were induced during the 4-h leptospiral infection ( same regulation trends at 1 h , 2 h , and 4 h ) , which did not include the instantaneous regulations at the 1-h or the 2-h time points . Based on the Gene Ontology ( GO ) biological process , the percentage of regulated genes in each GO biological process ( i . e . the number of regulated genes divided by the number of total genes in the pathway ) was calculated to study the differences of gene regulation in MPMs and HBMs infected by L . interrogans . The percentages of regulated genes in eight biological processes of MPMs were significantly greater than those of HBMs ( >2-fold ) , such as GO:0019882 , antigen processing and presentation ( 7 . 87-fold ) ; GO:0042391 , regulation of membrane potential ( 3 . 47-fold ) ; GO:0045087 , innate immune response ( 3 . 11-fold ) ; GO:0006919 , caspase activation ( 2 . 98-fold ) ; GO:0016477 , cell migration ( 2 . 62-fold ) ; GO:0007010 , cytoskeleton organization and biogenesis ( 2 . 29-fold ) ; GO:0006955 , immune response ( 2 . 18-fold ) ; and GO:0008152 , metabolism ( 2 . 12-fold ) ( Figure 2 ) . These differences suggested that HBMs were less activated , which could eventually contribute to L . interrogans evading the host immunity and favor establishment of the infection . Although signal transduction often occurs at the level of phosphorylation , not at the level of transcription , the time series gene regulation analysis revealed a series of signaling factor changes in this study . The percentage of up-regulated and down-regulated genes in each Kyoto Encyclopedia of Genes and Genomes ( KEGG ) signaling pathway ( i . e . the number of up- or down-regulated genes divided by the total genes in the pathway ) was used to evaluate the significance of gene regulation of the signaling pathway . For the up-regulations , the percentages of up-regulated genes in the PPAR signaling pathway and the TGF-beta signaling pathway of MPMs were much greater than those of HBMs ( >2-fold ) . The percentage of up-regulated genes in the Notch signaling pathway of HBMs was much greater than that of MPMs ( >2-fold ) . For the down-regulations , the percentage of down-regulated genes in the B cell receptor signaling pathway of MPMs was much greater than that of HBMs ( >2-fold ) . The percentages of down-regulated genes in the ErbB , GnRH , Hedgehog , mTOR , phosphatidylinositol , and Wnt signaling pathways of HBMs were much greater than those of MPMs ( >2-fold ) ( Figure 3 ) , which suggested that the cell differentiation and development signals that were closely related to MAPK and PI3K pathways may be less activated in HBMs . In summary , these KEGG pathway statistics primarily revealed that the signaling pathways of MPMs showed more up-regulation and less down-regulation than those of HBMs , and the signal transductions of MPMs may be more active than those of HBMs during L . interrogans infection . The main regulated infection-related biological processes and pathways are discussed in detail below . The host TLR signaling pathway plays an important role in identifying pathogens through their pathogen-associated molecular patterns ( PAMPs ) . At least 11 TLRs have been identified so far in immune cells . Most TLRs can be constitutively expressed in macrophages or macrophage cell lines [25] , [26] , and some of their expressions can be induced during infection [26] . Several leptospiral components , such as LPS , lipoproteins , and OMPs , have been verified to activate host cells by TLRs [3] , [6] , [15] . Leptospiral LPS only induces immune response through TLR2 in human but through both TLR2 and TLR4 in mouse [15] . Although the stimulating activity of leptospiral LPS in peripheral monocytes is at least 1000-fold less than that of the LPS of E . coli [10] , the TLR2 signal induced by leptospiral LPS may be very important in human leptospirosis , as shown by the extraordinarily high expression level of TLR2 in human monocytes [26] . In addition , leptospiral OMPs and lipoproteins also induce immune response through the TLR2 signaling pathway [6] . Previous research has shown that the leptospiral outer membrane has a relatively complex antigen profile compared to other pathogenic spirochetes [27] . Only the OMPs of pathogenic Leptospira , not those of saprophytic Leptospira , can mediate early inflammation in proximal tubule cells [6] . The genes involved in the TLR signaling pathway showed significant regulations during L . interrogans infection . The major pro-inflammatory effective cytokine genes , such as the genes of TNF-alpha , IL-1-beta , etc . , were up-regulated in both MPMs and HBMs ( Figure 4 ) . Though no significant regulation of TLR2/4 was observed during the 4-h leptospiral infection , the different expressions of the cytokines may partially be due to the different regulation of the other adaptors and receptors . For example , the TLR1 gene ( tlr1 ) was up-regulated in MPMs , but it was down-regulated in HBMs . Considering that TLR1 heterodimerizes with TLR2 to recognize triacetylated lipoproteins , and also participates in the recognition of leptospiral LPS in human cells [15] , the different regulation may be related with the differential activation by leptospiral lipoproteins and LPS . The LPS-binding protein gene ( lbp ) was only persistently down-regulated in MPMs infected by L . interrogans . These two different regulations between MPMs and HBMs were pathogenic Leptospira-specific regulations which was not happened in MPMs or HBMs infected by L . biflexa ( Figure S5 ) . In addition , myeloid differentiation primary response factor ( MyD88 ) , the key adaptor downstream of the TLR pathways , was down-regulated in HBMs; Two key factors of the MyD88-independent pathway , TRAM ( TIRP/TICAM-2 ) and TRIF ( TICAM-1 ) , were only down-regulated in HBMs . while the Toll-IL-1 receptor domain-containing adapter protein ( TIRAP ) controlling the activation of MyD88-dependent pathways downstream of TLR-4 [28] , was down-regulated in MPMs . Previous research has shown that the OMPs from pathogenic Leptospira and the purified LipL32 lipoprotein all up-regulated TLR2 expression [6] . However , the TLR2 gene expression in this study was not regulated in MPMs nor in HBMs; hence , this result may be due to down-regulation of the OMPs previously revealed in renal tubule and urine as well as our cell infection models [29] , [30] . In addition , the genes involved in the nucleotide-binding domain and leucine-rich repeat ( NLR ) containing signaling pathway showed few regulations during leptospiral infection . The NLRP10 ( NACHT , leucine rich repeat and PYD containing 10 ) gene was moderately up-regulated in MPMs . The NLRP14 ( NACHT , leucine rich repeat and PYD containing 14 ) gene was persistently up-regulated , while the NLRP13 ( NACHT , leucine rich repeat and PYD containing 13 ) gene was persistently down-regulated in HBMs . Though pathogenic Leptospira spp . is an extracellular pathogen , the intracellular signaling pathway should not be ignored due to the intracellular life cycle [14] and the NLRP3-dependent cytokine secretion [31] . NF-kappa B regulates the expression of many genes involved in immune and inflammatory response [32] . NF-kappa B heterodimers can migrate from cytoplasm to nucleus , and regulate different sets of target genes [33] . NF-kappa B is an anti-apoptosis factor because it can up-regulate the expression of cell death inhibitors; thus , inhibition of NF-kappa B promotes cell death [34] . It has been reported that NF-kappa B activation and p38 phosphorylation can be induced by Leptospira infection or exposure to partially purified leptospiral lipoproteins in microglial cells [35] , and induced by leptospiral LPS and lipoproteins in human and murine macrophage cell lines [3] , [15] . In this study , the up-regulation folds of the LPS-inducible NF-kappa B inhibitor alpha gene ( nfkbia ) and zeta gene ( nfkbiz ) in HBMs were significantly greater than those in MPMs , and the NF-kappa B 1 gene ( nfkb1 ) was only slightly up-regulated in HBMs . This gene regulation difference suggested that induction of the NF-kappa B signaling cascade in HBMs may be less than that in MPMs . The other signaling pathways , such as the p53 and MAPK signaling pathways , etc . , showed a few regulation changes in our cell infection models . However , the phosphorylation signaling cascades in these pathways deserve more attention in future leptospiral infection studies . Inflammatory cytokines are immunomodulatory proteins that help the host mount an immune response to diverse inflammatory processes . It has been reported that hamsters that died from leptospirosis had significantly greater expression levels of both pro- and anti-inflammatory mediators in comparison to the survivors [19] . The inflammation induced by pathogenic Leptospira is is always less than that induced by other Gram-negative pathogens [36] . Previous research has shown that L . interrogans can induce pro-inflammatory cytokines , such as TNF-alpha , IL-6 , etc . , in mouse peritoneal macrophages [10] , [16] . Though high expression levels of pro-inflammatory cytokines from macrophages were verified to correlate with the clearance of pathogenic Leptospira [10] , recent research has also shown that susceptible TLR-deficient mice infected with Leptospira died from both elevated levels of pro-inflammatory cytokines and high bacterial loads [21] . Furthermore , pathogenic Leptospira can induce production of type 1 cytokines involved in cellular immunity in a hamster infection model [17] . As expected , the genes of the major cytokines , including TNF-alpha , IL-1 alpha/beta , IL-10 , and inhibin beta-A , etc . , were significantly up-regulated both in MPMs and HBMs during leptospiral infection; while the expression levels of IL-2 and IFN-gamma did not change significantly in MPMs and HBMs ( Figure 4 ) . TNF-alpha induces monocytes to secrete other cytokines , such as IL-1 , IL-6 , and IL-8 , which are essential for controlling infection and cleaning LPS from blood , and is also involved in sepsis and tissue lesions [37] , [38] . Previous research has shown that L . interrogans induces greater TNF-alpha levels than those stimulated by Borrelia garinii and Treponema pallidum , but less than that activated by E . coli LPS in isolated rat liver macrophages ( Kupffer cells ) . The greater expression of TNF-alpha was mainly induced by leptospiral LPS , which indicated that TNF-alpha up-regulation may be related to severe hepatitis during leptospirosis [39] . In addition , other clinical research has shown that TNF-alpha is closely related to severe nephritis during human leptospirosis [40] . In this study , the expression levels and the fold change of TNF-alpha expression in HBMs were all greater than those in MPMs ( >2-fold , P<0 . 05 ) , both at mRNA and protein levels ( Figures 1 and S5 ) . However , in the L . biflexa infection control , the up-regulation folds of TNF-alpha protein expression in HBMs were also significantly higher than those in MPMs ( >2-fold , P<0 . 05 ) ( Figure 1 ) , which indicated that the different regulation of TNF-alpha during L . interrogans infection was not pathogenic Leptospira-specific . The high expression level of TNF-alpha in HBMs may be related to the severe pathological symptoms of human leptospirosis . Interestingly , possibly due to the divergent expression of TNF-alpha , IL-6 was only up-regulated in MPMs and IL-8 was up-regulated in HBMs ( Figure 4 ) . These results were primarily consistent with the previous comparative cytokine analysis in different macrophages that the IL-6 level in mouse cells rose more rapidly than it did in human cells [16] . A recent study showed that leptospiral LPS synergizes with glycolipoprotein to produce IL-1-beta [31] . In this study , IL-1 alpha/beta and its receptor ( IL-1R , ICE ) were up-regulated in both MPMs and HBMs infected by L . interrogans and L . biflexa , both at mRNA and protein levels ( Figures 1 and S5 ) . The upper activation pathways of IL-1 alpha/beta include the activation of pro-caspase-1 , a cytosolic protease , to become caspase-1 after inflammasome activation , and activated caspase-1 causes the proteolytic processing of pro-IL-1-beta , resulting in maturation and secretion of IL-1-beta [41] . IL-10 is a multifunctional anti-inflammatory cytokine that plays an important role in limiting inflammatory response and preventing tissue damage [42] . It can be secreted by macrophages , and it suppresses the release and function of other factors , such as IL-1beta , IL-6 , TNF-alpha , IL-12 , etc . [34] . TNF-alpha can induce expression of IL-10 , while IFN-gamma and IL-10 itself can inhibit the production of IL-10 . Our microarray data showed that the expression of TNF-alpha during L . interrogans infection was greater than that of IL-10 after 1-h , which was consistent with the inductive effect of TNF-alpha on IL-10 . However , the expression levels and regulation patterns of IL-10 were dramatically different in MPMs and HBMs infected by L . interrogans ( Figures 1 , 4 and S5 ) . The IL-10 expression in MPMs was detectable at a low level and not further up-regulated after 4-h , while IL-10 was persistently up-regulated ( more than 20-fold ) in HBMs , both at mRNA and protein levels . However , in the L . biflexa infection control , the IL-10 protein expressions in MPMs and HBMs were not induced at the 4-h time point ( Figures 1 and S5 ) . These results were different from the previous reports that leptospiral glycolipoprotein induces IL-10 production of human peripheral blood mononuclear cells [5] . In addition , the IL-10 receptor ( IL-10R ) gene was down-regulated in MPMs , which may further reduce the functional role of IL-10 in MPMs . A previous study on borrelial infection has shown that IL-10 is differentially expressed in macrophages from different murine models , suggesting that it may contribute to the control of inflammation in Lyme disease [43] . Therefore , it was reasonable to presume that the low expression level of IL-10 in MPMs may contribute to a high inflammatory response , which may help mice to reduce the leptospiral burden during infection . Furthermore , the IL-10/TNF-alpha ratio has been proposed as a prognosis indicator in sepsis during leptospirosis [44] . This ratio reflects a persistent secretion of IL-10 at a later stage in septic patients . In this study , the IL-10/TNF-alpha ratio in macrophages within this short time period may not be relevant to the outcome in mouse and human infection [19] . The interferon-gamma ( IFN-γ ) expression level has been verified to be very low in patients who have leptospirosis [16] , [45] . This cytokine , which is not produced by macrophages , is mainly produced by activated T helper 1 cells , T cells , and natural killer ( NK ) cells [46] . Therefore , it was not unexpected to find that IFN-gamma in MPMs and HBMs infected by L . interrogans or L . biflexa was not up-regulated ( Figure 1 ) . A previous in vitro study using human whole blood stimulated by heat-killed L . interrogans showed that the production of IFN-gamma is partially controlled by IL-12 secreted by macrophages [47] . In our live Leptospira cell infection model , the IL-12a gene of MPMs and the IL-12b gene of HBMs were persistently up-regulated during L . interrogans infection . However , up-regulation of the IL-12b gene of HBMs may not further induce IFN-γ expression in CD4+ T cells during human leptospirosis , since IL-10 in HBMs was also up-regulated and the high-level of IL-10 could suppress the immune response . IL-6 is a pleiotropic cytokine that influences antigen-specific immune responses and inflammatory reactions , and it is always treated as the best marker for severity of infectious stress [48] . Previous research has shown that leptospiral heat stable components other than LPS can stimulate mouse macrophage IL-6 expression [16] , and leptospiral infection can induce IL-6 secretion in a mouse model [10] . In this study , IL-6 was up-regulated in MPMs ( 5-fold at mRNA level and 3-fold at protein level ) , but it remained unchanged in HBMs , during L . interrogans infection . Therefore , it is debatable whether IL-6 should be used as a predictor in the early diagnosis of human acute leptospirosis . In the L . biflexa infection control , IL-6 was also only up-regulated in MPMs , which indicated that the different regulation of IL-6 in MPMs and HBMs may not be a pathogenic Leptospira-specific pattern ( Figures 1 and S5 ) . Chemokines are a type of chemotactic protein that can attract leukocytes to the sites of infection . More than 40 chemokines have been identified in human , most of which are a group of structurally related cytokines involved in immune responses [49] . Previously , pathogenic Leptospira induced different chemokine profiles in resistant BALB/c and susceptible C3H/HeJ mice models during a two-week infection; hence , these results indicate that the distinct chemokine profiles may be related to the different outcomes in chronic and acute leptospiral infections [18] . In this study , significantly different chemokine regulations between MPMs and HBMs infected by L . interrogans were also revealed by microarray analyses ( Figure 5 ) and partially by cytokine array analyses ( Figure 1 ) . The major difference in regulation between MPMs and HBMs was that six genes , including chemokine ( C-C motif ) ligand 5 ( CCL5 ) , chemokine ( C-C motif ) ligand 17 ( CCL17 , TARC ) , chemokine ( C-C motif ) ligand 25 ( CCL25 , TECK ) , chemokine ( C-X-C motif ) ligand 5 ( CXCL5 , ENA-78 ) , chemokine ( C-X-C motif ) ligand 10 ( CXCL10 , IP-10 ) , and chemokine ( C-C motif ) receptor 1 ( CCR1 ) , were only up-regulated in MPMs . However , compared with the protein expressions of the L . biflexa infection control ( Figures 1 and S5 ) , these regulations were not pathogenic Leptospira-specific chemokine differences . While , eight genes , including chemokine ( C-C motif ) ligand 1 ( CCL1 ) , chemokine ( C-C motif ) ligand 3 ( CCL3 , MIP-1alpha ) , chemokine ( C-C motif ) ligand 4 ( CCL4 , MIP-1beta ) , chemokine ( C-C motif ) ligand 20 ( CCL20 ) , chemokine ( C-X-C motif ) ligand 1 ( CXCL1 ) , chemokine ( C-X-C motif ) ligand 2 ( CXCL2 , MIP-2 ) , chemokine ( C-X-C motif ) ligand 9 ( CXCL9 , Mig ) , and chemokine ( C-C motif ) receptor 4 ( CCR4 ) , were only up-regulated in HBMs , in which the different mRNA or protein regulations of MIP-1 alpha , MIP-1 beta , MIP-2 should be pathogenic Leptospira-specific chemokine differences ( Figures 5 and S5 ) . Though pronounced chemotactic gene regulations , including up-regulations and down-regulations , were observed in MPMs ( Figure 5 ) , the gene expressions of the major chemokines , such as MIP-1-alpha/beta , and MIP-2 were only significantly up-regulated in HBMs , which possibly reflects that HBMs would attract leukocytes to the sites of leptospiral infection more efficiently . Unexpectedly , our result that MIP-1-alpha was not up-regulated in MPMs at both mRNA and protein levels seemed to be contradictory to previous animal infection models [18] , [50] , which showed that increases of MIP-1-alpha may contribute to host resistance to Leptospira infection in resistant BALB/c and Oncins France 1 mice . A reasonable explanation is that the expression levels of MIP-1-alpha can vary depending on the tissue and time after infection [18] , so the expression of MIP-1-alpha in macrophages within 4-h may not resemble those in various animal tissues after infection for several days . Moreover , the significant up-regulation of MIP-2 in HBMs may correlate to the severity and progression of human leptospirosis . The complement system consists of a large population of plasma and membrane proteins that can be produced by macrophages , intestinal epithelial cells , liver and spleen cells , etc . , and it plays important roles in both innate and adaptive immunity during infection [51] . Previous in vitro studies have shown that the complement system of healthy serum can kill saprophytic Leptospira , but not pathogenic Leptospira [52] , and it is also indispensable for the phagocytosis of human polymorphonuclear leukocytes [53] . Recently , pathogenic Leptospira have been shown to be resistant to complement-mediated killing due to the fact that the pathogen can directly disrupt the complement system by capturing its components ( e . g . factor H , C4BP , etc . ) [54] , [55] , [56] , [57] . In this study , the central component involved in both the classical and alternative pathways , the complement component 3 ( C3 ) gene [58] , was only up-regulated persistently in MPMs during L . interrogans infection ( Figure S5 ) . This different regulation was a pathogenic Leptospira-specific pattern . However , the negative complement regulator of the lectin and classical pathways , the C4BP gene , was also up-regulated by more than 10-fold in MPMs , while the C4BP-alpha gene was slightly down-regulated in HBMs . In addition , the C4B gene was only down-regulated in MPMs . This inconsistency made it difficult to understand the complement-mediated functions in MPMs . In summary , the complement components of host macrophages were easily regulated during the early stage of leptospiral infection , and they may be related with complement function and immune activation . Macrophages are an important antigen-presenting cell ( APC ) of host immunity , although the antigen presentation of macrophages is less efficient than that of dendritic cells ( DCs ) [59] . Considering that murine macrophages kill and degrade leptospiral cells more efficiently than do human macrophages [14] , the different regulations in the antigen processing and presentation pathways of MPMs and HBMs should be easily detected . As mentioned above , the percentage of regulated genes involved in antigen processing and presentation of MPMs was much greater than that of HBMs ( Figure 2 ) . In MPMs infected by L . interrogans , the cathepsin L gene and two histocompatibility-2 locus genes ( T region locus 24 and Q region locus 10 ) were up-regulated; while another four histocompatibility-2 locus genes ( class II antigen A alpha , class II antigen E alpha and beta , and O region beta locus ) and an Ia-associated invariant chain gene were down-regulated . In HBMs infected by L . interrogans , a nuclear transcription factor Y alpha gene and an IFN-alpha-8 gene were up-regulated; while a regulatory factor X-associated protein gene , a proteasome ( prosome , macropain ) activator subunit 1 ( PA28 alpha ) gene , a major histocompatibility complex class I , E gene , a CD4 antigen ( p55 ) , an IFN-alpha-6 gene , and a regulatory factor X , 5 ( influences HLA class II expression ) gene were down-regulated . The genes involved in antigen processing and presentation pathways in MPMs and HBMs were mainly down-regulated , and the down-regulations in HBMs were more significant than those in MPMs . Taken together , these results suggested that antigen processing and presentation in HBMs was weaker than that in MPMs . Pathogen invasion and internalization of bacterial components into a target cell by binding to a cell receptor can cause pathogen-induced apoptosis [60] . The apoptosis effect often occurs during the early stage of an infectious disease , and it can contribute to efficient colonization and diffusion of pathogens , initiation of inflammation in hosts , or defensive reactions in hosts [61] . It seems that macrophages are particularly susceptible to pathogen-induced apoptosis [62] . Previous research has revealed that pathogenic Leptospira can induce apoptosis in mouse macrophages by invasion or in hepatocytes by noninvasive mechanisms [11] , [63] . In addition , macrophage apoptosis occurs through caspase-8 and caspase-3 pathways [13] . In general , the genes involved in this pathway showed robust up-regulations both in MPMs and HBMs . Especially , the caspase-8 and Fas-associated protein with death domain ( FADD ) -like apoptosis regulator genes were significantly up-regulated , which supported previous caspase-8 and -3 pathway results [13] . The upper signaling pathway of caspase-8 and the FADD-like pathway seem to be closely related to TNF-alpha and its receptor ( TNF-R1 ) , since the genes of TNF-alpha ( Tnf ) , TNF ( ligand ) super family member 9 ( Tnfsf9 ) , TNF receptor super family member 1b ( Tnfrsf1b ) , TNF receptor super family member 9 ( Tnfrsf9 ) , and TNF receptor-associated factor 1 ( Traf1 ) in MPMs , and the genes of TNF super family member 2 ( Tnfsf2 ) , Tnfsf9 , and Tnfrsf1b were all up-regulated in HBMs . In addition to the highly consistent apoptosis of MPMs and HBMs , there were some differentially regulated genes . The caspase-3 , caspase-7 , and Bcl2-like 1 genes were only up-regulated in MPMs; while MYD88 and colony-stimulating factor 2 receptor beta 1 ( low-affinity , granulocyte-macrophage ) genes were only down-regulated in HBMs . Interestingly , the main anti-apoptotic gene , bcl-2 , was only down-regulated in MPMs , which was also a pathogenic Leptospira-specific regulation ( Figure S5 ) . A previous report has shown that bcl-2 is down-regulated and apoptosis is increased in macrophages after infection with Mycobacteria bovis BCG [64] . In addition , another BCL-2 family member , BCL2-related protein A1 , was also significantly up-regulated in HBMs ( >8-fold ) , which suggested that HBMs may antagonize cell apoptosis during the early stage of infection . In conclusion , these results were primarily consistent with previous results that MPMs and HBMs were all induced to become apoptotic by leptospiral infection , the apoptosis of MPMs occurred earlier than that of HBMs , and the proportion of early apoptotic cells in MPMs was significantly higher than that in HBMs during the 4-h leptospiral infection [14] . Pathogenic Leptospira can bind to the host cells by protein interactions between ECM components and leptospiral surface proteins [65] , [66] , [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , which should be closely related with the pathogenesis of leptospirosis . Recent studies also have shown that the OMPs of pathogenic Leptospira can induce ECM accumulation through a TGF-beta1/Smad-dependent pathway [75] . However , in our live Leptospira infection model , the up-regulations of ECM components , synthesis enzymes , and degrading enzymes made it difficult to confirm the ECM accumulation during infection ( Tables 1 and 2 ) . Especially , in HBMs , seven collagen components were significantly up-regulated , while matrix metalloproteinase 1 ( interstitial collagenase ) was dramatically up-regulated by more than 50-fold . Therefore , a reasonable explanation is that the host cells should maintain the balance of ECM synthesis and degradation . In addition , the ECM degradation may facilitate the spread of pathogenic Leptospira in the intercellular space . Further dynamic studies on leptospiral adhesion will uncover the function of ECM regulation during leptospiral infection .
Mature macrophages can phagocytize and kill pathogens , process and present antigens for the adaptive immune system , and secrete a series of cytokines and chemokines to regulate host immune response; these characteristics make macrophages an important and effective cell of host innate immunity . Macrophages express many surface receptors , such as TLRs , complement receptors , scavenger receptors , mannose receptors , etc . , which help the host recognize pathogens and present antigens for adaptive immunity [76] . In this study , several differences between murine and human macrophages infected by L . interrogans were revealed by transcriptomics and cytokine array methods; these findings somewhat reflected the differences of chronic infection in mice and acute infection in humans . Our previous research on Leptospira-macrophage interaction has shown that there is little difference of the transcriptomics of L . interrogans infecting murine and human macrophage cell lines [30] . In contrast , significant transcriptomics and cytokine differences of murine and human primary macrophages infected by L . interrogans were uncovered in this study , suggesting that different immune responses explain the major disparities in the murine and human Leptospira-macrophage infection models . However , live leptospiral cells can regulate their high-antigenicity antigens ( such as heat-shock proteins and flagellar proteins ) when they infect macrophages [35] . Hence , the different regulations of MPMs and HBMs may be partially due to the different regulations of the leptospiral genes . The major differences of the murine and human macrophages in this study were the dramatically different expression profiles of cytokines and chemokines . These differences partially reflected the different outcomes of chronic and acute leptospiral infections [17] , [19] . Considering that these cytokines can further regulate humoral immune and inflammatory responses , it is necessary to further investigate the relationship between cytokine secretion and immune response during leptospirosis on gene knockout cell or mouse models . In conclusion , this study uncovered a series of molecular changes in host immune cells , and the findings provide a foundation for further studies on different immune responses due to chronic and acute leptospiral infections .
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Although pathogenic Leptospira is not an obligate intracellular pathogen , recent studies have shown that phagocytosis and innate immunity play important roles in leptospirosis . The Leptospira-macrophage interaction is a common model used to elucidate the initial response in leptospiral infection . Our previous research has shown that there is little difference in the transcriptomics of pathogenic Leptospira infecting murine or human macrophage cell lines . Contrarily , in this study , we observed significant differences of murine and human primary macrophages infected by L . interrogans as shown in several processes , such as antigen processing and presentation , Toll-like receptor signaling pathway and innate immune response , complement and coagulation cascades , expression of major cytokines and chemokines , etc . These results suggested that different immune responses explain the major disparities in the murine and human Leptospira-macrophage infection models . This study added to the former leptospiral transcriptomics research on the Leptospira-macrophage interaction model and laid a foundation for further investigation in the pathogenesis of leptospirosis .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2013
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Responses of Murine and Human Macrophages to Leptospiral Infection: A Study Using Comparative Array Analysis
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Mycobacterium leprae , an obligate intracellular bacillus , infects Schwann cells ( SCs ) , leading to peripheral nerve damage , the most severe leprosy symptom . In the present study , we revisited the involvement of phenolic glycolipid I ( PGL I ) , an abundant , private , surface M . leprae molecule , in M . leprae-SC interaction by using a recombinant strain of M . bovis BCG engineered to express this glycolipid . We demonstrate that PGL I is essential for bacterial adhesion and SC internalization . We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor ( MR/CD206 ) in infected cells in a peroxisome proliferator-activated receptor gamma ( PPARγ ) -dependent manner . Of note , blocking mannose recognition decreased bacterial entry and survival , pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve . Moreover , an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets ( LDs ) formation and prostaglandin E2 ( PGE2 ) , previously described as fundamental players in bacterial pathogenesis . Finally , this pathway was shown to induce IL-8 secretion . Altogether , our study provides evidence that the entry of live M . leprae through PGL I recognition modulates the SC phenotype , favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation .
The most serious consequence of leprosy is the peripheral nerve damage that occurs in all clinical forms of the disease . Nerve damage results from the capacity of M . leprae , an obligate intracellular bacillus , to infect SCs , the glial cells of the peripheral nervous system ( PNS ) . SCs show remarkable plasticity and contribute to the regenerative capacity of the adult PNS even after severe injury has occurred . M . leprae-nerve fiber colonization results in loss of sensation , an early symptom of the disease . While multidrug therapy ( MDT ) treats the infection , it may be unsuccessful in either preventing or arresting the nerve damage responsible for disfigurement and disabilities [1 , 2] . In-depth investigation of M . leprae-nerve interactions objectifying the development of new strategies for the prevention and treatment of leprosy-related nerve impairments is , therefore , of utmost importance . M . leprae is easily seen inside vacuoles in the non-myelinating and myelinating SC cytoplasm in nerve specimens of leprosy patients [3 , 4] and , as a consequence , the three physiological functions of nerves–sensory , motor and autonomic–are affected . However , the first symptoms of leprosy are related to loss of temperature sensation and decreased touch sensation , functions provided by non-myelinating fibers , indicating their early invasion by the leprosy bacillus during the natural course of the disease [5] . Thus , the use of non-myelinating SCs as an in vitro model of infection is physiologically relevant and may reveal early fundamental aspects of M . leprae neuropathogenesis . The tropism of M . leprae to the peripheral nerves has been attributed to its capacity to bind to the globular domain of the α2 chain of laminin-2 [6] , a laminin isotype with restricted tissue distribution constituting a major component of the basal lamina surrounding SC-axon units [7–9] . Two M . leprae adhesins named Hlp and PGL I have been characterized as laminin-binding molecules responsible for attachment to SC . Shimoji et al . [10] and Marques et al . [11] have described the 21-kDa histone-like protein ( Hlp ) , a conserved molecule among species of mycobacteria , as a laminin-binding protein ( also called ML-LBP21 ) . The other molecule , PGL I [12] is an abundant lipid composing the outermost layer of the M . leprae envelope [13] . PGLs are present in other species of mycobacteria , but differ among themselves in their carbohydrate moieties [14–16] ( Fig 1 ) . The PGL I trisaccharide is highly specific to M . leprae [13 , 17] , having been shown to bind exclusively to the G domain of the laminin-2 α2 chain [12] . It has been proposed that this interaction most convincingly explains the specific neural tropism displayed by M . leprae since Hlp can also bind the α1 and β1 chains that make up other laminin isotypes [10 , 11 , 18 , 19] such as laminin-1 with a wide range of tissue distribution [7] . It has been speculated that , due to its abundance , PGL I would then initiate the specific M . leprae-SC interaction while Hlp would increase avidity binding by enacting a secondary role [12] . Upon infection , M . leprae seems to evoke drastic metabolic and phenotypic changes in SC , abetting infection and bacterial persistence in the host . In previous studies , we were able to show that M . leprae induces the production of insulin-like growth factor 1 ( IGF-1 ) to advantage host cell survival [20] while triggering drastic changes in the SC lipid and glucose metabolism that promote bacterial persistence [21–23] . Moreover , recent data have shown the capacity of M . leprae to induce SC reprogramming to a progenitor/stem-like cell stage , most probably increasing the spread of infection [24] . However , the molecular mechanisms underlying these events and the contributions of such M . leprae constituents as PGL I continue to be poorly understood . Multiple attempts to grow M . leprae in axenic or tissue cultures have been in vain so that infected nine-banded armadillo and athymic nude mouse still constitute the major bacterial sources for both biochemical and functional studies [2] . Since mutated strains of M . leprae are yet unobtainable , a genetically-engineered M . bovis BCG strain producing and secreting PGL I ( BCG PGL I ) instead of its own PGL was created as an alternative tool to better decipher the role of this glycolipid in leprosy pathogenesis [15] . Tabouret et al . [15] used the recombinant BCG strain to analyze the role of PGL I in the interaction of the leprosy bacillus with macrophages and dendritic cells . As a result , previous data were confirmed and new insights into the molecular bases of PGL I to down modulate the innate immune response were provided . Here , the recombinant BCG PGL I was used as an alternative tool to elucidate the role of PGL I along with the molecular mechanisms by which it participates in the M . leprae-SC interaction .
As a first step , the critical role of PGL I on M . leprae adhesion and SC internalization was addressed by comparing the capacity of the recombinants BCG PGL I , BCG PGL TB—a recombinant BCG strain that produces PGL from M . tuberculosis ( BCG PGL TB ) instead of PGL bovis [25]—and wild type BCG ( BCG WT ) , their parental strain , to infect the human schwannoma cell line ST8814 . The recombinant bacteria were labeled with the green fluorescent dye PKH67; and association ( % ) was determined after 4 h , 24 h and 48 h of incubation with SC at MOI 50:1 by flow cytometry . Association ( adhesion + internalization ) values showed that the PGL I-expressing recombinant strain acquired a high capacity to interact with these cells as opposed to the lesser capacity of the BCG WT strain ( histogram plot-Fig 2A ) . Interestingly , BCG PGL TB behaved similarly to the wild-type strain , indicating that the observed effect was mediated by the unique trissacharide moiety of M . leprae PGL ( Fig 2A ) . For BCG PGL I , a time-dependent association between the bacterium and SCs was observed , with about 80% of cells evidencing associated bacilli after 48 h of incubation ( Fig 2A ) . At this time point , most of the bacilli had been internalized , as monitored by quenching extracellular bacteria with Trypan Blue ( S1A Fig ) . Moreover , S1B Fig delineates that , in assays performed at 4°C to inhibit internalization , PGL I was shown to mediate the adhesion step . We also made use of GFP expressing recombinant BCG PGL I or PGL TB and the parental BCG strain to confirm their differential capacity to associate to SCs ( S1C Fig ) . Since host cell internalization could be an active process dependent on bacterial viability , we next compared the ability of live versus lethally-irradiated bacterial cells to invade SCs ( Fig 2B ) . For this purpose , the degree of internalization was determined by quenching the fluorescence of adhered bacteria with Trypan Blue ( Fig 2B , S2A Fig ) . M . leprae was included in these assays with results equivalent to those obtained with BCG PGL I . Both dead BCG PGL I and dead M . leprae measured roughly 50% less internalization capacity in contrast to live BCG PGL I and live M . leprae , respectively ( dead BCG PGL I 30 . 33 ± 5 . 21% and dead M . leprae 40 . 66 ± 4 . 41% ) . S2B Fig shows that the percentage of SCs with internalized bacteria augmented in accordance with an increasing multiplicity of infection ( MOI ) . In comparing dead with live bacilli at a MOI of 10:1 and 50:1 , it was seen that when dead bacteria were used the SC population with internalized bacteria fell about 50% . Interestingly , however , at a MOI of 100:1 , both live and dead bacilli were present in nearly 100% of the cell population . Nevertheless , regardless of the MOI used , in analyzing the Median Intensity of Fluorescence ( MFI ) of the cell population , the use of live bacteria resulted in significantly higher values , indicating that the number of bacilli per cell was always higher in the ones infected with live bacilli ( S2C Fig ) . The importance of PGL I in SC invasion was confirmed by fluorescence microscopy ( Fig 2C ) . After 48 h of infection , the percentage of ST8814 cells with associated BCG PGL I was more elevated ( 88 . 33 ± 3 . 79% ) than that found for either BCG WT ( 10 . 17 ± 2 . 47% ) or BCG PGL TB ( 10 . 50 ± 1 . 00% ) . M . leprae was used as a positive control , resulting in the presence of associated bacteria in 88 . 67 ± 3 . 51% of the cells . Also , the influence of viability on SC invasion was corroborated since the association capacity of dead BCG PGL I and dead M . leprae was about 50% less in comparison to live BCG PGL I and live M . leprae , respectively ( dead BCG PGL I 41 . 00 ± 6 . 00% and dead M . leprae 47 . 33 ± 2 . 52% ) while , when compared to live mycobacteria , dead BCG WT and BCG PGL TB maintained the same levels . Since ST8814 is a tumor cell line , similar experiments were repeated with Primary Human Schwann Cells ( PHSC ) . As shown in Fig 2D , the critical role played by PGL I in the bacterial SC invasion was validated in that the association values attained by BCG PGL I were similar to those for M . leprae itself . Likewise , as seen with ST8814 cells , the internalization of live bacteria was significantly higher than that found for dead bacilli in the context of both BCG PGL I and M . leprae . Moreover , PGL I-covered beads showed a higher internalization rate than uncovered ones ( Fig 2E ) , attesting to the importance of PGL I in mycobacterium SC internalization . Altogether , these results confirm that the unique phenolic glycolipid produced by M . leprae is a key molecule involved in bacterial adhesion and SC internalization and that bacterial viability strongly facilitates entry . Intracellular pathogens are known to modulate host-cell phagocytic receptors as a way to facilitate their entrance into host cells . We , therefore , turned to investigating whether M . leprae or BCG PGL I modulate the SC phagocytic phenotype . A series of experiments using different combinations of pre- and secondary stimuli were conducted with ST8814 SC . The first stimulus consisted of unlabeled bacteria or latex beads in a 10:1 proportion . The second stimulus with a 50:1 proportion consisted of the addition of PKH67 ( green ) -labeled bacteria or green fluorescent latex beads 1 hour after the initial stimulus . Cells were then incubated for 48 h and analyzed via flow cytometry or fluorescence microscopy . Surprisingly , pre-infection with BCG PGL I or M . leprae led to a significant internalization of BCG WT ( live or dead ) in SCs ( Fig 3A and 3B ) . Contrariwise , dead M . leprae and BCG PGL I were unable to instigate a major upgrade in SC internalization ( Fig 3C ) . PGL I-covered latex beads were likewise unable to mimic the effect of PGL I-expressing bacteria ( Fig 3C ) . Alternatively , even the direct addition of purified PGL I to the medium had no tangible effect on the SC phagocytic response ( S3A Fig ) . Moreover , the expanded phagocytic capacity induced by M . leprae and BCG PGL I on SC was specific to BCG WT since the degree of internalization of non-pathogenic M . smegmatis ( Fig 3D ) and the uptake of green fluorescent latex beads remained unchanged ( S3B Fig ) . To summarize , these results suggest that live PGL I-producing mycobacteria modulate the expression in SCs of phagocytic receptors that recognize cell wall envelope components specifically expressed by pathogenic or slow growing mycobacteria . LAM , present in the mycobacterial cell envelope , is an abundant glycolipid shown to play a key role in mycobacteria-host cell interaction [26 , 27] . In pathogenic mycobacteria , this molecule , denominated ManLAM [28] , is mannose capped . It has been demonstrated that the terminal mannose residues of ManLAM are of critical importance in macrophage recognition of M . tuberculosis , M . leprae and M . bovis BCG [29 , 30] via the capacity of these residues to bind to the host cell mannose receptor ( MR/CD206 ) [31] . In contrast , M . smegmatis produces a structurally different LAM that is capped with phosphatidyl-myo-inositol residues that do not bind to CD206 [32] . Based on this knowledge , we postulated the candidacy of the mannose receptor as possible target of upregulation by PGL I-expressing mycobacteria in infected SCs . To validate this hypothesis , the effect of excess free mannose on BCG WT internalization in SCs pre-infected with M . leprae or BCG PGL I by flow cytometry was evaluated . This competitive assay showed that the presence of mannose at 100 μg/mL or 1000 μg/mL significantly reduced BCG WT internalization induced by pre-infection with BCG PGL I or M . leprae ( Fig 4A ) . Under the condition of 1000 μg/mL of mannose , the degree of BCG WT internalization decreased by about 40% . Fluorescence microscopy also showed decrease in association degree ( S4 Fig ) , suggesting the recognition of LAM mannose caps during BCG WT internalization . To further explore this idea , green fluorescent latex beads were covered with M . leprae-purified ManLAM and used as the second stimulus in SCs pre-infected with live BCG PGL I or M . leprae . ManLAM-covered beads showed an increased uptake as seen for BCG WT , pointing to the upregulation of a mannose receptor in pre-infected cells ( Fig 4B ) . Since CD206 was previously shown to be expressed by SCs [33 , 34] , we then explored the ability of M . leprae or BCG PGL I to modulate this receptor . The mrc1 gene transcriptional levels in SCs were assayed at different time points by quantitative RT-PCR . Fig 4C shows the relative expressions of normalized mrc1 ( delta delta Ct ) in BCG PGL I- , M . leprae- , or BCG WT-infected SCs at 4 h , 24 h and 48 h . Results are presented in terms of a fold change after normalization with ribosomal protein L13 ( RPL13 ) mRNA . It was found that M . leprae and BCG PGL I were capable of inducing mrc1 transcription in SCs . It was also seen that the upregulation of mrc1 occurred at an early stage in the infection in that , a mere 4 h later , the transcription level had reached a peak . Of note , BCG WT also induced mrc1 gene upregulation but at a 50%-or-less efficacy rate as compared to the bacilli-expressing PGL I . It was , however , solely in the presence of M . leprae or BCG PGL I that upregulation was sustained until 24 h post-infection ( Fig 4C ) . Accordingly , flow cytometry assays showed an increased expression of CD206 upon M . leprae or BCG PGL I but not BCG WT infection ( Fig 4D ) . Comparable results were observed by fluorescence microscopy analysis ( Fig 4E ) , indicating that the expression of CD206 is upregulated in SCs infected with PGL I-expressing bacilli . That M . leprae and BCG PGL I infection led to the upregulation of CD206 and its subsequent involvement in the uptake of BCG WT was confirmed by mrc1 knockdown experiments . Treatment with mrc1 siRNA targeting the mrc1 gene coding for CD206 , led to a significant reduction in CD206 expression in BCG PGL I and M . leprae-infected SC , as compared to cells transfected with the control siRNA ( Fig 5A ) . As monitored by flow cytometry ( Fig 5B ) and fluorescence microscopy ( Fig 5C ) , mrc1 knockdown resulted in a significant reduction in the degree of BCG WT internalization . Of note , mrc1 knockdown also decreased M . leprae internalization ( Fig 5B and 5C ) . Taken together , these results show that live PGL I-producing mycobacteria induce the expression of the mannose receptor CD206 in SCs , a mechanism that may promote M . leprae internalization by an alternative pathway . Previous reports have shown a reciprocal regulation between CD206 and the transcription factor peroxisome proliferator-activated receptor gamma ( PPARγ ) [35 , 36] . Furthermore , the induction and activation of PPARγ by pathogenic mycobacteria following macrophage infection has been linked to their capacity to persist in these cells [34 , 36 , 37] . We thus analyzed the potential involvement of PPARγ in inducing CD206 in M . leprae and BCG PGL I-infected SCs . Fig 6A indicates that M . leprae and BCG PGL I infection induce PPARγ expression in contrast to BCG WT , which does not . Moreover , cells pre-treated with GW9662 , an irreversible antagonist of PPARγ , showed reduced CD206 levels in response to M . leprae and BCG PGL I monitored 24 h post-infection by both flow cytometry ( Fig 6B ) and fluorescence microscopy ( Fig 6C ) . At this time point , the percentage of SCs with internalized bacteria remained unchanged in GW9662-treated cells as compared to untreated ones ( S5A Fig ) . However , after 48 h of infection , GW9662 pre-treated cells demonstrated a lower number of M . leprae bacilli per cell , comparable to that observed after the mrc1 knockdown ( S5B Fig ) . Additionally , cell pre-treatment with GW9662 combined with pre-infection with PGL I -expressing mycobacteria were unable to increase ManLAM-covered bead uptake ( Fig 6D ) , confirming the involvement of PPARγ in the upregulation of CD206 in SCs infected with mycobacteria expressing PGL I . An evaluation was then carried out to determine whether bacterial recognition via CD206 was involved in PPARγ induction and activation . To test this hypothesis , live bacteria were incubated with cells in the presence of an excess of free mannose to block bacterial recognition by CD206 . The induction and activation of PPARγ was monitored via immunofluorescence . As shown in Fig 7A , significantly lower levels of PPARγ induction and activation were observed in the presence of free mannose , suggesting the involvement of bacterial recognition via CD206 in the capacity of M . leprae to activate this nuclear receptor/transcriptional factor in SCs . Since PPARγ has been implicated in LD biogenesis induced by mycobacteria [37] , we next evaluated whether this was the case in the context of M . leprae infected SCs . Inhibition of PPARγ with GW9662 abolished the induction of LDs ( Fig 7B ) by M . leprae . Moreover , inhibiting signaling from MR/CD206 by infecting the cells in the presence of excess of free mannose reduced LDs levels significantly ( Fig 7C ) . In previous reports we showed that M . leprae- induced LDs constitute sites of ( PGE2 ) synthesis [22] . Next , we investigated the role of MR/PPARγ crosstalk in PGE2 production . PPARγ inhibition decreased PGE2 production to baseline levels in response to M . leprae ( Fig 7D ) . A similar effect was observed when cells were infected in the presence of excess of free mannose ( Fig 7E ) . Experiments were then conducted to monitor the effect of mrc1 knockdown on PGE2 production in response to M . leprae . However , the high background of PGE2 production in the uninfected cells transfected with the mrc1 siRNA prevented reaching a reliable conclusion ( S6 Fig ) . It was then determined to examine if the recognition of M . leprae by CD206 causes an effect on subsequent bacterial intracellular survival since LDs formation was previously shown to promote M . leprae persistence in infected cells [23] . To block recognition of M . leprae by CD206 , an excess of free mannose ( 100 μg/mL ) was added to SCs followed by the monitoring of bacterial viability . Fig 7F shows a 27% ( *p<0 . 05 ) decrease in cellular bacterial viability upon 48 h of infection when bacterial recognition by CD206 was inhibited . These results provide evidence that CD206-PPARγ crosstalk promotes bacterial survival in M . leprae-infected SCs . The connection of CD206 to PPARγ and its role as an important regulator of macrophage immune response to M . tuberculosis has recently been reported [35 , 38] . In that study , a signaling pathway involving recognition of Man-LAM by CD206 followed by PPARγ expression and activation terminating in IL-8 induction was described [35] . In this context , our next step involved examining whether this pathway was active in mycobacterium-infected SCs . To answer this question , the effect of mrc1 knockdown or inhibition of PPARγ activity on IL-8 was investigated . Our results demonstrated that M . leprae infection enhanced IL-8 production and that knocking down mrc1 caused a decrease on IL-8 production ( Fig 8A ) . Moreover , infection in the presence of an excess of free mannose evoked a similar inhibitory effect of IL-8 secretion ( Fig 8B ) . Also , treatment with GW9662 decreased IL-8 production indicating the involvement of PPARγ ( Fig 8C ) . Altogether , these results suggest that there is an active crosstalk between PPARγ and CD206 that links lipid metabolism with the downstream innate immune response in SCs infected with PGL I-producing mycobacteria . The ability of M . leprae to induce the expression of CD206 in SCs cultures prompted us to further examine leprosy nerve specimens to corroborate the in vitro findings with in situ evidence . To verify if CD206 was expressed by SCs , tissue sections of patients with leprosy and non-leprosy neuropathies were also stained with anti-S100 , a specific SC marker ( Fig 9 , S7 Fig , S8 Fig ) . Nerve biopsies previously known to be bacilli positive ( AFB ( + ) ) were chosen for this analysis , as shown by Wade's [39] staining in Fig 9A . Fig 9B and 9C show the staining profile of S100 and the mannose receptor CD206 , respectively . Cells with a SC morphology expressing CD206 and S100 were observed in leprosy patients ( Fig 9D ) . CD206-expressing SCs can be better visualized in the insets ( 1 , 2 ) with a magnified view . Nerve biopsies from other 4 patients were analyzed generating similar results ( S8 Fig ) . Nerve biopsies obtained from patients with non-leprosy neuropathies showed no CD206/S100 colocalization ( Fig 9E–9G ( insets 3 and 4 ) , S7 Fig ) . In order to confirm that SCs expressing CD206 were harboring M . leprae , leprosy nerve tissue sections were labeled with anti-CD206 , anti-Myelin Basic Protein ( MBP ) , a SC marker , and anti-Liporabinomannan ( LAM ) for mycobacterium staining . Fig 10 shows most cells in the field double stained for CD206 and MBP , confirming the presence of SCs expressing CD206 in leprosy patients nerves . Since one Schwann cell may spread over hundreds of μm long nerve , a serial slices analysis would probably be necessary to confirm the presence of bacteria in a single cell . Nevertheless , examining a single cross-sectioned slice of the nerve biopsy we were able to detect M . leprae inside half of the SCs . Altogether , these data strongly suggest that M . leprae induces the expression of CD206 in in vivo-infected SCs reproducing our in vitro findings .
The most severe symptoms of leprosy are caused by nerve infection . Thus , deciphering the molecular basis of the early events of mycobacterial peripheral nerve infection is a crucial step towards acquiring a basic understanding of nerve pathogenesis with the potential to generate new tools for its prevention and treatment . The capacity of M . leprae to bind to laminin-2 , a major component of the SC basal lamina , has been described as a fundamental feature of the bacterial predilection for the peripheral nervous system [6] . Moreover , the PGL I and Hlp/LBP-21 molecules produced by the leprosy bacillus located on the bacterial surface have been implicated as likely adhesins involved in this interaction [12] . In the present study , we revisited the involvement of PGL I in M . leprae-SC interactions by using a recombinant strain of M . bovis BCG engineered to express PGL I . We demonstrate for the first time that PGL I is essential for mycobacterium adhesion and SC internalization . We were also able to confirm that the unique trisaccharide moiety of PGL I mediates the specific M . leprae-SC interaction since other phenolic glycolipids with identical lipid moieties proved incapable of mediating bacterial internalization into these cells . Of note , it was found that live PGL I-producing mycobacterium induces the activation of a crosstalk between the endocytic receptor MR/CD206 and the transcriptional factor PPARγ , allowing bacterial recognition and entry by way of this alternative pathway . Bacterial sensing via mannose recognition was shown to be essential for the induction of LD formation , organelles previously shown to play a key role in M . leprae-SC interaction [22 , 23] . Finally , the detection of strongly positive CD206 SCs in leprosy nerve sections suggests that M . leprae induces CD206 expression in in vivo-infected SCs , which may be critical in the development of bacterial pathogenesis in the nerve . The results herein presented firmly indicate that PGL I is the key molecule responsible for capacitating M . leprae to successfully invade SCs . In this process , PGL I imposes a secondary role on other potential adhesins such as Hlp/LBP-21 , a well-conserved , histone-like protein present on the surface of several species of mycobacteria , including BCG [19] . Recombinant Hlp binds in vitro to the laminin-2 globular domain; and latex beads covered with the protein have been seen to display a greater capacity to adhere to and be internalized by SC [10 , 11] . Indeed , other species of mycobacteria were shown to bind laminin-2 and adhere to SCs in the presence of soluble alpha2-laminin [40] . However , most evidence suggesting a role for Hlp in M . leprae–SC interactions have been drawn from assays conducted with the isolated protein alone in the absence of PGL I . Moreover , previous studies on M . leprae adhesion to SC were always performed with killed bacteria that may in the end display altered cell-wall structures , and , therefore , lead to misleading conclusions regarding the relative importance of the different bacterial molecules involved in host-cell binding [12 , 31 , 41] . The use of a PGL I-expressing BCG strain made it possible to decipher the essential role played by PGL I in this process in view of the fact that the BCG WT strain expressing Hlp , but not PGL I , was unable to invade SC . In any case , it is deemed worthwhile to investigate the chance that , subsequent to the initial PGL-I-mediated M . leprae-SC interaction , the Hlp binding to laminin may provide the bacilli with higher avidity . The present study likewise showed that CD206 , an important mycobacterial recognition receptor [28 , 32 , 42–45] , is significantly upregulated at both the mRNA and protein levels at early time points after SCs infection by PGL I-producing mycobacteria . When stimulus occurred with dead bacteria or PGL I-covered latex beads , this effect was not detected , suggesting that while PGL I binding to SC receptors is necessary , it is insufficient in terms of inducing this phenotypic change in SCs . This observation indicates that the status of bacilli ( live or dead ) is a key aspect in the interplay between M . leprae and SC and that live bacteria might modulate several pathways that go beyond the binding of PGL I to laminin 2 in these cells . Indeed , in previous studies , we have shown that only live M . leprae was able to induce the accumulation of lipids leading up to the formation of a foamy phenotype in infected SCs [23] . More recently , the capacity of M . leprae to modulate host-cell glucose metabolism and activate the IFN type I response in SCs has also been demonstrated to primarily depend on bacterial viability [21 , 46] . CD206 or MR is a member of the C-type lectin family that binds mannose and fucose with the highest affinity [47] . MR is a pattern-recognition receptor ( PRR ) performing a key role in binding to microbial pathogens and facilitating their uptake by innate immune system cells . CD206 was shown to play a major role in the interaction of pathogenic mycobacteria with human macrophages , via recognition of Man-LAM abundantly present on their cell surface [45 , 48–50] . Our findings suggest that the involvement of CD206 in the first wave of M . leprae entry in SCs is probably minimal , increasing in importance at later time points of infection . The observance of a lower M . leprae-invasion rate into SCs in which mrc1 was silenced is indicative that the ManLAM-MR binding may participate in bacterial recognition and uptake after PGL I-mediated internalization . Moreover , infection of SCs for 48h in the presence of excess of free mannose reduced M . leprae internalization by about 50% ( S9 Fig ) . Also , the induction of CD206 only by live M . leprae may at least partially explain the higher efficiency of live bacteria in comparison to dead organisms to enter into SCs . Although we observed a clear upregulation of CD206 in infected SCs , the induction by M . leprae of additional receptors such as Dectin-2 or the dendritic cell-specific adhesion molecule 3-grabbing nonintegrin ( DC-SIGN or CD209 ) cannot be ruled out since they also bind to ManLAM [51 , 52] . Teles et al . [52] have furnished evidence that human SCs may express CD209 , both in vitro and in neural leprosy lesions . Although M . leprae was also found in CD209 ( - ) SCs , it was shown to contribute to M . leprae-SC binding and be consistent with the detection of M . leprae-specific antigens in vitro and in situ in CD209 ( + ) SCs . The capacity of M . leprae to induce DC-SIGN expression is a topic deserving of further study . We also showed the involvement of the nuclear receptor/transcriptional factor PPARγ in the MR induction by PGL I-producing mycobacteria [36 , 53] . Moreover , a positive loop between CD206 and PPARγ was detected since blocking M . leprae recognition with excess of free mannose inhibited PPARγ activation . PPARγ is a master transcriptional factor regulating multiple cellular functions , including lipid metabolism and foam-cell generation [54–57] . The role of PPARγ and subsequent lipid-droplet biogenesis in mycobacterium-infected macrophages has also been described [37 , 55] . Since M . leprae induces LD formation [23 , 58] , an obvious subsequent investigation was the involvement of PPARγ in LD formation . Of note , an important finding of the current study was that interference in the CD206/ PPARγ crosstalk , either by inhibiting bacterial recognition by mannose receptors or by blocking PPARγ activation completely abolished M . leprae- induced lipid droplets in SCs . Next , we identified a dependence of PGE2 production on PPARγ activity in SCs . This observation , in combination with the known role of this nuclear receptor on LD formation [55] , is in agreement with our previous studies indicating that PGE2 synthesis occurs in lipid droplets and its secretion is in correlation with the lipid droplet levels in M . leprae infected SCs [23] . Actually , in that same study we had identified a link between lipid metabolism through LD formation and the innate immune response triggered by live M . leprae in SCs . Besides PGE2 , M . leprae was able to induce IL-10 that was abolished when LD formation was inhibited . Moreover , SCs even started producing IL-12 in the absence of LD formation . Interestingly , the same phenotype ( decreased IL-10 and increased IL-12 ) was observed when NS-398 , a COX-2 inhibitor , was used , suggesting that PGE2 may contribute to the negative modulation of the innate immune response toward intracellular infection . This goes in line with the immunomodulatory properties of PGE2 , which has been shown to inhibit Th1 response and the microbicidal mechanisms of macrophages [59–62] . Furthermore , Schreiber et al . [63] found evidence that the mannose receptor biosynthesis is up-regulated by E-series prostaglandins . The hypothesis that a PGE2 autocrine loop may participate in raising MR expression levels in M . leprae-infected SC , needs more scrutiny . In previous studies , we detailed the capacity of M . leprae to induce lipid accumulation in both macrophages and SCs [23 , 58 , 64] . In SCs , this effect was only observed with live bacteria , being in agreement with the findings here presented . Moreover , lipid droplets were shown to be recruited to mycobacterium-containing phagosomes , and both blockage of this recruitment or inhibition of LD formation lead to bacterial killing [23] . Moreover , as mentioned above , M . leprae-induced LD biogenesis and PGE2 production play a role in the generation of an innate immune response that may be permissive for bacterial persistence and proliferation . Thus , the decreased M . leprae viability in cells in which bacterial recognition through MR became inhibited after adding an excess of free mannose , can be explained by the blockage of LD formation and the secondary effects as a consequence of this inhibition . In addition to aiding in bacterial internalization , signaling through MR/CD206 has also been seen to play an important role in M . tuberculosis pathogenesis [31 , 50] . In infected macrophages , the recognition of ManLAM , abundantly present on the M . tuberculosis cell surface , by MR has been detected directing live bacilli to a phagosomal compartment with limited lysosomal fusion . This process was seen to be MR-ManLAM-specific because entry via another C-type lectin PRR , DC-SIGN , did not mediate this effect . Neither did PILAM , LM , nor any other mannosilated glycolipids present in the mycobacterial cell envelope [31] . In a recent study , the signaling cascade triggered by MR/CD206 to limit phagosome-lysosomal fusion was described [65] . In a previous report , we showed an active avoidance of phagolysosomal fusion in SCs by viable , but not heat-killed M . leprae [66] . Assuming that MR/CD206 triggers a similar signaling cascade in SCs , this could be explained by the fact that MR induction only occurs in the presence of live bacteria . However the link between M . leprae sensing via the CD206 and the limited phagosome maturation observed in M . leprae infected SC needs confirmation . Our results imply that besides PGE2 , PPARγ also regulates IL-8 production by SCs in response to M . leprae . Indeed , it has also been reported that the IL-8 promoter contains a PPARγ response element whose expression is regulated by PPARγ synthetic ligands [67] . Also , it was previously shown that lepromatous leprosy patients tend to have an up-regulated IL-8 response [68] and that SCs produce higher levels of IL-8 after contact with M . leprae antigens [69] . Moreover , our findings are in connection with a previous study that described that PPARγ induction by a virulent M . tuberculosis strain in human macrophages was seen to mediate the subsequent production of IL-8 and PGE2 by these cells [35] . IL-8 is a classic pro-inflammatory mediator that has been linked to demyelination and neurodegeneration [70] . Interestingly , M . leprae-induced demyelination as a strategy for intracellular survival has previously been described . The demyelination process induces de-differentiation of SCs and de-differentiated Schwann cells have been described as highly susceptible to M . leprae invasion [71] . IL-8 may also be taking part in cell recruitment and inflammation in these nerves . MR expression may potentially influence multiple cellular functions due to the presence of an intronic miRNA , miR-511 , embedded in and co-expressed with the mrc1 gene in macrophages [42 , 72] . miR-511-3p , the active strand of miR-511 , is co-regulated with the MR mRNA and protein . Of note , it is predicted that miR-511-3p is in control of a wide range of genes involved in multiple cellular processes , including cellular morphogenesis , metabolism , protein localization , and gene transcription [73] . Interestingly , miRNA-511 presents a target sequence within the 3’ UTR of the human PPARγ gene [74] . It is recommended that further studies be carried out to explore the regulation and impact of miRNA-511 on M . leprae-infected SC and leprosy pathogenesis . In summary , the present along with the reported data in the literature strongly suggest that M . leprae actively modulates SC functions in order to establish a safe bacterial intracellular niche . A key fundamental aspect of M . leprae pathogenesis seems to be the induction of LDs . In the proposed model represented in Fig 11 , signals from at least three recognition pathways contribute to LD formation: i ) PGL-I , via binding to laminin-2 , appears as the key molecule necessary for bacterial entry . Internalization was previously shown to be mandatory for LD formation [23]; ii ) bacterial recognition via TLR6 was previously shown to play a role in M . leprae internalization and to be crucial for LD biogenesis , although the mycobacterial component that binds to TLR6 remains to be determined [22 , 58]; and iii ) signals generated from CD206 are also critical for LD induction . Binding of bacterial mannose enriched molecules to baseline levels of CD206 expressed by SCs is probably sufficient to initiate PPARγ activation , starting the reciprocal regulation with CD206 and their subsequent upregulation . The amplified signals originated from the CD206/ PPARγ crosstalk promotes the accumulation of host-derived lipids in infected cells . This is followed by LD recruitment to bacterium-containing phagosomes . The high levels of CD206 expression allows bacterial entry by this alternative pathway at later time points of infection . LD formation leads to PGE-2 and IL-10 secretion and inhibition of IL-12 production [22] , which favors the inhibition of SC´s microbicidal mechanisms . Moreover , PPARγ activation induces the secretion of IL-8 that may contribute to demyelination as well as to the recruitment of immune cells to the site of infection and subsequent inflammation and tissue damage in infected nerves . The data presented in this study disclose fundamental aspects of M . leprae pathogenesis in the nerve , pointing to the expression of PGL-I and the activation of the CD206/PPARγ crosstalk as central aspects in this process . The importance of CD206 in mycobacterial infection is further corroborated by evidence that CD206 polymorphisms are associated with an enhanced susceptibility to M . tuberculosis [75] or M . leprae infection [76] . Moreover , our results support the idea that SCs are immunocompetent cells that play active role during peripheral nerve injury . Finally , the disclosed data could prove useful in developing alternative interventions to prevent and treat leprosy neuropathy , based on the blockage of PGL-I- mediated bacterial internalization and/or inhibition of PPARγ activation and PG production .
The ST8814 tumor cell line generously donated by J . A . Flechter ( Dana Farber Cancer Institute , Boston , MA , USA ) originated from malignant schwannomas ( neurofibromatosis type 1 ) of patients with neurofibromatosis type I . The cells were grown in RPMI 1640 medium ( LGC , Biotecnologia ) supplemented with 100 U/mL of penicillin , 100 μg/mL of streptomycin , 2 mM l-glutamine ( LGC , Biotecnologia , SP , Brazil ) , and 10% fetal calf serum ( FCS ) ( Cripion Biotecnologia LTDA ) in a humidified 5% CO2 incubator at 37°C . They were then plated in complete RPMI medium in culture dishes ( Nunc A/S , Roskide , Denmark ) or in 24-well plates ( Falcon , Franklin Lakes , NJ , USA ) at a density of 40 , 000 cells per well and maintained in humidified air in an atmosphere of 5% CO2 at 37°C for 24 h . Prior to infection or stimulation , the cells received new RPMI medium supplemented with 2% FCS but no antibiotics . Assays with primary cultures of human Schwann cells ( PHSC ) were carried out with cells isolated from human spinal nerves that were purchased from ScienCell , Carlsbad , CA , USA ( Cat . no . 1700 ) . The PHSC were maintained in Schwann cell medium ( SCM , Cat . no . 1701 , ScienCell ) supplemented with Schwann cell growth supplement ( SCGS , Cat . no . 1752 , ScienCell ) according to the suppliers’ recommendations . M . leprae Thai-53 purified from athymic BALB/c ( nu/nu ) mouse footpads was donated by the Lauro de Sousa Lima Institute , Bauru , São Paulo , Brazil . Recombinant M . bovis BCG strains with plasmids expressing PGL I , PGL TB , or the BCG Wild Type with an empty plasmid were kindly provided by Dr . Christophe Guilhot ( Institut de Pharmacologie et Biologie Structurale , Toulouse , France ) . Also , GFP expressing recombinant strains were provided . Culture was processed as described by Tabouret et al . [15] . Briefly , M . bovis BCG Pasteur 1173P2 was cultured in Middlebrook 7H9 broth ( DIFCO laboratories , USA ) and supplemented with 10% ADC ( 0 . 2% dextrose , 0 . 5% bovine serum albumin fraction V , 0 . 0003% beef catalase ) and 0 . 05% Tween 80 under constant agitation on a magnetic plate until the exponential phase or on solid Middlebrook 7H11 broth containing ADC and 0 . 005% oleic acid ( OADC ) ( Becton Dickinson , Sparks , USA ) . For recombinant strains , kanamycin was added to the medium at the final concentration of 50μg/mL . For GFP expressing recombinant strains , kanamycin ( Km ) and hygromycin ( Hyg ) were added to the medium at the final concentration of 40μg/ml and 50μg/ml respectively . M . smegmatis ( mc2 155 ) was cultured in Middlebrook 7H9 broth ( supplemented with 10% ADC and 0 . 05% Tween 80 ) . For in vitro infections , all bacteria were resuspended in RPMI1640 medium before use . Part of each lot of mycobacteria received lethal gamma irradiation of 15 kilogray ( Aceletron—Acelétrica Comércio e Representações Ltda . , Rio de Janeiro , RJ , Brazil ) , as previously recommended [44] . Bacilli were counted according to Shepard and McRae [77] criteria; and bacillar viability was corroborated by way of LIVE/DEAD BacLight Bacterial Viability Kits ( Invitrogen , USA ) , according to the manufacturer’s instructions . Mycobacteria were labeled with a red ( PKH26 ) or green ( PKH67 ) fluorescent dye according to the manufacturer´s instructions ( PKH26GL , PKH67GL , Sigma-Aldrich ) . For the phagocytosis assays , PKH-labeled or GFP expressing bacilli were added to the ST8814 cells in 24-well plates and maintained in a humidified 5% CO2 incubator at 33°C for 48 h in an antibiotic-free medium . SCs were infected with recombinant BCG strains , M . smegmatis or M . leprae at , unless otherwise stated , a multiplicity of infection of 50 ( MOI 50:1 ) . Phagocytosis was evaluated using flow cytometry and fluorescence microscopy . Cells were harvested with Trypsin 1x ( LGC , Biotecnologia ) , washed , and fixed with 1% paraformaldehyde . For PKH67 , green-labeled bacilli flow cytometry was carried out using the Accuri C6 ( Accuri Cytometers , Inc . ) FL1-A ( green ) channel and analyzed via CFLow Plus . To distinguish between adhered and internalized bacilli , the fluorescence of the externally-adhered ones was quenched with Trypan Blue ( Sigma-Aldrich ) . For the microscopic assays , cells were plated ( Corning , Fisher Scientific , USA ) on cover slips in 24-well plates . Fluorescence microscopy used the Axio Observer Z1 ( Carl Zeiss ) microscope and AxioVision Rel . 4 . 8 software ( Zeiss , Göttingen , Germany ) . The mean fluorescent signal value was quantified via open-source ImageJ software . ( https://imagej . nih . gov/index . html; Research Services Branch , National Institute of Mental Health , National Institutes of Health , Bethesda , MD , USA ) . Protocol for PKH labeling available at: dx . doi . org/10 . 17504/protocols . io . phcdj2w . Protocol for phagocytosis assays available at: dx . doi . org/10 . 17504/protocols . io . pnpdmdn . Green fluorescent polystyrene beads ( Polysciences , Warrington , PA ) ( 1μM diameter ) were coated with 100 μg/mL ManLAM derived from M . leprae ( NR-19348 , BEI Resources ) in sodium bicarbonate buffer ( pH 9 . 6 ) . After washing , the beads were blocked with 2% BSA in PBS . Alternatively , the beads were coated with 100 μg/mL PGL I derived from M . leprae ( NR-19342 , BEI Resources ) . The coating protocol was based on Ng , et al . [12] ManLAM or PGL I-coated beads were incubated with SC cultures for 48 h . The degree of association or internalization was determined by flow cytometry and visualized by fluorescence microscopy . Procedure for coating of beads with glycolipids available at: dx . doi . org/10 . 17504/protocols . io . pmrdk56 . The first stimulus , consisting of unlabeled bacteria or non-fluorescent latex beads , was always carried out with a low ( 10:1 ) multiplicity of infection ( MOI ) or proportion ( beads:cell ) one hour prior to the second stimulus . In these assays , live and dead bacteria were tested as pre-stimuli . When latex beads or dead bacilli were added , the term “proportion” was applied . The second stimulus consisted of PKH67- or PKH26-labeled bacteria or green fluorescent latex beads at a MOI or proportion of 50:1 . All experiments were carried out during a 48 h period ( unless otherwise stated ) and analyzed using flow cytometry or fluorescence microscopy . In some cases , compounds like mannose at 100 μg/mL or 1000 μg/mL ( 112585 Sigma-Aldrich , USA ) and GW9662 at 5 μM ( CAS 22978-25-2 , Cayman Chemical , USA ) were added to the culture medium before the first stimulus . GW9962 is an irreversible antagonist of the transcription factor PPARγ . Optimal concentration of GW9662 to inhibit lipid body biogenesis was determined empirically for ST8814-SC . Based on previous reports [37] three concentrations ( 1 μM , 5μM and 10μM ) of the drug were tested . Using fluorescence microscopy and Oil Red O labeling , we did not detect any effect on lipid body formation with 1 μM . The 5 μM concentration had a visible negative effect on lipid body formation . And with the 10 μM we readily observed a cytotoxic effect upon microscopic evaluation . To assess cell viability after using GW9662 , cytotoxicity testing via MTT tetrazolium was performed . The 5 μM concentration showed no cytotoxic effect . Results are shown as S10 Fig . CD206 or PPARγ expression was evaluated using flow cytometry and/or fluorescence microscopy . For the former , ST8814 cells were harvested using EDTA 5mM and then washed and fixed with 1% paraformaldehyde . Mannose receptor labeling was carried out with antibody anti-CD206 -FITC ( clone15-2 , Mouse IgG1 , κ1 , Biolegend ) . Negative controls were labeled with an FITC-conjugated isotype antibody . CD206 detection was performed 24 h post-infection using the Accuri C6 ( Accuri Cytometers , Inc . ) FL1-A channel . For fluorescence microscopy , the cover-slip cells were fixed and permeabilized with PBS containing paraformaldehyde 1% and saponin 0 . 1% . PPARγ detection was analyzed 48 h post-infection by way of the specific rabbit polyclonal antibody ( H-100 ) SC-7196 ( Santa Cruz Inc . , USA ) followed by incubation with goat IgG anti-rabbit conjugated with Alexa Fluor 555 A-21428 ( Molecular Probes , USA ) for immunofluorescence detection . Nuclei were evidenced by DAPI ( 4 , 6-diamidino-2-phenylindole ) staining ( Sigma-Aldrich ) ; and slides were mounted with Permafluor ( Thermo Scientific , Waltham , MA ) and analyzed via the AxiObserver Z1 Colibri microscope . Immunofluorescence was quantified using Open-Source ImageJ1 software ( Research Services Branch , National Institutes of Health , Bethesda , MD , USA ) . Protocol for antigen labeling of eukaryotic cells and detection by flow cytometry available at: dx . doi . org/10 . 17504/protocols . io . pg8djzw . Protocol for antigen labeling of eukaryotic cultured cells and detection by fluorescence microscopy available at: dx . doi . org/10 . 17504/protocols . io . phadj2e . For gene-expression knockdown , ST8814 cells were plated in complete RPMI medium at a density of 30 , 000 cells in a 24-well plate . Cells were transfected with a pre-designed Silencer Select siRNA for the mrc1 gene ( siRNA ID 106809 or siRNA ID 279717 , # AM16708 , Ambion Life Technologies , USA ) or the control siRNA ( # 4390874 , Ambion ) . Lipofectamine 2000 ( Life Technologies ) was used as a vehicle in line with the manufacturer’s instructions . The cells were transfected with 20 pmol of siRNA in a 100μl transfection mix of Opti-MEM ( Gibco ) and lipofectamine and 400μl of complete RPMI medium under a protocol in compliance with the manufacturer’s instructions ( Life Technologies ) . Twenty-four hours after transfection , the cells were infected with live M . leprae for 48 h in RPMI medium+ 2% FCS , followed by extraction of the nucleic acids and measurement of the mRNA by qRT-PCR . Cellular lysates were obtained using a cold “RIPA” buffer ( 50mM Tris pH 7 . 5; 1% Nonidet p40; 0 . 25% sodium deoxycholate; 0 . 1% sodium dodecyl sulfate ) containing protease inhibitors ( Complete Inhibitor Cocktail Tablets Roche , USA ) . Proteins were quantified via BCA assay ( Thermo Scientific , USA ) . A total of 20μg of protein/well were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) and transferred to nitrocellulose membranes ( Bio-Rad , Hercules , CA , USA ) . The antibodies PPARγ ( H-100 ) SC-7196 ( rabbit ) and GAPDH ( H2114 ) SC-32233 ( mouse ) , both from Santa Cruz Inc . ( USA ) , were diluted in 5% ( w/v ) nonfat milk dissolved in Tris-Tween-buffered saline ( TTBS; 20 mM Tris-HCl buffer , pH 7 . 6 , containing 137 mM NaCl [v/v] 0 . 05% Tween 20 ) . Results were visualized via the enhanced chemiluminescence detection system—ECL ( Amersham Biosciences , NJ , USA ) . Protocol for Western Blot available at: dx . doi . org/10 . 17504/protocols . io . pnrdmd6 . After stimulation , culture supernatants were harvested , centrifuged , and stored at -70°C . IL-8 levels in the ST8814 culture supernatant were evaluated by ELISA using the DuoSet kit ( R&D Systems ) . PGE2 concentration was measured in cell-free supernatants via an EIA kit ( Cayman Chemical Co . , Ann Arbor , MI , USA ) . The assays were conducted according to the manufacturer’s protocol . Schwann cells adhering to coverslips were fixed in 4% paraformaldehyde , and the area occupied by LDs were analyzed using fluorescent Oil red O ( ORO , Cat . N° O0625 SIGMA ) . Coverslips were mounted , the morphology of the fixed cells was observed , and LDs areas were enumerated in 500 consecutively scanned cells . Nuclei were stained with 2 mM DAPI ( Sigma-Aldrich ) at room temperature for 5 min . The ORO images ( taken with a 40-objective lens ) were transformed into black and white pictures and analyzed via Open-Source ImageJ1 software . The spots were determined by automatic spot detection; and the total area and average size of fluorescent LDs was obtained for each field and divided by the number of cells in the respective field . Fluorescence microscopy used the Axio Observer Z1 ( Carl Zeiss ) microscope and AxioVision Rel . 4 . 8 software ( Zeiss , Göttingen , Germany ) . Protocol for Oil Red O staining available at: dx . doi . org/10 . 17504/protocols . io . phbdj2n . Total RNA was extracted from stimulated SCs using TRIzol ( Thermo Fisher Scientific ) according to the manufacturer’s instructions . Total RNA was converted to cDNA using the RevertAid first-strand cDNA synthesis kit ( Thermo Fischer Scientific ) ; and samples were stored at −20°C until further use . A total of 10ng of cDNA was used for qRT-PCR that was performed by StepOnePlus ( Applied Biosystems , Foster City , CA , USA ) and the SYBR Green PCR master mix ( Applied Biosystems ) . Specific primers for mrc1 were used ( Forward: TGGTGGAAGAAGAAGCAGTC/ Reverse: TAGTCAAGGAAGGGTCGGAT ) . Thermal cycling conditions comprised an initial incubation at 95°C for 10 min , 40 cycles of denaturation at 95°C for 15 s , and annealing and extension at 60°C for 1 min . To normalize the relative mrc1 expression , rpl13 ( Forward: GACAAGAAAAAGCGGATGGT /Reverse: GTACTTCCAGCCAACCTCGT ) was used as an endogenous control whereas the relative expression values ( mrc1/rpl13 ) were obtained by converting the cycle threshold ( Ct ) values according to the following formula: expression value = 2 ( −ΔΔCt ) . Protocol for RNA extraction available at: dx . doi . org/10 . 17504/protocols . io . pg7djzn . Protocol for qPCR assays available at: dx . doi . org/10 . 17504/protocols . io . pnqdmdw . M . leprae viability was determined by qRT-PCR using the protocol previously described by Martinez , et al [78] . Briefly , ST8814 cells infected for 48 h with M . leprae and pre-incubated with mannose were submitted to RNA and DNA extraction with TRIzol ( Thermo Fisher Scientific ) . DNA was removed from the RNA preparations using the DNA-free turbo kit ( Ambion ) . RNA was reverse transcribed via a random primer and GoScript kit following the manufacturer’s instructions ( Promega ) . The levels of 16S rRNA were determined and normalized against 16S DNA . PCR efficiency for each experiment was gauged via LinRegPCR software [79]; and normalization was performed considering efficiency corrections , as described above [80] . The same set of primers for the rRNA 16S was used for both cDNA and DNA: sense 5’- GCATGTCTTGTGGTGGAAAGC -3’ and antisense 5’- CACCCCACCAACAAGCTGAT -3’ . cDNA and DNA were measured by TaqMan real-time PCR assay ( Taqman probe 16S 5’- CATCCTGCACCGCA -3’ ) . M . leprae viability was determined by the comparative Ct method [78]; and 100% viability was arbitrarily assumed for the infected control samples . Other values were normalized as a percentage of the control . Reactions were incubated in the ABI StepOne Plus Sequence Detection System ( Applied Biosystems ) . Protocol for determining M . leprae molecular viability by qPCR available at: dx . doi . org/10 . 17504/protocols . io . pmwdk7e . Histological sections of the peripheral nerves of five patients with pure neural leprosy ( PNL ) were included in the present study . One of them was a relapse of lepromatous leprosy . Diagnosis of PNL was carried out according to Jardim et al . [81] and all included cases presented acid-fast bacilli ( AFB ) in the biopsy . Also , three patients with axonal neuropathies not related to leprosy were included as controls . All patients were in attendance at the Leprosy Outpatient Unit of the Oswaldo Cruz Foundation , Rio de Janeiro , RJ , Brazil . After informed consent , nerve biopsy specimens were obtained and used for immunofluorescence testing . Tissue sections were thawed on sylane pre-coated slides and then rehydrated and stained with haematoxylin-eosin to evaluate the inflammatory infiltrate and cellularity . In accordance with standard protocols , the tissue sections were then stained with Gomori's trichrome to assess fibrosis and nerve structure [82] and subsequently submitted to Wade [39] staining to detect acid-fast bacilli . Immunofluorescence of cryostat tissue sections was performed initially , blocking the unspecific binding sites with 5% normal goat serum ( NGS; Life technologies ) with 2% bovine serum albumin ( BSA; Sigma-Aldrich ) in PBS containing 0 . 3% Triton X-100 ( Amersham Biosciences ) at room temperature for 4 h . After blocking , the tissue sections were incubated overnight with antibodies against S100 ( rabbit polyclonal; Sigma-Aldrich ) or anti-Myelin Basic Protein ( anti-MBP Rat monoclonal; Millipore ) , two specific SC markers , lipoarabinomannan for M . leprae labeling ( anti-LAM Rabbit polyclonal NR1809 provided by Bei Resources ) , and CD206 ( clone19 . 2 , Mouse IgG1 , ; BD Bioscience , San Jose , CA ) , a mannose receptor marker , followed by incubation for 2 h with goat anti-rabbit IgG conjugated with Alexa Fluor 568 A-11011 , goat anti-mouse IgG1 conjugated with Alexa Fluor 488 A-21121 ( Molecular Probes , Carlsbad , CA ) and goat anti-rat IgG conjugated with Alexa Fluor 594 ( Abcam , Cambridge , UK ) . As negative control the tissue sections were incubated only with secondary antibody , omitting the primary antibody . Nuclei were stained with DAPI ( Sigma Aldrich ) . The immunofluorescence of peripheral nerve sections was examined using a Zeiss Axio Observer microscope ( Carl Zeiss , Thornwood , NY , USA ) equipped with Plan Apo 40 and 100 objectives and coupled with Apotome optical sectioning system to generate optical sections of the fluorescent images ( Apotome . 2- Carl Zeiss ) . The system was coupled to a CoolSNAP-Pro CF digital camera in conjunction with Axion Vision Version 4 . 7 . 2 software ( Carl Zeiss ) . The images were edited via AxioVision software and their contrast was enhanced via Adobe Photoshop 14 ( Adobe Systems ) . Protocol for immunohistochemistry for frozen sections: dx . doi . org/10 . 17504/protocols . io . pkmdku6 . Assessment of the toxic effect of GW9662 on SCs was performed using an MTT assay . MTT , 3- ( 4 , 5-dimethylthiazol-2-yl ) -2 , 5-diphenyl tetrazolium bromide ( Sigma-Aldrich ) . The cells were seeded in 96-well plates at a density of 3000 cells per well and allowed to attach overnight . After a 44 h culture 10 μl MTT ( 5 mg/mL ) was added to each well for 4 h . The reactions were terminated by removing all culture medium and adding 100 μL de SDS 10% ( Bio-Rad ) to each well . Following uniform oscillation for 10 min to ensure complete solubilization of the purple formazan crystals , the absorbance values were determined at 570 nm with a plate reader . Assessment of the toxic effect of SC infection with BCG or BCG PGL I at MOI 50:1 for 48h at 33°C was also performed . The results are shown in S10 Fig . For the use of human samples , written informed consent was obtained from all patients and the procedures described were approved by the Ethics Committee of the Oswaldo Cruz Foundation ( Approval number 546/10 ) . These samples were obtained specifically for this study . Results are presented as the mean ± standard error of the mean ( SEM ) of independent experiments at a minimum in triplicate . Data were analyzed using the GraphPad Prism 5 Project ( GraphPad Software , La Jolla , CA , USA ) . Unless otherwise stated , analyses were performed by applying one-way ANOVA and the Bonferroni post-test to compare the different groups . A p-value <0 . 05 was considered statistically significant .
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Nerve damage is the most severe symptom of leprosy , an ancient disease that continues to be a major health problem in several countries . Nerve damage is due to the ability of Mycobacterium leprae , the etiologic agent , to invade SCs , the glial cells of the peripheral nervous system . Understanding the molecular basis of M . leprae–SC interaction is essential for the creation of new tools aiming to treat and , above all , prevent leprosy neuropathy . This study demonstrates the critical role of PGL I , an M . leprae-abundant specific cell wall lipid , in establishing infection . PGL I is not only a prerequisite in initiating bacterial adhesion to and subsequent invasion of SCs , but also for changing the repertoire of cell surface proteins to allow for the entrance of bacteria via alternative pathways . These new invasive pathways induced by PGL I involve recognition of other bacterial cell surface glycolipids that , in turn , evoke functional changes in the infected cell , including the accumulation of host cell-derived lipids , which favor bacterial survival . These pathways also promote the secretion of inflammatory mediators that may contribute to nerve damage . In an era of translational-oriented research , exploring these receptors in depth could lead to the development of attractive strategies to ensure the targeted intracellular delivery of therapeutics aiming to prevent neuropathy .
|
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2018
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PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves
|
The ability of neurons to differentially respond to specific temporal and spatial input patterns underlies information storage in neural circuits . One means of achieving spatial specificity is to restrict signaling molecules to particular subcellular compartments using anchoring molecules such as A-Kinase Anchoring Proteins ( AKAPs ) . Disruption of protein kinase A ( PKA ) anchoring to AKAPs impairs a PKA-dependent form of long term potentiation ( LTP ) in the hippocampus . To investigate the role of localized PKA signaling in LTP , we developed a stochastic reaction-diffusion model of the signaling pathways leading to PKA activation in CA1 pyramidal neurons . Simulations investigated whether the role of anchoring is to locate kinases near molecules that activate them , or near their target molecules . The results show that anchoring PKA with adenylyl cyclase ( which produces cAMP that activates PKA ) produces significantly greater PKA activity , and phosphorylation of both inhibitor-1 and AMPA receptor GluR1 subunit on S845 , than when PKA is anchored apart from adenylyl cyclase . The spatial microdomain of cAMP was smaller than that of PKA suggesting that anchoring PKA near its source of cAMP is critical because inactivation by phosphodiesterase limits diffusion of cAMP . The prediction that the role of anchoring is to colocalize PKA near adenylyl cyclase was confirmed by experimentally rescuing the deficit in LTP produced by disruption of PKA anchoring using phosphodiesterase inhibitors . Additional experiments confirm the model prediction that disruption of anchoring impairs S845 phosphorylation produced by forskolin-induced synaptic potentiation . Collectively , these results show that locating PKA near adenylyl cyclase is a critical function of anchoring .
Synaptic plasticity , the activity-dependent change in the strength of neuronal connections , is a cellular mechanism proposed to underlie memory storage . One type of synaptic plasticity is long term potentiation ( LTP ) , which displays physiological properties that are highly suggestive of information storage . Because of the role of the hippocampus in memory , LTP in the hippocampus is studied as a model of cellular properties underlying memory [1] . The induction of long-lasting forms of LTP requires interaction among calcium-activated pathways and metabotropic-receptor-activated pathways , but the interactions among these pathways depend on the extent to which signals are spatially restricted to subcellular compartments . The production of diffusible second messengers facilitates interactions , but interferes with signaling specificity [2] . Nonetheless , an increasing number of experiments have shown that the compartmentalization of critical proteins provides downstream signaling specificity [3] . For example , a PKA-dependent form of hippocampal LTP requires not only PKA activation , but also the appropriate localization of PKA [4] , [5] . Two basic mechanisms have been proposed for compartmentalization of signaling molecules: diffusional barriers and organization into multi-enzyme signaling complexes . Diffusional barriers in neurons are best exemplified by dendritic spines [6] , which compartmentalize calcium due to the small size of the spine neck [7] , [8] . Other synaptically activated , yet diffusible signaling molecules involved in synaptic plasticity , such as cAMP [9] and Ras [2] , can spread to multiple synapses that are in close proximity on a dendrite . A second mechanism for compartmentalization is to colocalize enzymes that work together . This organization is mediated by anchoring proteins , which are structural proteins that contain binding sites for various enzymes . PKA is compartmentalized to different subcellular locations through interaction with A-Kinase Anchoring Proteins ( AKAP ) [10] . Binding between the PKA regulatory subunit and the AKAP produces signaling specificity of the diffusible catalytic subunit of PKA [11] . Different AKAPs , such as AKAP5 , gravin , and MAP2 , anchor PKA to different locations , such as to the spine or the dendrite . In addition to binding PKA , various AKAPs bind other enzymes such as adenylyl cyclase , calmodulin , phosphodiesterase , or calcineurin [12]–[14] . Though PKA-dependent LTP requires an anchored pool of PKA [4] , [5] , it is unknown whether the critical function of anchoring is to place PKA near adenylyl cyclase , the source of cAMP that activates PKA , or near target molecules , such as GluR1 . To investigate this question , we perform simulation experiments using a novel , multi-compartment model of postsynaptic signaling pathways underlying PKA-dependent LTP in CA1 pyramidal neurons of the hippocampus . Furthermore , predictions from the model are confirmed experimentally using electrophysiological and biochemical approaches .
Prior to exploring PKA location , we first investigated the effect of adenylyl cyclase location on cAMP gradients . Simulations were performed with the dopamine D1 receptor and adenylyl cyclase colocalized either to the spine head or to the dendrite submembrane region , these two locations being suggested by ultrastructual analysis of dopamine receptors [17] and anchoring of adenylyl cyclase [12] , [18] . G proteins were colocalized with both the receptor and the adenylyl cyclase for all simulations [19] . Simulations show that localization of dopamine D1 receptor and adenylyl cyclase in the spine leads to higher cAMP in response to stimulation ( Figure 3B ) . Though calcium influx occurs in both spine and dendrite , calcium concentration is elevated in the spine head as compared to the dendrite ( Figure 3A ) , similar to that measured experimentally [20] . This calcium gradient produces a greater calmodulin-activated adenylyl cyclase when it is located in the spine head as compared to the dendrite . Localizing adenylyl cyclase in the spine also produces a large gradient of cAMP from spine head to dendrite ( Figure 3B ) . No gradient from the dendrite to the spine is apparent when adenylyl cyclase is in the dendrite , because cAMP diffuses easily to other parts of the dendrite; also , diffusion of a few molecules from the larger dendritic volume into the smaller spine volume is sufficient to raise the spine concentration . PKA is compartmentalized to different subcellular locations through interaction with various A-kinase anchoring proteins [10] . A pool of PKA in dendritic spines is created by synaptically localized AKAPs such as AKAP5 and Yotiao [13] , [21] . Recent evidence suggests that both Yotiao and AKAP5 also bind to adenylyl cyclase producing colocalization of PKA with its source of cAMP [12] , [22] . Other experiments demonstrate that PKA is enriched in the dendrite via MAP2 anchoring [23] . The PKA anchored to these different locations may serve different functions . To explore whether anchoring PKA near its activators or near its targets is more important in the induction of four-train LTP at Schaffer collateral CA1 synapses , PKA was either localized to the spine head , or placed in a focal region of the dendrite submembrane . We simulated these two spatial variations of PKA with the two spatial variations of adenylyl cyclase ( Figure 2 ) . The first case had PKA and adenylyl cyclase in the spine head , thus PKA was co-localized with both the source of cAMP and the AMPA receptor target . The second case placed PKA in the spine head but adenylyl cyclase in the dendrite submembrane , thus PKA was near the AMPA receptors but apart from adenylyl cyclase . The third case had PKA and adenylyl cyclase in the dendrite submembrane , thus PKA was co-localized with the source of cAMP , but separated from the AMPA receptors . The fourth case placed PKA in the dendrite submembrane and adenylyl cyclase in the spine head , thus PKA was apart from both its source molecules and its target . The four cases in this 2×2 experimental design ( Figure 2 ) allowed assessment of the role of PKA location relative to its source , cAMP , or one of its targets , the AMPA receptor GluR1 subunit , separately and in combination . With adenylyl cyclase located in the spine , PKA anchored in the spine produces a greater activity than PKA anchored in the dendrite ( Figure 4A ) . The quantity of PKA catalytic subunit is small because it has high affinity for each of its many binding partners ( type 4 phosphodiesterases , inhibitor-1 , AMPA receptor GluR1 subunits and PKA regulatory subunits ) , consequently the fluctuations are large . With adenylyl cyclase located in the dendrite , the effect of PKA colocalization is not apparent ( Figure 4B ) because of the large fluctuations . When the results are averaged over five simulation trials ( which used different random number seeds ) , a difference between these two cases emerges ( Figure 4C ) . The quantity of free PKA catalytic subunit is greater when PKA is colocalized with adenylyl cylcase in the dendrite than when PKA is separated from the adenylyl cyclase . These differences due to location of PKA and adenylyl cyclase were confirmed with statistical analysis ( SAS ) using the procedure GLM ( F = 238; P<0 . 0001; n = 20 ) . Colocalizing PKA and adenylyl cyclase in the spine produces greater PKA activation than colocalizing PKA and adenylyl cyclase in the dendrite ( P<0 . 001 ) , which produces greater PKA activation than either of the cases with PKA and adenylyl cyclase apart from each other ( P<0 . 001 ) . Another measure of PKA activity is phosphorylation level of downstream targets , of which four ( out of many ) are included in the model . Three of the PKA targets , phosphodiesterase types 4B and 4D , and inhibitor-1 , are distributed throughout the neuron , whereas one of the PKA targets , the AMPA receptor GluR1 subunit , is located exclusively in the post-synaptic density of the spine . Inhibitor-1 is important because its phosphorylation level increases with LTP induction [24] , and subsequent inhibition of protein phosphatase 1 enhances CaMKII phorphorylation . The AMPA receptor GluR1 subunit is phosphorylated on Ser845 by PKA , which enhances AMPA channel function and leads to increased AMPA channel expression [25] , [26] . Therefore , to evaluate the role of PKA anchoring on PKA activity , we quantify the phosphorylation levels of GluR1 on Ser845 , phosphodiesterase types 4B and 4D , and inhibitor-1 . The location of PKA and adenylyl cyclase modulates the level of phospho-inhibitor 1 ( Figure 5A; F = 68 . 8; P<0 . 0001 , n = 20 ) . The differences in phospho-inhibitor-1 levels due to anchoring are larger than the differences in free PKA catalytic subunit . When adenylyl cyclase is in the spine head , mean phospho-inhibitor-1 is greater when PKA is colocalized with adenylyl cyclase in the spine head than when PKA is in the dendrite ( P<0 . 001 ) . Similarly , when adenylyl cyclase is in the dendrite , mean phospho-inhibitor-1 is greater when PKA is colocalized with adenylyl cyclase in the dendrite than when PKA is in the spine ( P<0 . 001 ) . Thus , colocalizing PKA with its source molecules is one critical function of anchoring . Impeded diffusion of the PKA catalytic subunit from the spine to the dendrite , where most of the inhibitor-1 is located , does not prevent phosphorylation by PKA because the phosphorylation reactions are slow compared to diffusion . Two additional PKA targets in the model are phosphodiesterase types 4B and 4D . Anchoring PKA produces a change in phosphorylation of phosphodiesterases ( Figure 5B ) similar to that observed with phosphorylation of inhibitor-1 . Namely , colocalization of PKA with adenylyl cyclase in the spine head produces the greatest phosphorylation of phosphodiesterases . In addition , higher phosphorylation is observed when PKA is colocalized with adenylyl cyclase in the dendrite , as compared to when PKA is separated from adenylyl cyclase . Statistical analysis reveals a significant effect of PKA and adenylyl cyclase location ( F = 158 . 6; P<0 . 0001; n = 20 ) , and post-hoc tests confirm that the colocalized cases produce significantly greater phosphorylated phosphodiesterase than the non-colocalized cases ( P<0 . 001 for each adenylyl cyclase location ) . To better assess the importance of PKA proximity to source versus target molecules , GluR1 phosphorylated on Ser845 is analyzed because it is confined to the post-synaptic density of the spine . Figure 5C illustrates that the fraction of GluR1 phosphorylated on Ser845 depends on PKA and adenylyl cyclase location ( F = 9 . 1; P = 0 . 001; n = 20 ) . Phosphorylation by PKA is greatest when PKA is colocalized both with cAMP production in the spine and with the AMPA receptor GluR1 subunit ( P<0 . 001 ) . More importantly , GluR1 phosphorylated on Ser845 is greater when PKA is co-localized with adenylyl cyclase in the dendrite than when PKA is apart from adenylyl cyclase but colocalized with GluR1 ( P<0 . 05 ) . The large fluctuations in GluR1 phosphorylation ( Figure 6 ) suggests that not all synapses will be potentiated during experimental LTP induction . In conclusion , simulations reveal that PKA localization close to the source of cAMP is more important than localization near the target molecule . These results are robust to variations in parameters . Diffusion constants in vivo are substantially slower than those measured or calculated in vitro [27] . To demonstrate that our results are not dependent on the estimated in vivo values , we repeated a subset of simulations using faster diffusion constants , closer to the in vitro measurements . Even with these faster diffusion constants , a greater PKA activity is observed when PKA is anchored with adenylyl cyclase in the spine , than when PKA is anchored in the dendrite away from adenylyl cyclase ( Figure S1A ) . Because the rates for phosphorylation and dephosphorylation of GluR1 are not well constrained , we also evaluated the role of anchoring using a slower rate for dephosphorylation of phospho GluR1 by protein phosphatase 1 . This change in rate constants similarly does not change the effect of colocalization on PKA activity ( Figure S1B ) . Spine morphology varies widely in hippocampal CA1 dendrites [6] , and the diffusional barrier of the spine-neck geometry is an important determinant of NMDA receptor-dependent calcium signaling in the dendrite [28] . Thus , to evaluate whether spine morphology plays a role with more diffusible molecules such as cAMP , simulations were repeated using a spine with either a longer ( 1 . 0 µm ) or shorter spine neck ( 0 µm ) , representing the range of experimentally measured values . The longer spine neck produces a greater cAMP concentration and larger gradient from spine to dendrite ( Figure 7A ) . The smaller spine neck has the opposite effect , reducing the cAMP concentration . This change in cAMP due to spine neck propagates downstream to produce analogous changes in PKA activity ( Figure 7B; F = 568 , P<0 . 0001 , n = 10 ) , phosphorylation of inhibitor-1 ( Figure 7C; F = 7 . 7; P = 0 . 02 , n = 10 ) and phosphorylated PDE4s , reinforcing the role of PKA colocalization with adenylyl cyclase ( as opposed to target molecules ) . Experiments show that long-lasting LTP induced with four spaced trains of synaptic stimulation is impaired in the presence of Ht31 peptide , which competes for PKA anchoring [4] , [5] . To evaluate this experimental observation , we simulated PKA activity when PKA is uniformly distributed as produced by disruption of anchoring by Ht31 peptide . The simulation shows that the amount of free PKA catalytic subunit , phosphorylation of inhibitor-1 and GluR1 phosphorylated on Ser845 are reduced by 30–40% ( Figure 8A ) when PKA is uniformly distributed . The differences between PKA colocalized with adenylyl cyclase in the spine , and PKA uniformly distributed are statistically significant ( F = 256 , P<0 . 0001 for PKA; F = 54 . 3 , P<0 . 0001 for phospho-inhibitor-1; F = 11 . 9 , P = 0 . 0018 for GluR1-S845; n = 14 ) . This finding supports experimental studies showing that long-lasting LTP is blocked when anchoring is disrupted by Ht31 peptide . This finding further leads to the prediction that both phospho-inhibitor-1 and GluR1 S845 phosphorylation after experimental LTP induction will be smaller in the presence of Ht31 peptide . Collectively , the simulation results suggest that PKA needs to be near adenylyl cyclase , to be surrounded by a high concentration of cAMP , because phosphodiesterase activity lowers the cAMP concentration as it diffuses away from the adenylyl cyclase [3] . This leads to the prediction that PDE inhibition should rescue LTP when PKA anchoring is blocked . To test the computational prediction that PKA anchoring close to the source of cAMP is critical in synaptic plasticity , we use forskolin to induce synaptic plasticity in mice expressing Ht31 peptide in the hippocampus and in wildtype controls . Previous research shows that forskolin , which elevates cAMP by direct activation of adenylyl cyclase , induces PKA-dependent LTP in wildtype mice [29] . Figure 8B1 shows that forskolin-induced potentiation is impaired two hours after the drug treatment in Ht31-expressing transgenic mice ( P = 0 . 012 ) . The model predicts that inhibition of phosphodiesterases would rescue the LTP deficit caused by disruption of PKA anchoring . Indeed , in the presence of IBMX ( 3-isobutyl-1-methylxanthine ) , a non-specific PDE inhibitor [4] , inhibition of anchoring with Ht31 peptide does not impair forskolin-induced synaptic plasticity ( Figure 8B2 , P = 0 . 65 ) . In a second set of slices , the quantity of phosphorylated S845 on GluR1 , relative to total GluR1 levels , was measured 15 minutes after bath application with forskolin , both in wildtype and Ht31 mutant mice ( Figure 8C ) . No difference in S845 phosphorylation was detected when slices were incubated with vehicle ( P = 0 . 79 ) . In contrast , the forskolin-mediated increase in GluR1 S845 phosphorylation was reduced in slices from mice expressing Ht31 ( P = 0 . 03 ) . Thus , as predicted by the model , disruption of PKA anchoring decreases GluR1 phosphorylation on S845 produced by activation of adenylyl cyclase . Spatial specificity of signaling and synaptic plasticity is critical for information processing , in particular for a neuron to discriminate between different patterns of input . To address whether cAMP and PKA activity exhibit spatial specificity , simulations were repeated in a 20 µm long dendrite with multiple spines ( Figure 1C ) . Both PKA and adenylyl cyclase were colocalized in the spine head , and two of the spines , located on one end of the dendritic segment , were stimulated . Figure 9 shows that spatial specificity decreases as molecule activation is propagated downstream in the signaling pathways . Calcium activation in the spine head ( Figure 9A2 ) leads to cAMP microdomains both in the spine head ( Figure 9A1 ) and in the dendrite ( Figure 9B1 ) ; however , the cAMP microdomains extend farther , e . g . 6 µm distant from the stimulated spines , than the calcium microdomains . In contrast , PKA activity does not exhibit spatial specificity in terms of phospho-inhibitor-1 ( Figure 9B2 ) or free PKA catalytic subunit ( results not shown ) . In addition , phospho-inhibitor-1 shows a cumulative increase with subsequent stimulus trains , though cAMP does not . This result demonstrates the importance of inactivation mechanisms for producing spatial specificity . Degradation of cAMP by phosphodiesterase prevents cAMP from diffusing throughout the dendrite , but the lack of inactivation mechanisms for PKA permits PKA to diffuse throughout the dendrite and phosphorylate inhibitor-1 far from the site of stimulation .
PKA is one of the key molecules in the intracellular signaling networks mediating a long lasting form of LTP in the CA1 region of the hippocampus induced by four spaced trains of high frequency stimulation . AKAPs spatially restrict PKA signaling pathways through the organization of macromolecular complexes that effectively colocalize activators and effectors of enzymes . Compartmentalization of signaling microdomains by AKAPs may be one mechanism allowing spatial specificity of LTP . We investigated whether the critical function of AKAPs is to localize PKA near target proteins or near the source of cAMP , using a multi-compartmental stochastic reaction-diffusion model of the signaling pathways leading to PKA activation in hippocampal CA1 pyramidal neurons . Simulations show that PKA anchoring near the source of cAMP and near specific targets both enhance PKA activity; however , anchoring near the source of cAMP dominates . PKA phosphorylation of GluR1 was greater when the PKA holoenzyme was colocalized with adenylyl cylcase in the dendrite than when the PKA holoenzyme was colocalized in the spine with GluR1 , but apart from the dendritic adenylyl cyclase . Experiments confirmed this model prediction by demonstrating that forskolin-induced GluR1 phosphorylation was greater in wildtype mice than in mice which express Ht31 peptide . The ideal test of the model prediction would be imaging of cAMP concentration and PKA activity simultaneously ( e . g . using both an Epac FRET probe and an AKAR FRET probe [30] ) during LTP induction using spatially specific synaptic stimulation . Demonstration that cAMP and PKA activity had different spatial microdomains , and that Ht31 disrupted the PKA gradient without altering the cAMP gradient awaits further development of live cell imaging techniques . PKA anchoring near adenylyl cyclases by AKAPs is crucial for PKA signaling due to phosphodiesterases , which produce microdomains of cAMP near the adenylyl cyclase . In other systems , phosphodiesterases produce microdomains of elevated cAMP near the adenylyl cyclase , and prevent the widespread elevation of cAMP elsewhere in the cell [15] , [31] , [32] . This leads to preferential activation of PKA that is colocalized with the adenylyl cyclase . The Ht31 peptide , however , disburses PKA outside the range of elevated cAMP , which results in the observed failure of long-lasting forms of LTP . Consistent with this concept , inhibition of phosphodiesterase disrupts spatial gradients of cAMP [31] and allows a cell-wide elevation of cAMP to overcome the effect of PKA mislocalization by Ht31 peptide , and rescues forskolin-induced synaptic potentiation . The rescue of LTP with phosphodiesterase inhibitors emphasizes the importance of inactivation mechanisms as opposed to diffusional barriers for signaling specificity . The characteristic decay length of a molecule's concentration gradient is governed by the diffusion constant of the molecule as well as the inactivation rate [33] , [34]; thus , gradients are stronger when the rate of inactivation is faster than the rate of diffusion . Though cAMP diffuses , increased levels of cAMP only extend to within ∼6 µm of the stimulated spines because of the strong degradation by phosphodiesterases . The PKA catalytic subunit diffuses more slowly than cAMP , yet no gradients of phospho-inhibitor-1 are observed because inactivation of PKA is rather slow , allowing it to diffuse a greater distance . Thus , this result demonstrates that also in stochastic systems the spatial gradient depends on the balance of inactivation and diffusion . Based on prior work , PKA gradients would be expected if a much longer dendrite were included in the model [35] . This suggests that spatial specificity of PKA activity on a smaller spatial scale requires additional mechanisms for PKA inactivation . The presence of a large pool of PKA anchored to MAP2 [23] available to bind to free catalytic subunit throughout the neuron may speed inactivation and provide spatial specificity of PKA activity . Though our simulations anchored all of the PKA in a single location , these different pools of anchored PKA probably coexist in a single neuron . Diverse pools of PKA may phosphorylate different proteins in different compartments of the neuron , including the nucleus . Thus , PKA anchored in the spine may phosphorylate proteins in the spine head , of which GluR1 S845 is an example used in our simulations . Similarly , PKA anchored in the dendrite may be more important for phosphorylating molecules in the dendrite; and PKA anchored near the nucleus may be critical for control of gene transcription . The purpose of simulating the anchoring of PKA in a single location was to evaluate whether individual pools of PKA are spatially restricted . Similarly , anchoring proteins have some degree of mobility , and not all of the PKA or adenylyl cyclase is anchored to AKAPs . The mobility of the AKAP bound to both adenylyl cyclase and PKA will not alter the colocalization of PKA with adenylyl cyclase . Nonetheless , allowing for partial mobility may decrease the effect of anchoring near PKA targets in the model and may decrease the difference between colocalized and non-colocalized cases . Therefore , the rationale for completely immobilizing all of the PKA and adenylyl cyclase was to delineate which function of anchoring is most critical . One additional assumption in these simulations is that Ht31 peptide produces a uniform distribution of PKA . Imaging of PKA location in the AKAP5 knockout [36] reveals that PKA is redistributed to the soma . If this redistribution were simulated in the model by locating all the PKA at one end of the dendrite , then PKA activity in the presence of Ht31 peptide would be even lower . The targets of PKA activity included in the model are a subset of known proteins phosphorylated by PKA . Phosphorylation of GluR1 , either on S845 by PKA or on S831 by other kinases , is sufficient to support enhanced AMPA receptor conductance [26]; however , there is no evidence that GluR1 phosphorylation on Ser845 is increased after induction of LTP [37] . On the other hand , PKA phosphorylation is required for trafficking of AMPA receptors [38] . The mutation of Ser845 to Ala in GluR1 does not impair the early phase of LTP , but the effects of this mutation on long-lasting forms of LTP or forskolin-induced potentiation have not yet been examined [26] . Other PKA targets include RIM1α [39] , [40] , adenylyl cyclase [41] , CREB [42] , and constituents of the mitogen-activated-protein-kinase ( MAPK ) pathway . In particular , PKA phosphorylation of CREB and molecules in the MAPK pathway , either in the dendrite or in the soma and nucleus , are important for the protein synthesis and gene transcription required for long-lasting forms of LTP . Because colocalizing PKA with adenylyl cyclase enhanced the phosphorylation of all PKA targets in the model , it is expected that anchoring PKA with adenylyl cyclase also would enhance phosphorylation of these PKA targets needed for transcription and translation that are not included in the model . Because the induction of LTP involves complex networks of intracellular signaling pathways , computational models have been developed to gain an understanding of events leading to LTP . Many of these models explain the temporal sensitivity of long term potentiation and depression , but very few have investigated spatial specificity or sensitivity to spatial pattern [43] . Two previous models investigated the mechanisms underlying spatial profiles of cAMP and MAPK in the dendrites . Neves et al . [35] demonstrated that both inactivation mechanisms and cell morphology contribute to the size of the cAMP and MAPK microdomains . Ajay and Bhalla [44] explained that the broad spatial profile of MAPK phosphorylation experimentally measured in the dendrite cannot be explained by diffusion , but requires distributed dendritic stimulation . Unlike the present study , neither of these models investigated the role of subcellular location of signaling proteins . Furthermore , the inclusion of spines in our model necessitates a stochastic simulation technique , because the number of molecules in the spine head is small , and inaccurately represented using concentration . Spatial , stochastic simulations were critical to the results presented . In particular , the simulations revealed that GluR1 phosphorylation on Ser845 exhibits large fluctuations , both within trials , and between trials . In general , the stochastic fluctuations were large relative to the mean when the number of molecules was low , as compared to molecules which had a high concentration . Thus , the fluctuations in GluR1 phosphorylation on Ser845 and PKA activity were greater than the fluctuations in phospho-inhibitor 1 . The large within trial variation also lead to a variation between trials: in some trials GluR1 phosphorylation on Ser845 increased , and in some trials it decreased . The average over multiple trials reduced the stochastic variation , and better represented the results that would obtain when measuring hundreds of synapses using field potentials . The variability in individual trials may correspond to the variability observed in experiments when measuring few synapses [45] . Because LTP involves spatially-restricted biochemical reactions , spatial modeling was required to investigate the effect of molecule anchoring on enzyme activation . The locally high calcium concentration in the spine was due to the diffusional barrier of the spine neck coupled with strong inactivation mechanisms . Diffusion was required for interaction between the catalytic subunit of PKA and inhibitor-1 , and , in some cases , cAMP activation of PKA . Though diffusion coefficients are difficult to determine precisely ( range for cAMP of 100–700 µm2/sec in vitro ) due to the difficulty of measurements in vivo , we demonstrated that out simulation results are robust to variations of the diffusion coefficients . Evidence suggests that anchoring of several other molecules is important for synaptic plasticity . Anchoring of calcineurin by AKAP5 plays a role in LTD [13] . Calcium-calmodulin dependent protein kinase II ( CaMKII ) anchors to PSD95 and either the NMDA receptor or voltage-gated calcium channels [46] depending on its state . Some experiments suggest that anchored , phosphorylated CaMKII is not accessible to dephosphorylation by protein phosphatase 1 [47] . Though this effect might play an important role in maintaining GluR1 phosphorylation on Ser 831 and controlling AMPA receptor cycling , the role of CaMKII anchoring is beyond the scope of the present research . Nonetheless , the technique and software used to investigate PKA anchoring could be applied to investigate CaMKII anchoring . Additional evidence suggests that PKA is critical for synaptic tagging [4] , [48] , which provides the synaptic specificity important for information processing . The synaptic tag theory proposes that plasticity related proteins required for long-lasting forms of LTP can only be captured and utilized at synapses that have been tagged by previous activity [49] . Our results suggest that PKA anchored with adenylyl cyclase in the spine would preferentially phosphorylate tag proteins in the spine , not just GluR1 . Therefore , our study of the effects of PKA spatial location on PKA activity may provide additional insights about synaptic tagging and synaptic specificity . Future simulations will explore how anchoring influences PKA phosphorylation of other substrates in other compartments , such as CREB in the nucleus , needed for production of plasticity related proteins .
All research with animals was consistent with NIH guidelines and approved by the IACUC at the University of Pennsylvania . The multi-compartment , computational model ( Figure 1A ) , consists of signaling pathways known to underlie synaptic plasticity in hippocampal CA1 pyramidal neurons . Calcium influx through the NMDA receptor leads to calcium-calmodulin activation of adenylyl cyclase types 1 and 8 [50] , phosphodiesterase type 1B , protein phosphatase 2B ( PP2B or calcineurin ) [51] and CaMKII [52] . In addition to autophosphorylation , CaMKII can phosphorylate AMPA receptor GluR1 S831 [53] , and is itself dephosphorylated by protein phosphatase 1 . Dopamine binds to the Gαs coupled D1/D5 type receptors [54] , which are expressed in CA1 [55] . Gαs synergistically activates adenylyl cyclase [56] , which produces cAMP . After binding 4 molecules of cAMP , the two catalytic subunits ( PKAc ) dissociate from the regulatory subunit dimer ( PKAr ) and become active [57] . Targets of PKA include inhibitor-1 , AMPA receptor GluR1 S845 [58] , and phosphodiesterase types 4B and 4D [59] . Phosphorylated inhibitor-1 binds to and inhibits protein phosphatase 1 ( PP1 ) [24] , thereby decreasing both CaMKII and GluR1 dephosphorylation . Because calcium is crucial for activation of adenylyl cyclase , calcium dynamics were adjusted to emulate experimental observations [7] , [60] , [61] . Calbindin and two submembrane calcium pumps were included to approximate calcium dynamics in the neuron [28] , [62]–[65] . One pump had an affinity similar to the PMCA , the other pump had a lower affinity , similar to the NCX . Molecules were either diffusible , non-diffusible that were evenly distributed , or non-diffusible that were anchored to specific regions . The diffusible molecules included cAMP , ATP , all forms of calmodulin , CaMKII , inhibitor-1 and the catalytic subunit of PKA . The anchored molecules included the dopamine D1 receptor , G protein , adenylyl cyclase , PKA , phosphodiesterases and AMPA receptors . Because G proteins have limited mobility in the membrane [66] , they were colocalized with both the receptor and the adenylyl cyclase for all simulations , as suggested experimentally [19] . To simulated anchoring , the diffusion coefficient of anchored molecules was set to 0 . The molecules were anchored in specific regions by initializing the concentration to zero in all but the anchored regions in this morphology . To specifically evaluate which function of PKA anchoring was more important ( near the source of cAMP or near target proteins ) , D1R , G proteins , adenylyl cyclase , and PKA were anchored either in the spine head , or in a focal dendritic region which had a volume that produced a local concentration equal to that when D1R , G proteins and adenylyl cyclase were located in the spine head . A set of chemical reactions ( Tables 1–4 ) , with concentrations of chemical species as variables , were constructed to implement these pathways in the model as illustrated in Figure 1A . Rate constants used in this model were obtained from the biochemical literature . Initial concentrations are provided in Table 5 . In addition , the diffusional movements of molecules depended not only on the morphology , but also the diffusion constants , which are summarized in Table 6 . The multi-compartment morphology ( as default ) included a 5 µm long segment of dendrite ( 0 . 6 µm wide by 0 . 4 µm depth ) with a single spine . The spine consisted of spine head ( 0 . 6 µm diameter ) , neck ( 0 . 2 µm diameter and 0 . 3 µm long ) and post-synaptic density ( PSD; Figure 1B ) [6] . The approximation of a cylindrical dendrite as a rectangular cuboid captured the essential axial and radial diffusion of molecules , as well as the correct surface to volume ratio . The morphology was subdivided into multiple compartments in order to simulate the reactions and diffusion mesoscopially . The dendrite was subdivided into 200 subvolumes of dimension 0 . 12×0 . 125×0 . 4 µm3 , allowing 2-dimensional diffusion The spine was subdivided into 0 . 1 µm cylindrical or conical slices , yielding 3 spine neck subvolumes , 2 spine head subvolumes and 1 PSD subvolume , permitting 1-dimensional diffusion . One layer of dendritic subvolumes on either edge of the dendrite was considered to be the submembrane region . The 0 . 12 µm width submembrane region with 0 . 36 µm width cytosol gave the same ratio of submembrane to cytosol volume as a cylinder with 0 . 07 µm width submembrane region . For simulations of the 20 µm long dendrite with 11 spines , all molecules were anchored in the spine head; thus the dendrite was subdivided into 300 subvolumes of dimension 0 . 2×0 . 2×0 . 4 µm3 and two spines located at one end of the dendrite were stimulated . Empirically , these subvolume sizes were both large enough to meet the well-stirred criterion [67] and smaller than the length scale of observed concentration gradients . Stimulation for long-lasting LTP induction consisted of four 1 sec trains of 100 Hz stimulation . Each stimulation pulse consisted of a 0 . 7 msec influx of calcium ( 62 . 5 molecules/msec ) which accumulated during the train to approach a plateau [20] . Calcium influx occurred at two places in the model ( Figure 1B ) : one was the PSD region and the other was the dendrite . These two areas represent influx through NMDA receptor channels [8] , [68] in the spine , and voltage dependent calcium channels in the spine and dendrite [69] . An 80 sec inter-train interval was used because this interval produces PKA-dependent LTP experimentally [16] . In addition to the calcium influx , each 100 Hz train is accompanied by a 1 sec , ∼1 uM increase in dopamine [70] ( 0 . 8 molecules/msec ) , which binds to the Gαs coupled dopamine receptors . We used a computationally efficient , Monte Carlo ( stochastic ) reaction-diffusion algorithm , called NeuroRD [15] , for modeling these signaling pathways . The stochastic algorithm was required because many of the molecular populations were small; thus , the assumption of continuous concentration of molecules was incorrect . NeuroRD was used because the large numbers of molecules in the morphology described ( Figure 1B , Table 5 ) made tracking individual molecules in microscopic stochastic simulators computationally prohibitive . The NeuroRD algorithm integrates the tau-leap stochastic reaction algorithm of Gillespie [71] with a computationally-efficient , stochastic diffusion algorithm [72] that , similar to tau-leap , allows multiple diffusion events at each time step . The leaping approach maintains accuracy while dramatically reducing the number of time-steps required for a simulation [73] as compared to spatial extensions of the Gillespie exact stochastic simulation algorithm [74] . Even with this accelerated algorithm , the 600 sec of simulation time ( using a time step of 5 µsec ) required ∼4 days for the 5 µm long dendrite and ∼8 days for the 20 µm long dendrite . Both the simulation software and the files used for the model simulations are freely available from modelDB ( http://senselab . med . yale . edu/ModelDB/ ) and the authors website ( http://krasnow . gmu . edu/CENlab/ ) . We used 3–5 months old Ht31 ( 1 ) mice which express Ht31 peptide in the hippocampus [5] and wildtype littermates to verify modeling predictions . All experiments were conducted according to National Institutes of Health guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania . Mice were sacrificed by cervical dislocation and hippocampi were quickly collected in ice cold , oxygenated artificial cerebrospinal fluid ( aCSF ) containing 124 mM NaCl , 4 . 4 mM KCl , 1 . 3 mM MgSO4 , 1 mM NaPO4 , 26 . 2 mM NaHCO3 , 2 . 5 mM CaCl2 and 10 mM D-glucose bubbled with 95% O2/5% CO2 . Transverse hippocampal slices ( 400 µm ) were made by McIIwain tissue chopper and placed in an interface recording chamber at 28°C ( Fine Science Tools , Foster City , CA ) . Slices were equilibrated for at least 2 hours in aCSF ( pH 7 . 4 ) constantly perfused over slices at 1 ml/min . A bipolar nichrome stimulating electrode ( 0 . 5 mm; AM Systems , Carlsborg , WA ) was positioned in stratum radiatum of area CA1 . A glass micropipette ( AM Systems , Carlsborg , WA ) filled with aCSF with a resistance of 2–5 MΩ was placed next to the stimulating electrode to record field EPSPs ( fEPSPs ) . Data were acquired using Clampex 9 . 2 and a Digidata 1322 A/D converter ( Axon Instruments , Molecular Devices , Union City , CA ) at 20 KHz and low pass filtered at 2 KHz with a 4-pole Bessel filter . Maximum 30 V stimulation was given to the stimulating electrode and slices that have maximum amplitude responses of more than 5 mV were used . The stimulus strength was set to elicit 40% of the maximum fEPSP amplitude . An adenylyl cyclase activator forskolin ( Molecular grade FSK , Sigma ) was prepared as a 50 µM solution and applied to slices for 15 minutes to induce chemical LTP as described before [29] and LTP was recorded for 3 hours . Synaptic strength was measured by the initial slope of fEPSP . The first 20 minute baseline values were averaged and the average was used to normalize each initial fEPSP slope . For measurements of GluR1 phosphorylation , directly after forskolin or vehicle treatment , mouse hippocampal slices were flash frozen and stored at −80°C . Slices were lysed in buffer containing 50 mM Tris , pH 9; 1% Sodium deoxycholate , 50 mM sodium fluoride , 20 mM EDTA , 40 µM β-glycerophosphate , and 1∶100 dilutions of protease and phosphatase inhibitors . After adding NuPAGE LDS Sample Buffer ( Invitrogen ) , 20 µg of protein was resolved using NuPAGE 4–12% Bis-Tris gels and NuPAGE MOPS Running Buffer ( Invitrogen ) for 2 hrs at 120 V . The separated proteins were transferred to PVDF membranes ( Invitrogen ) at 30 mA over night at 4°C . After blocking with 5% milk in Tris-buffered saline containing 0 . 1% ( v/v ) Tween-20 ( TBST ) for 1 hr with gentle shaking , membranes were incubated with antibodies directed specifically against beta-tubulin ( Sigma , 1∶10 , 000 , mouse ) and phospho-S845 ( Millipore , 1∶1 , 000 , rabbit ) over night at 4°C . The membranes were washed 3 times for 10 minutes in TBST . Horseradish peroxidase ( HRP ) -conjugated anti-rabbit or anti-mouse ( Santa Cruz Biotechnology ) were added 1∶1 , 000 in 5% milk in TBST and incubated for 2 hrs at 4°C . The membranes were washed as previously described , then incubated with Amersham ECL Western Blotting Detection Regents ( GE Healthcare ) for one minute . Excess ECL substrate was blotted away and the signal was detected on film ( Kodak BioMax ) for several time points ranging from 5 seconds to 15 minutes . Afterwards , the membranes were stripped using 10 mL Restore Western Blot Stripping Buffer ( Thermo Scientific ) for 20 minutes at room temperature . The membranes were washed 3 times for 10 minutes in TBST and blocked with 5% milk in TBST for 1 hr . The membranes were then incubated with total GluR1 antibody ( Millipore , 1∶1000 , mouse ) over night at 4°C . Membranes were incubated with HRP-conjugated anti-mouse , incubated with ECL and developed following the procedure as described above . Densitometry was performed using mean gray values on ImageJ software . The model simulations and the role of anchoring were evaluated from the total quantity ( representing both amplitude and duration of the elevation ) of the enzymes PKA catalytic subunit , phosphodiesterase type 4B , and phosphodiesterase type 4D , and the mean quantity of phosphorylated inhibitor-1 , and GluR1 phosphorylated on Ser845 . Simulations were repeated due to stochastic variability , and the procedure General Linear Models ( SAS ) was employed for statistical analysis of the simulation results . In order to protect against an elevated type I error due to multiple comparisons , post-hoc tests used planned comparisons only . The effect of PKA anchoring disruption by Ht31 peptide in FSK induced chemical LTP was analyzed using the last 20 minutes of the experimental recordings . The two-sided t-test procedure was used , including tests for equality of variance , separately for the IBMX condition and for the no IBMX condition . Western blots were analyzed by first calculating the quantity of GluR1 phoshorylation on S845 relative to total GluR1 , and then using the procedure General Linear Models ( SAS ) followed by planned contrasts . For both experiments and model simulations , data were first tested for normality using the procedure univariate ( SAS ) , and P>0 . 05 was considered not significant . Both the bar graphs summarizing model simulations and the fEPSP versus time traces display mean and S . E . M .
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The hippocampus is a part of the cerebral cortex involved in formation of certain types of long term memories . Activity-dependent change in the strength of neuronal connections in the hippocampus , known as synaptic plasticity , is one mechanism used to store memories . The ability to form crisp and distinguishable memories of different events implies that learning produces plasticity of specific and distinct subsets of synapses within each neuron . Synaptic activity leads to production of intracellular signaling molecules , which ultimately cause changes in the properties of the synapses . The requirement for synaptic specificity seems incompatible with the diffusibility of intracellular signaling molecules . Anchoring proteins restrict signaling molecules to particular subcellular compartments thereby combating the indiscriminate spread of intracellular signaling molecules . To investigate whether the critical function of anchoring proteins is to localize proteins near their activators or their targets , we developed a stochastic reaction-diffusion model of signaling pathways leading to synaptic plasticity in hippocampal neurons . Simulations demonstrate that colocalizing proteins with their activator molecules is more important due to inactivation mechanisms that limit the spatial extent of the activator molecules .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology",
"computational",
"biology",
"neuroscience"
] |
2011
|
Colocalization of Protein Kinase A with Adenylyl Cyclase Enhances Protein Kinase A Activity during Induction of Long-Lasting Long-Term-Potentiation
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Human T-cell leukemia virus type 1 ( HTLV-1 ) infects mainly CD4+CCR4+ effector/memory T cells in vivo . However , it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells . Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells . Therefore , we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 ( STLV-1 ) , whose virological attributes closely resemble those of HTLV-1 . Although the tax transcript was detected only in certain tissues , Tax expression was much higher in the bone marrow , indicating the possibility of de novo infection . Furthermore , Tax expression of non-T cells was suspected in bone marrow . These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow . To explore the possibility that HTLV-1 infects hematopoietic stem cells ( HSCs ) , we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis ( HAM/TSP ) and a HTLV-1 carrier using the high-throughput sequencing method . Identical integration sites were detected in neutrophils , monocytes , B cells , CD8+ T cells and CD4+ T cells , indicating that HTLV-1 infects HSCs in vivo . We also detected Tax protein in myeloperoxidase positive neutrophils . Furthermore , dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells , indicating that infected monocytes are implicated in viral spreading in vivo . Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points , indicating that infected HSCs persist and differentiate in vivo . This study demonstrates that HTLV-1 infects HSCs , and infected stem cells differentiate into diverse cell lineages . These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo .
Human T-cell leukemia virus type 1 ( HTLV-1 ) is the causal agent of adult T-cell leukemia-lymphoma ( ATL ) and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis ( HAM/TSP ) [1–4] . HTLV-1 is a unique retrovirus since this virus transmits only by cell-to-cell infection [5–7] . The infectivity of free HTLV-1 virions is very inefficient whereas this virus transmits efficiently through cell-to-cell contact [8 , 9] . Therefore , HTLV-1 induces proliferation of infected cells to increase the chance of transmission [10–12] . There are two different ways to increase the number of HTLV-1-infected cells in vivo: proliferation of infected cells ( mitotic division ) and de novo infection [7] . It is thought that mitotic division is predominant in the chronic infection of this virus . HTLV-1 is a member of the primate T-cell leukemia virus type 1 ( PTLV-1 ) group , which contains simian T-cell leukemia virus type 1 ( STLV-1 ) [13] . Based on phylogenetic analyses , HTLV-1 is thought to be derived from STLV-1 by interspecies transmission [14] . Old World monkeys are infected with STLV-1 while New World monkeys are not infected [15] . It was reported that the seroprevalence of STLV-1 in Japanese macaques ( JMs ) was high [16] . We have reported that STLV-1 induces clonal proliferation of CD4+ T cells in vivo , and development of T-cell lymphoma was observed in a STLV-1 infected JM [17] . STLV-1 encodes Tax in the plus strand and STLV-1 bZIP factor ( SBZ ) in the minus strand . STLV-1 Tax and SBZ possess similar functions to HTLV-1 Tax and HTLV-1 bZIP factor ( HBZ ) . Therefore , the STLV-1 infected JM is a good model for HTLV-1 infection [17] . However , the frequency of expression of viral genes in various organs and tissues in vivo remains unknown . The receptors for HTLV-1 are glucose transporter 1 ( GLUT-1 ) and neuropilin 1 , which are expressed on various types of cells [18] . Therefore , this virus can infect different types of cells in vitro [19–22] . However , the HTLV-1 provirus is mainly detected in CD4+ T cells , in particular , CADM1+CCR4+CD45RO+ T cells in vivo [23–26] . This suggests that HTLV-1 either modulates the immunophenotype of T cells , or preferentially infects this subpopulation . Since the cellular receptors of this virus do not absolutely define the specificity of target cells , it is possible that hematopoietic stem cells ( HSCs ) are also infected by HTLV-1 . Although Tax expressing cells were found in the bone marrow [27] , previous studies reported that HTLV-1 did not infect HSCs in ATL patients [28 , 29] . In this study , we analyzed expression of tax and SBZ genes in various organs and tissues of STLV-1 infected JMs , and found that expression of SBZ were higher than those of the tax gene , while the tax gene was highly expressed in the peripheral blood and bone marrow , suggesting that infectious cycle replication of STLV-1 occurs in the bone marrow . To explore the possibility that HTLV-1 infects HSCs , we analyzed integration sites of HTLV-1 in the different hematopoietic cells . The same integration sites of HTLV-1 proviruses were detected in neutrophils , monocytes , B cells , CD8+ T cells and CD4+ T cells in HAM/TSP patients , suggesting that HTLV-1 infects HSCs . This study uncovers a new aspect of HTLV-1 infection and spread in vivo .
To explore in vivo viral gene expression of virus-infected cells , we analyzed the proviral loads ( PVLs ) and transcripts of the tax and SBZ genes in STLV-1-infected JMs . To reduce contamination of peripheral blood lymphocytes in organs and tissues , three monkeys were perfused ( JM1 , JM2 and JM3 ) . PVL was presented as the percentage of infected cells in total cells . The limit of detection was 100 copies per sample as described in Materials and Methods . The PVL varied between tissues and organs although it was high in peripheral blood , lymph nodes and spleen ( S1 Table ) , indicating that infected cells are abundant in lymphatic tissues and lymphocytes . Next , we quantified tax and SBZ transcripts per provirus of these tissues and organs . The expression levels of tax and SBZ were measured by real-time PCR with the ddCt algorithm using an STLV-1 infected cell line , Si-2 , as a reference . In order to compare the expression of tax and SBZ in each sample , the absolute amount of them in Si-2 was determined by the standard curve method , and the expression values in all samples were normalized to the expression in Si-2 cells . Details of this calculation are described in Materials and Methods . In general , the level of SBZ expression was much higher than that of tax . SBZ was expressed in most tissues and organs , although the level of expression was variable ( Fig 1 ) . However , tax transcripts were detected in very limited tissues and organs . In particular , the tax transcript was highly expressed in peripheral blood and bone marrow . It has been reported that Tax-expressing cells were abundant in the bone marrow of HAM/TSP patients [27] . Likewise , this study showed that higher Tax expression was found in the bone marrow cells of STLV-1-infected JMs ( Fig 1 ) . Since Tax is essential for viral replication and transmission , the presence of Tax-expressing cells suggests that de novo infection of HSCs with HTLV-1 occurs in the bone marrow . To address this question , we analyzed Tax expression in bone marrow cells of two STLV-1-infected JMs ( JM4 , 5 ) and an uninfected JM ( JM6 ) . Twenty-four hours after removal of CD8+ T cells from the bone marrow cells , Tax expression was measured by flow cytometry . As shown in Fig 2 , both CD3+ and CD3- bone marrow cells expressed Tax . Tax positive cells were also found in CD4- or CD8- cells . These data indicate that non-T cells are infected by STLV-1 . On the other hand , Tax expression was not detected in a non-infected monkey ( JM6: S2 Fig ) . Further analyses suggested that stem cells ( CD4-CD34+ ) , myeloid cells ( CD4dimCD33+ or CD4-CD33+ ) and B cells ( CD4-CD19+ ) express Tax in vivo ( Fig 2 and S2 Fig ) . These data indicate the possibility that not only T cells but also non-T cells are infected by STLV-1 in bone marrow . In view of the similarity between STLV-1 and HTLV-1 , we speculated that HTLV-1 also infects hematopoietic precursor cells in bone marrow . To test this possibility , we analyzed the genomic integration sites of HTLV-1 in peripheral blood mononuclear cells ( PBMCs ) and neutrophils of a HAM/TSP patient ( HAM/TSP#1 ) using high-throughput sequencing . Contamination of infected T cells is a serious problem to identify the integration sites of provirus in various hematopoietic cells . Since neutrophil is abundantly present in the peripheral blood , the level of T-cell contamination is low . Contamination of lymphocytes in isolated neutrophils was morphologically confirmed , and their percentages were 0 . 2–0 . 9% ( 0 . 92% for HAM/TSP#1 , 0 . 20% for HAM/TSP#2 , and 0 . 26% for HAM/TSP#3 ) . We observed certain integration sites in both PBMCs and neutrophils ( HAM/TSP#1 ) ( S2 Table ) , suggesting that HTLV-1 infects HSCs in vivo . However , since most proviruses are present in T cells in vivo , the risk of contamination of T cells cannot be excluded in this experiment . To examine further the possibility of HTLV-1 infection in HSCs , we isolated various lineages of hematopoietic cells ( CD4+ T cells , CD8+ T cells , B cells , monocytes , and neutrophils ) from two HAM/TSP patients ( HAM/TSP#2 and #3 ) and a HTLV-1 carrier , and then analyzed integration sites of the HTLV-1 provirus in each lineage ( S3 Table ) . The PVLs of HTLV-1 in each of these lineages are shown in S4 Table . To avoid the contamination of detected sequences , we analyzed each sample using the Ion PGM machine on a separate chip . The observation that a given proviral integration site is present at a higher abundance in non-T cell lineages than in CD4+ or CD8+ T cells , argues against the possibility of T-cell contamination . Further , the presence of such integration sites in different cell lineage suggests that HTLV-1 infects HSCs . Tables 1–3 show the 15 most abundant clones in each cell lineage from two HAM/TSP patients and a HTLV-1 carrier . We repeatedly identified identical integration sites in different cell types , suggesting that HTLV-1 infects HSCs in the bone marrow and subsequently differentiate in vivo . The purity of each cell type was not perfect , raising the question whether certain detected integration sites were derived from contaminating T cells . However , certain integration sites that were frequently observed in neutrophils , B cells or monocytes were rarely detected in CD4+ T cells . Conversely , certain integration sites observed in high-abundance CD4+ T cell clones were not detected in cells of other lineages ( Tables 1 , 2 and 3 ) . These data suggest that in non-T cells and HSCs are infected with HTLV-1 . All integration site data in all lineage cells are summarized in S5 Table . Next , we analyzed the proportion of HTLV-1 infected cells that share integration sites with other lineage cells and are derived from infected HSCs . The percentages of infected cells with the same integration sites with other lineage cells were generally high in neutrophils , monocytes and B cells ( Fig 3 ) , suggesting that these cells were infected as precursor cells in the bone marrow . A substantial number of CD4+ T cells ( 16 . 0% for HAM/TSP#2 , 16 . 7% for HAM/TSP#3 and 35 . 9% for a HTLV-1 carrier ) possessed integration sites observed in other hematopoietic cells . This indicates that some HTLV-1 infected CD4+ T cells are derived from infected HSCs . These data indicate that HTLV-1 infects HSCs in vivo . To confirm the presence of HTLV-1 infection in neutrophils , we tried to detect Tax protein in neutrophils using immunofluorescent staining . Tax protein was detected in the neutrophils from HAM/TSP patients along with myeloperoxidase ( Fig 4A ) , which confirmed HTLV-1 infection of neutrophils . It has been reported that HTLV-1 infected DCs spread virus to T cells via a virological synapse [30] . When the monocytes that are infected in the bone marrow differentiate in vivo , infected DCs may subsequently disseminate the virus . To check this possibility , we differentiated monocytes from HAM/TSP patients to DCs using GM-CSF and IL-4 in the presence of azidothymidine ( AZT ) , and the differentiated DCs were then co-cultured with Jurkat cells stably transfected with plasmid that encodes the tandem dimer Tomato ( tdTomato ) under the control of the Tax responsive element ( JET WT35 ) . Differentiation to DCs was confirmed by expression of CD11c and CD209 , and loss of CD14 expression ( S3 Fig ) . Treatment by AZT or raltegravir partially reduced tdTomato positive cells of JET WT35 co-cultured with an ATL cell line , HPB-ATL-2 , indicating that JET WT35 could detect both de novo infection and Tax expression ( via soluble Tax or cell fusion ) ( Fig 4B ) . As shown in Fig 4C , tdTomato positive JET WT35 cells showed de novo infection or Tax production from the differentiated DCs . The quantitative data was shown in Fig 4D . These data suggest that DCs derived from HTLV-1-infected monocytes facilitate in vivo infection of this virus . The next question is whether HTLV-1-infected HSCs persist in vivo . To answer this question , we analyzed HTLV-1 integration sites in neutrophils at different time points in the same HAM/TSP patients . Neutrophils with the same integration site as observed in other hematopoietic cells were detected after one year in two HAM/TSP patients ( HAM/TSP#2 and HAM/TSP#3 ) ( Fig 5 ) . Since half-life of neutrophil in blood is about seven hours [31] , this data suggests that HTLV-1-infected HSCs can survive and generate infected neutrophils in vivo . Data on the integration sites is summarized in S6 Table . Interestingly , approximately half of the clones detected in neutrophils were present at the first analysis ( 96 of 179 clones ( 54% ) in HAM/TSP#2 , and 9 of 20 clones ( 45% ) in HAM/TSP#3 ) , again suggesting that HTLV-1-infected clones can persist in vivo .
It has been reported that HTLV-1 can infect various types of cells in vitro [19–22] . Furthermore , the provirus was detected in various hematopoietic cells in vivo [32] . However , it remains uncertain whether HTLV-1 infects HSCs in vivo . It was thought that HTLV-1 infects mature lymphocytes , macrophages and dendritic cells in the periphery . Indeed , previous studies reported that HSCs were not infected by HTLV-1 [28 , 29] . In these studies , HSCs were isolated from patients with ATL , in which most of the HTLV-1-infected cells were leukemic cells that frequently do not express Tax [3] . On the other hand , Tax was relatively highly expressed in peripheral blood of HAM/TSP patients , and Tax-expressing cells were also found in the bone marrow of HAM/TSP patients [27 , 33] . These observations raise the possibility that bone marrow is a reservoir of HTLV-1 [34] . It has been reported that CD4+ memory T cells specific for cytomegalovirus , tetanus toxoid , measles , mumps and rubella are enriched in the bone marrow [35] . It is possible that such memory T cells are infected by HTLV-1 and express Tax in the bone marrow . Tax is an essential protein for HTLV-1 replication , and the observed Tax expression suggests that de novo infection occurs in the bone marrow in HAM/TSP patients . Indeed , we first presented the evidence that HTLV-1-infected hematopoietic cells of different lineages have the same integration sites in vivo , indicating that this virus infects HSCs . It is critical to show that these commonly identified integration sites are not derived from contamination with HTLV-1-infected lymphocytes during separation of each cells . It is almost impossible to completely exclude contamination of lymphocytes from isolated cells in every case due to predominance of CD4+ T cells in infected cells . However , it is unlikely that all of the integration sites identified in different cell lineages were derived from contaminated cells , for the following reasons . First , approximately 90% of HTLV-1 provirus is detected in CD4+ T cells [23] . Therefore , the contaminating cells are likely to be CD4+ T cell clones with high abundance . However , the abundance of a given integration site in non-CD4+ T cells was frequently higher than that in CD4+ T cells ( Tables 1–3 ) . Second , some integration sites were identified only in non-T cells ( in HAM/TSP#2 , clone ID 5328 , 4300 , 3210 , 4336 , 3489 , 3751 , and 4082 in monocytes; ID 4675 and 4360 in neutrophils: in HAM/TSP#3 , clone ID 6420 and 7133 in monocytes; 6598 in neutrophils: in a carrier , clone 11253 in B cells; ID 10488 , 9664 in monocytes; ID 11415 , and 11692 in neutrophils ) ( Tables 1–3 ) . These data indicate that HTLV-1 infects HSCs in vivo . However , the percentage of infected cells derived from infected HSCs might be over or underestimated . As shown in S6 Table , identified integration sites in neutrophil were frequently found only in CD4+ T cells one year ago , suggesting that frequency of infected cells derived from infected HSCs is underestimated . At the same time , contamination of infected CD4+ T cells might cause overestimation of this frequency especially when only a cell with the integration site was found in non-T cell lineage . Is HTLV-1 infection of HSCs beneficial for this virus ? Viral transmission needs expression of viral antigens ( Env , Gag , Pol , Tax , and Rex ) to form viral particles . Therefore , cytotoxic T lymphocytes tend to attack infected cells during transmission . In this regard , HTLV-1-infected cells that differentiate from HSCs can reduce necessity to express viral antigens in vivo . It might be a strategy of HTLV-1 to decrease viral replication , in order to avoid immune attack by the host . After transmission via breast-feeding or sexual intercourse , it is speculated that HTLV-1 infects many T cells as shown in bovine leukemia virus infected cow [36] . During this stage , some infected T cells might migrate into the bone marrow . It is possible that hypoxic condition in the bone marrow enables infected T cells to express Tax , which causes de novo infection [37] . This scenario should be analyzed in the future studies . It is intriguing that a fraction of infected CD4+ T cells appear to be derived from infected HSCs , suggesting that infected pre-T cells in the bone marrow migrate to the thymus and differentiate to CD4+ and CD8+ T cells . It has been well recognized that HTLV-1-infected cells and ATL cells possess specific surface markers including CD4 , CD25 , CCR4 , and CADM1 [24–26 , 38 , 39] . There are two possible scenarios . First , HTLV-1 targets this specific subpopulation . Secondly , viral proteins modulate phenotypes of infected cells . Our finding that HTLV-1 infected HSCs can differentiate to mature CD4+ T cells in vivo suggests that viral proteins convert infected cells to cells with specific markers , which supports the second hypothesis . It has been reported that HBZ induces expression of Foxp3 while Tax suppresses its expression [40 , 41] . Recently , we have reported that HBZ induces expression of CCR4 , T cell immunoglobulin and ITIM domain ( TIGIT ) , and PD-1 , which are expressed on ATL cells and HTLV-1-infected cells [42] . Thus , HBZ is considered to control the immunophenotype of infected cells and ATL cells during differentiation from HSC . Analyses of integration sites at different time points demonstrated that identical integration sites were frequently detected in neutrophils and other lineage cells ( Fig 5 ) , indicating that HTLV-1 infected HSCs can persist in vivo for at least one year . It is noteworthy that approximately half the observed integration sites in neutrophils were detected in other lineage cells one year earlier . These findings suggest that most of infected HSCs persist in vivo . It has been reported that the risk of cancer is influenced by the number of stem cell divisions [43] . If HTLV-1-infected HSCs survive for a long time , persistent HTLV-1 infection in HSCs might predispose to leukemogenesis by HTLV-1 . It has been shown that HTLV-1 infects DCs , which likely transmits viruses to T cells [21 , 30 , 44] . This study reveals that at least , some HTLV-1 infected monocytes are derived from infected HSCs in vivo . It is thought that DCs derived from infected monocytes efficiently transmit virus to T cells through virological synapses formed between DCs and T cells . De novo infection requires expression of Tax and other viral proteins . However , infected monocytes derived from HSCs do not need to express viral proteins until transmission occurs at the periphery . This strategy might therefore enable infected cells to evade the host immune responses in vivo . In this study , we demonstrate that HTLV-1 infects HSCs , which then differentiate to multiple lineage hematopoietic cells in vivo . This study suggests that HTLV-1-infected HSCs form a persistent reservoir of HTLV-1 infection , which has implications for viral propagation and possibly leukemogenesis .
Neutrophils ( > 99% ) and PBMC were isolated using 3% dextran and density gradient media; Ficoll-Paque PLUS ( GE Healthcare Bio-Science ) . CD4 T lymphocytes ( >98% ) , CD8 T lymphocytes ( >98% ) , monocytes ( > 97% ) and B lymphocyte ( >97% ) were subsequently isolated by positive selection with BD IMag ( BD Bioscience ) . Purity of cells is shown in parenthesis . To obtain whole blood , bone marrow aspirates and organs from Japanese macaques ( Macaca fuscata ) , four animals were euthanized with Pentobarbital ( 50mg/kg ) [17] . Appropriate procedures were utilized in order to reduce potential distress , pain and discomfort . We obtained whole blood , bone marrow aspirates and organs for this study . Before sampling of organs , monkeys were perfused with phosphate buffered saline ( PBS ) to get rid of the contamination of PBMC in solid organs . Blood samples from adult patients with HAM/TSP and a HTLV-1 carrier were collected after the written informed consent was obtained in accordance with the Declaration of Helsinki . These experiments were approved by the Institutional Ethics Committee of Kyoto University ( approval number G311 ) . Six Japanese monkeys ( Macaca fuscata ) were used for this study . All monkeys were supplied from colonies in the Primate Research Institute . The monkeys were reared in outdoor group cages with wooded toys provided as environmental enrichment . They were fed with apple , potato and commercial monkey diet . They were able to access to water ad libitum . They had own health record from birth with yearly health checkup . Blood samples were obtained from the macaques under ketamine anesthesia with medetomidine , followed by administration of its antagonist atipamezole at the end of the procedure . At euthanasia , ketamine anesthesia to the macaques was followed by injection of pentobarbital sodium at a dose of ≥25 mg/kg . Then they were perfused with phosphate buffered saline ( PBS ) to get rid of the contamination of blood cells in solid organs before necropsy for this study . The animal experiment was approved by the Animal Welfare and Animal Care Committees of Kyoto University ( approval number R11-11 , R12-01 , R13-01 , R14-01 and R15-01 ) , and was carried out in accordance with the Guidelines for Care and Use of Nonhuman Primates ( Version3 ) by the Animal Welfare and Animal Care Committee of KUPRI . This guideline was prepared based on the provisions of the Guidelines for Proper Conduct of Animal Experiments ( June 1 , 2006; Science Council of Japan ) as well as Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions [Notice No . 71 of the Ministry of Education , Culture , Sports , Science and Technology dated June 1 , 2006] , in accordance with the recommendations of the Weatherall report , “The use of non-human primates in research”: http://www . acmedsci . ac . uk/more/news/the-use-of-non-human-primates-in-research/ . HTLV-1 transformed human T-cell line ( MT4 , HPB-ATL-2 ) and HTLV-1 negative human T-cell lines ( Jurkat , CCRF-CEM ) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum ( FBS ) ( Biowest ) and antibiotics . MT-4 and HPB-ATL-2 cells were gifts from Dr . Isao Miyoshi ( Kochi University ) and Dr . Shigeru Morikawa ( Shimane University ) respectively . Jurkat cells were obtained from Dr . Shimon Sakaguchi ( Osaka University ) . JET WT35 is a subline of Jurkat cell expressing tdTomato under the control of 5 times tandem repeat of Tax responsive element ( TRE ) [45] . They were cultured with RPMI 1640 medium supplemented with 10%FBS , antibiotics and G418 ( 250 μg/mL ) for selection . 5x104 JET WT35 cells were co-cultured with 1x104 cells of either HPB-ATL-2 or CEM cell lines in 12 wells plate in presence of either DMSO , azidothymidine ( AZT ) ( 5 μM ) , or raltegravir ( RAL ) ( 5 μM ) . 1 . 5x105 DCs that were differentiated from monocytes of HAM/TSP patients were co-cultured with 1 . 5x105 JET WT35 cells . After 48 hours , total number of tdTomato positive cells was counted . Images represent overlay of differential interference contrast and tdTomato channels . Proviral load was measured by real-time PCR as previously described [17 , 23] . Briefly the copy number of the pX region and RAG1 gene in genomic DNA was quantified . HTLV-1 proviral load was calculated with relative quantification method by using TL-Om1 of which proviral load is 100% . STLV-1 proviral load was calculated with absolute quantification method using plasmid DNA that contains STLV-1 sequence . We used serially diluted plasmid DNAs ( the limit of detection is 100 copies ) for standard curve . Therefore , we defined proviral load lower than 100 copies as under detection level ( UD ) in S1 Table . The sequences of primers for RAG1 and pX were reported before [17] and newly constructed ones were as follows; tax primer ( human ) 5’-GAAGACTGTTTGCCCACCACC-3’ ( sense ) and 5’-TGAGGGTTGAGTGGAACGGA-3’ ( anti-sense ) ; pX probe ( Human ) was 5’-CACCCGTCACGCTAACAGCCTGGCAA-3’ . The reaction conditions were 50°C for 2 minutes , 95°C for 10 minutes and 45 cycles of 15 seconds at 95°C , followed by 60seconds at 60°C . Total RNA was extracted using Trizol reagent ( Thermo Fisher Scientific ) . The tissues of JMs were treated with RNA later ( Thermo Fisher Scientific ) to prevent RNA degradation . Reverse transcription was performed using random primer and SuperScript III reverse transcriptase ( Thermo Fisher Scientific ) . The transcripts of SBZ and those of STLV-1 tax were measured by real time PCR . GAPDH mRNA was measured as internal control . The primers and probes for GAPDH were previously described [46] . Others were as follows; stax primers; 5’-ATCCCGTGGAGGCTCCTC-3’ ( sense ) and 5′-CCAAATACGTAGACTGGGTATCCAT-3′ ( anti-sense ) ; stax probe; 5′-ACCAACACCATGGCCCACTTCCC-3′; SBZ primers; 5'-AGAGCGCAACTCAACCGG-3' ( sense ) and 5'-GCAGGGAACAGGTAAACA TCG-3' ( anti-sense ) ; SBZ probe; 5'-TGGATGGCGGCCTCAGGGCC-3' . The sequence of GAPDH primers and probe for JMs were same as those for humans . The amplification condition was 50°C for 2 min , 95°C for 10 min , 45 cycles of 95°C for 15sec and 60°C for 1 min . For the comparison of the expression level of tax with that of SBZ in each sample , we normalized the values of tax and SBZ per infected cell . Briefly , the relative expression levels of SBZ and tax were quantified by ddCt method using Si-2 , which is an STLV-1-infected cell line , as a reference sample . Next , we determined absolute copy number of tax and SBZ transcripts in Si-2 , and found that the copy numbers of tax and SBZ transcripts were 24 . 7 and 1 , respectively . To normalize the expression levels of SBZ and tax in primary JM tissues , the value of tax was multiplied by 24 . 7 ( S1 Fig ) . Bone marrow mononuclear cells from two STLV-1 infected JMs and an uninfected JM were prepared by density gradient centrifugation using Ficoll-Paque PLUS ( GE Healthcare Bio-Science ) . CD8 T lymphocytes were removed by positive selection with BD IMag ( BD Bioscience ) . CD8 T lymphocytes depleted cells were cultured for 24 hours with RPMI 1640 medium supplemented with 10% fetal bovine serum ( FBS ) and antibiotics . The following antibody was used for cell surface staining: anti-CD4 ( OKT4 ) , CD34 ( 561 ) ( all from BioLegend ) , CD8 ( RPA-T8 ) , CD3 ( SP34-2 ) , CD14 ( M5E2 ) , ( all from BD Bioscience ) , CD19 ( J3-119 ) ( from Beckman Coulter ) , CD33 ( AC104 . 3E3 ) ( from Miltenyi Biotec ) . After cell surface staining , cells were fixed and permeabilized with Fixation/Permeabilization working solution ( eBioscience ) , and Tax was stained by anti-Tax monoclonal antibody ( MI73 ) ( 39 ) . Samples were analyzed on a FACSVerse with FACSuite software ( BD Biosciences ) and data was analyzed with Flow Jo software ( FlowJo , LLC ) . Monocytes from HAM/TSP patients were isolated using positive selection with BD IMag systems ( BD Bioscience ) . Then , monocytes were cultured in AIMV medium ( Thermo Fisher Scientific ) supplemented with 5% human AB serum , IL-4 ( 10 ng/ml ) and GM-CSF ( 10 ng/ml ) . To avoid de novo infection of HTLV-1 , raltegravir ( 10 μM ) or azidothymidine ( 5 μM ) was added . After culture with the antiviral drugs for 5 days , cells were washed by RPMI supplemented with 10% FBS . Cells were then co-cultured with JET WT35 in RPMI 1640 medium supplemented with 10% FBS and antibiotics without G418 . The JET WT35 cells are indicator Jurkat cells stably transfected with a plasmid that encodes the tdTomato under the control of the Tax responsive element . After 48 hours , tdTomato expression in co-cultured cells was observed with the EVOS FL fluorescence microscope ( Thermo Fisher Scientific , 20× objective lens ) , and images were acquired by a CCD camera with which microscopy is equipped and built-in software . Cells were fixed using 2% paraformaldehyde for 15min and fixed on poly-D-lysine coated glass or FRONTIER-coated slides ( Matsunami-glass ) by centrifugation . Fixed cells were permeabilized with 0 . 2% Triton X-100 for 15 min , blocked by overnight incubation in blocking solution ( 10% Blocking One and 5% Normal Goat Serum in PBS , both from Nakalai tesque , Japan ) at 4°C , and then incubated with anti-Tax antibody ( clone: MI73 ) ( 1:1000 ) for 3 days at 4°C [46] . After incubation , cells were gently washed five times with PBS , treated with secondary antibody ( 1:500 dilution , Alexa Fluor 488-conjugated goat anti-mouse IgG , abcam ) for 1 h at room temperature , and subsequently rinsed five times with PBS . After re-blocking as described above , myeloperoxidase was stained to identify neutrophils . Cells were incubated with anti-myeloperoxidase antibody ( 1:200 dilution , abcam ) for 1h at room temperature , rinsed five times with PBS , incubated with secondary antibody ( 1:500 dilution , Goat Anti-Rabbit IgG H&L ( Alexa Fluor 568 ) , abcam ) for 1 h at room temperature , and washed five times with PBS . Then they were mounted with ProLong Gold antifade reagent with DAPI ( Thermo Fisher Scientific ) for staining of the nuclei . Images were observed using a confocal microscopy ( FV1000-D IX81 , Olympus , 40× objective lens ) at room temperature and obtained with FV10-ASW software . Brightness and contrast were adjusted by ImageJ software ( National Institutes of Health , http://imagej . nih . gov/ij/index . html ) . Integration sites were amplified with ligation mediated PCR and high-throughput sequencing was performed as previously reported with some modifications using Ion Torrent Personal Genome Machine ( Ion PGM , Thermo Fisher Scientific ) or Miseq ( Illumina ) [17 , 47] . Genomic DNA was extracted with phenol/chloroform method and sheared by sonication with a Covaris M220 instrument ( Covaris ) . After end-repair and linker ligation , nested PCR was performed to amplify the integration sites using the primers specific for viral and linker sequences . Amplicons were size-selected with E-Gel SizeSelect Agarose Gel ( Thermo Fisher Scientific ) to generate libraries for Ion PGM . Templates were prepared by Ion PGM Hi-Q OT2 Kit or Ion PGM Template OT2 400 Kit , and then sequencing was performed on Ion 318 Chip Kit v2 using Ion PGM Hi-Q Sequencing Kit or Ion PGM Sequencing 400 Kit ( Thermo Fisher Scientific ) . For Miseq , additional steps were needed after nested PCR . TruSeq DNA PCR-Free Sample Prep Kit ( Illumina ) was used to ligate the adaptor specific for Miseq according to the manuscripts but without fragmentation because samples were already fragmented . PCR products after nested PCR were used as input DNA in that case . High throughput sequencing was performed according to the manufacturer’s instructions . For Illumina pair-end sequencing , the obtained reads were trimmed with a quality threshold of 20 on the Phred scale and minimum length of both pairs of 20 bp in order to remove low quality reads and short reads using Trim Galore ! ( http://www . bioinformatics . babraham . ac . uk/projects/trim_galore/ ) . The end of viral sequence ( “TTTAGTACACA” was used as a marker ) and that of linker sequence ( “TCGCTCTTCCGATCT” was used as a marker ) were removed from Read1 and Read2 . Trimmed reads were arranged as Read1 including sequence started from the beginning base of integration site and Read2 including sequence started from the end base of shear-site and aligned to human genome reference ( UCSC hg38 ) using Burrows-Wheeler Aligner ( BWA ) [48] . The reads were filtered by mapping quality , removing supplementary reads and excluding un-paired reads with SAMtools software package [49] . Minus strand sequences were converted into complementary sequencing to count the number of clones and PCR duplicates . Sequence similarity for the longest sequence in each integration site was evaluated through the program ClustalW ( version 2 ) in order to remove twin integration sites arising from mismapping of some duplicates [50] . With regard to the pair of clones with high homology score ( >85 ) , the clone which has the smaller number of shear sites was removed . When the number of shear sites was same , we used total read number including the number of PCR products in addition to the number of shear sites . Furthermore , both clones were excluded when the pair of clones has the same shear-sites number and the same number of reads . For Ion PGM single end sequencing , data analysis was done as previously reported with some modifications [17] . When extracting the host genomic sequences , the viral 3’ LTR sequence ( TAGTACACA ) and the linker sequence ( AGATCGGAA ) were regarded as the tags and removed . After the reads were mapped to human genome reference ( UCSC hg38 ) by BWA , they were filtered by the mapping quality . The length of host genomic sequences was calculated using the start position and cigar codes in the SAM files to count the number of clones and PCR duplicates . Sequence similarity was assessed as described earlier .
|
HTLV-1 primarily infects peripheral CD4+CCR4+CADM1+ T cells in vivo . In this study , we show that HTLV-1 infects HSCs , which differentiate into multiple lineages of hematopoietic cells , and likely act as viral reservoir , giving rise to infected neutrophils , monocytes and B cells . Infected monocytes are thought to spread virus in vivo . Infected T cells in the periphery are chimeric in origin: T cells newly infected in the periphery , and infected T cells differentiated from infected HSCs . This observation suggests that viral genes such as HBZ and tax are responsible for converging the molecular differentiation program into a single direction with the characteristic immunophenotype associated with the expression of CCR4 and CADM1 . It has been believed that HTLV-1 infects target cells in the periphery . However , this study reveals a new strategy of HTLV-1 spreading in vivo . These findings have implications for understanding of HTLV-1 pathogenesis as well as treatment of HTLV-1 associated diseases .
|
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2017
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Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo
|
Satellite RNAs ( satRNAs ) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread . Despite many decades of research , the origin of satRNAs remains unknown . In this study we show that a β-glucuronidase ( GUS ) transgene fused with a Cucumber mosaic virus ( CMV ) Y satellite RNA ( Y-Sat ) sequence ( 35S-GUS:Sat ) was transcriptionally repressed in N . tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence . This repression was not due to DNA methylation at the 35S promoter , but was associated with specific DNA methylation at the Y-Sat sequence . Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat , suggesting that the N . tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation ( RdDM ) to the Y-Sat sequence in the 35S-GUS:Sat transgene . Consistent with this , Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat , suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA , a DNA feature associated with 24-nt sRNAs . Our results point to a host genome origin for CMV satRNAs , and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs .
Satellite RNAs ( satRNAs ) are among the smallest RNA pathogens in plants and depend on associated viruses ( helper viruses ) for replication , encapsidation and movement inside the host plant [1] , [2] . Their RNA genomes range from 220 to 1500 nucleotides ( nt ) in size and can form compact secondary structures by intra-molecular base-pairing that can be resistant to degradation by ribonucleases . SatRNAs are classified into three classes [3] . Class 1 satRNAs include large mRNA satellites that are 800 to 1500 nt in length and contain a single open reading frame that encodes at least one non-structural protein . SatRNAs belonging to class 2 are linear , less than 700 nt in size and possess no mRNA activity so do not encode any protein . SatRNAs of this class , including the Cucumber mosaic virus ( CMV ) satRNAs [4] , occur most frequently . SatRNAs of class 3 are circular , around 350 to 400 nt in length and also do not exhibit mRNA activity . SatRNAs normally accumulate at high levels in infected host plants relative to their helper viruses , presumably because of the small size and ribonuclease-resistant structure of their RNA genome . A previous study shows that a CMV satRNA , unlike the CMV helper virus , is resistant to host RNA-dependent RNA polymerase-mediated antiviral silencing in Arabidopsis [5] , which may also contribute to the high level accumulation of satRNAs . Whereas high-level replication and systemic infection of satRNAs depend on helper virus-encoded proteins , recent studies on CMV satRNAs indicate that satRNAs can be imported into the nucleus and transcribed there by host plant proteins independently of helper viruses [6] , [7] . satRNAs are not required for the life cycle of their helper viruses , but participate in helper virus-host interactions by modulating the level of helper virus accumulation and the severity of helper virus-induced symptoms [8] . In addition , satRNAs can induce disease symptoms in the host plants that are distinct from helper virus-caused symptoms [4] . Recent studies indicate that such satRNA-induced symptoms are due to silencing of host genes directed by satRNA-derived small interfering RNAs ( siRNA ) [9] , [10] . Like all plant viruses and subviral agents , the origin of satRNAs remains unclear . Two main origins of satRNA have been suggested: the genome of the helper virus or that of the host plant . However , unlike defective interfering RNAs , a group of subviral RNAs derived from truncated forms of the helper virus genome , satRNAs usually possess little or no sequence homology with their helper viruses [1] , which argues against the helper virus genome as their origin . One exception is the virulent satRNA strain of Turnip crinkle virus , which contains a long ( 166-nt ) segment that is homologous to the 3' end of the helper virus genome [11] . A number of studies have suggested satRNA emergence from the host genome . Sequence similarity has been observed between nucleotide stretches of the Arabidopsis genome and CMV satRNAs [1] . SatRNAs , such as CMV satRNAs that occur widely in Nicotiana species and some other Solanaceae species , are more commonly detected in experimental systems than in the wild or nature [1] . A number of studies have reported de novo emergence of satRNAs on serial passaging plants with the helper virus under controlled environmental conditions [12] , implicating the host genome as the origin of satRNAs . Another report showed structural similarities of Peanut stunt virus ( PSV ) satRNAs with cellular introns of nucleus , mitochondria and plant viroids [13] . However , in spite of these suggestions , no intact plant genome sequence with similarities to satRNAs has been reported . RNA silencing is an evolutionarily conserved gene regulation mechanism in eukaryotes mediated by 20-25-nt small RNAs ( sRNAs ) [14] , [15] . These sRNA are processed from double-stranded ( ds ) or hairpin ( hp ) RNA by Dicer or Dicer-like ( DCL ) protein . To induce silencing , one strand of a sRNA is loaded into an Argonaute ( AGO ) protein to form the RNA-induced silencing complex ( RISC ) , and guides the RISC to bind to complementary single-stranded RNA and cleave the RNA . Plants have three basic types of sRNA , 20-24-nt microRNA ( miRNA ) , 21-22-nt siRNA , and 24-nt repeat-associated siRNA ( rasiRNA ) [16] , [17] . MiRNAs induce posttranscriptional degradation or translational repression of mRNAs that encode regulatory proteins , such as transcription factors , and play a key role in plant development [17] , [18] . The 21-22-nt siRNAs direct degradation of viral RNA and some endogenous mRNA , and are important in plant defence against viruses and in the control of some endogenous genes [17] , [19] . The 24-nt rasiRNAs are unique to plants , and are involved in RNA-directed DNA methylation ( RdDM ) which is important for maintaining genome stability by silencing transposons and repetitive DNA sequences [17] , [20] , [21] . RdDM is highly sequence specific , and can be induced by both endogenous siRNAs and siRNAs derived from infecting viral agents including satRNAs [20] , [22] . During the analysis of a transgene ( 35S-GUS:Sat ) containing a β-glucuronidase ( GUS ) sequence transcriptionally fused at the 3′ end with the CMV Y-satellite RNA ( Y-Sat ) sequence in Nicotiana tabacum , we observed that the Y-Sat sequence was specifically methylated . This led us to hypothesize that 24-nt siRNAs homologous to Y-Sat may exist in N . tabacum inducing RdDM at the Y-Sat sequence of the transgene . Subsequent analyses revealed the existence of both 24-nt sRNAs and multiple DNA fragments in Nicotiana plants that showed sequence homology to Y-Sat , suggesting that CMV satRNAs originate from repetitive regions in the Nicotiana genome .
Three 35S promoter-driven GUS constructs were created , two of which had a 3′ fusion of a full-length 369-nt Y-Sat sequence [23] in either the sense ( sSat ) or antisense ( asSat ) orientation ( Fig . 1A ) . These were transformed into tobacco and multiple independent transgenic lines obtained for each . Plants transformed with the 35S-GUS:Sat fusion constructs showed reduced levels of GUS protein in comparison to those transformed with just the 35S-GUS construct lacking the Y-Sat sequence ( Fig . 1B , 1C ) . This reduction in GUS activity occurred for both the sense and antisense orientations of the Y-Sat sequence ( Fig . 1B ) . The low level of GUS activity was relatively uniform across the independent primary ( T0 ) transformants ( Fig . 1B ) and persisted in the subsequent ( T1 and T2 ) generations ( Fig . 1C ) . The MUG assay results were confirmed by northern blot hybridization , which showed that the GUS:sSat and GUS:asSat transcripts accumulated at a much lower level than the GUS transcript in the respective transgenic plants ( Fig . 1D ) . The repressed expression of the 35S-GUS:Sat transgenes could either be due to transcriptional ( TGS ) or posttranscriptional ( PTGS ) gene silencing or to transcript instability caused by the fused Y-Sat sequence . Since both the 35S-GUS:sSat and 35S-GUS:asSat transgenes showed similar reduction in expression , the secondary structures of the fusion sequence were not likely to be responsible for the repression , as sense and antisense Y-Sat sequences are predicted to form different secondary structures . It also implied that RNA instability was not the main cause for the repressed GUS:Sat trangene expression . This was supported by results from an Agrobacterium infiltration ( agro-infiltration ) assay where both TGS and PTGS were negated , which showed that the large difference in expression levels between the 35S-GUS and 35S-GUS:sSat transgenes observed in stably transformed plants ( ∼6 fold; Fig . 1 ) was dramatically reduced in the agro-infiltrated tissues ( ∼1 . 0–2 . 4 fold; S1 Fig . ) . We next investigated if PTGS or TGS was responsible for the repressed expression of the 35S-GUS:Sat transgenes . PTGS of a transgene is associated with 21-nt siRNAs corresponding to the transcribed region [17] . Northern blot hybridization failed to detect GUS- or Y-Sat-specific 21-nt siRNAs in three of the four 35S-GUS:Sat transgenic lines analysed ( Fig . 2A ) , suggesting that TGS , but not PTGS , was the main cause of transgene repression . Consistent with this , nuclear run-on assay showed that the repressed 35S-GUS:sSat and 35S-GUS:asSat transgenes generated much reduced RNA signals in comparison to the highly expressed 35S-GUS transgene ( Fig . 2B , C ) , indicating that they are transcriptionally repressed . As DNA methylation at promoter sequences can cause TGS [24] , we investigated if DNA methylation occurred in the 35S promoter of the 35S-GUS:sSat transgene using McrBC digestion-PCR . McrBC is a methylation-dependent restriction enzyme that recognizes DNA containing two or more methylated cytosine residues , separated by 30–2000 base pairs , and cleaves the DNA at multiple sites close to one of the methylated cytosines . Differences in PCR-amplified McrBC-digested and undigested DNA can provide a measurement of DNA methylation levels . McrBC digestion did not result in clear reduction in the amplification of a 380-bp amplicon , derived from the the 35S promoter near the transcription start site , which contains a total of 175 cytosines from both strands ( Fig . 3 ) . This indicated that the 35S promoter in the 35S-GUS:sSat transgene was not methylated , and that the reduced transgene expression was not due to promoter methylation . We extended the DNA methylation analysis to the GUS and Y-Sat sequence of the transgene . Similar to the 35S promoter sequence , four different regions of the GUS coding sequence showed no clear methylation , as indicated by strong amplification of McrBC-digested DNA ( Fig . 3 ) . In contrast to the 35S and GUS sequences , McrBC digestion strongly reduced PCR amplification of the Y-Sat sequence in the 35S-GUS:sSat transgene ( Fig . 3 ) , indicating that it was highly methylated . Furthermore , the degree of this methylation , as judged by the extent of reduction in PCR amplification upon McrBC digestion ( Fig . 3B and C , left ) , appeared to be inversely correlated with the level of GUS activity in the 35S-GUS:sSat plants ( Fig . 3C , right ) . McrBC PCR also indicated Y-Sat-specific DNA methylation in the 35S-GUS:asSat transgene that contains an antisense Y-Sat sequence ( S2 Fig . ) . Both the 35S and GUS sequences showed no difference in PCR amplification between McrBC-digested and undigested DNA , whereas the Y-Sat sequence showed a clear reduction in amplification upon McrBC digestion . Furthermore , the expression level of the 35S-GUS:asSat transgene also appeared to be inversely correlated with the extent of Y-Sat sequence methylation ( S2 Fig . ) . The McrBC PCR results were validated using bisulfite sequencing , which determines DNA methylation at a single cytosine nucleotide level due to the ability of bisulfite to convert unmethylated , but not methylated , cytosines to uracils [25] . Three regions of the 35S-GUS:sSat transgene were amplified from bisulfite-converted DNA , including the 35S promoter , the 35S-GUS junction , and the Y-Sat sequence ( S3A Fig . ) . We sequenced the bisulfite PCR product as a mixed DNA population , and determined the DNA methylation level based on the ratio between the peak heights of cytosine ( C ) and thymine ( T ) residues in the sequencing trace files , which has proven to be an effective way for measuring overall DNA methylation levels in a specific plant sample [26] . Consistent with the McrBC PCR result , the 35S and GUS sequences showed no significant methylation of cytosine residues as indicated by the lack of cytosine ( blue ) peaks at the cytosine positions in the trace files , which were instead replaced by thymine ( red ) peaks ( S3B-C Fig . ) . In contrast , the Y-Sat sequence showed strong methylation in all four DNA samples analyzed , especially at the CG and CHG sites ( H = A , C or T nucleotides ) ( Fig . 4 ) . Two pairs of 35S-GUS:sSat transgenic lines were analyzed by bisulfite sequencing , and in each pair the plants that showed lower GUS expression level displayed a higher degree of cytosine methylation , particularly at the CHG and CHH sites ( Fig . 4 ) . Taken together , the DNA methylation analyses indicated that the Y-Sat sequence of the 35S-GUS:Sat transgenes was specifically targeted for methylation in transgenic N . tabacum plants , and that this methylation appeared to correlate with the repression of the transgenes . The sequence-specific DNA methylation detected in the Y-Sat sequence of the 35S-GUS:Sat transgenes raised the possibility that the Y-Sat sequence might be subject to RNA-directed DNA methylation ( RdDM ) . RdDM can occur at all cytosine contexts ( CG , CHG and CHH ) , and is directed by the 24-nt size class of siRNAs [17] , [20] , [21] . sRNAs of 24 nt , but not of 21–22 nt , were readily detectable in both transgenic and wild-type Nicotiana plants by northern blot hybridization using the Y-Sat sequence as a probe , especially in the flowers ( Fig . 5A ) , a tissue known to contain relatively high abundance of 24-nt siRNAs [27] , [28] . Importantly , the 24-nt sRNA signals were not affected by the presence of the 35S-GUS:Sat fusion transgenes ( Fig . 5A and 2A ) , indicating that they are generated by the host plant genome and not by the transgene . Northern blot hybridization also showed that these 24-nt Y-Sat-like sRNAs are present in all three Nicotiana species analysed ( Fig . 5B ) . These results indicated that Nicotiana species generate 24-nt sRNAs with sequence homology to the Y-Sat , and suggested that the DNA methylation of the Y-Sat sequence in the fusion transgenes was likely induced by these 24-nt sRNAs . Detection of 24-nt Y-Sat-like sRNAs in wild-type Nicotiana plants using northern blot hybridization prompted us to identify the nucleotide sequences of these sRNAs using Illumina sequencing . To avoid possible contamination by CMV Y-Sat ( used in our Canberra laboratory ) , leaf samples from uninfected N . tabacum cv . Xanthi nc ( Nt-Xanthi ) grown under insect-proof conditions ( in our Beijing laboratory where the CMV Shandong strain ( SD-CMV ) was used ) were collected for sRNA extraction and sequencing . To investigate if CMV infection might affect the accumulation of Y-Sat-like sRNAs , we also sequenced sRNAs isolated from Nt-Xanthi infected with SD-CMVΔsatR , an infectious CMV clone devoid of SD-satRNA [29] . Approximately 17 and 23 million clean reads of sRNAs were obtained from the uninfected and SD-CMVΔsatR-infected plants , respectively , with 73 . 6% and 39% mapping perfectly to the uncompleted N . tabacum genome ( ftp . sgn . cornel . edu ) ( Table 1 ) . These N . tabacum-matching sRNAs were dominated by the 21 and 24-nt size classes ( Table 1 ) , consistent with the size distribution expected for plant sRNAs [30] . A large number of sRNAs matching the SD-CMV genome ( 27% ) were identified in the sRNA sequencing data from SD-CMVΔsatR-infected plants , but none from the uninfected plants ( Table 1 ) . The majority ( 85 . 1% ) of these SD-CMV-derived sRNAs was 21-22-nt in size , consistent with previous reports of sRNA distribution patterns from RNA viruses [19] . To identify Y-Sat-like sRNAs produced from the tobacco genome , the total sRNA reads were compared against the Y-Sat genome using BLASTN with varying statistical significance determined by E-values ( i . e . the lower the E-value the greater the statistical significance of the match ) . Two additional CMV satRNA sequences ( satCMV110 and SD-satRNA ) were also used for comparison along with three randomly chosen similar sized ( ∼360 nt ) non-satRNA sequences ( SD-CMV RNA1 , Influenza A virus subtype H1N1 and Rice stripe virus RNA3 genome sequences ) as negative controls . The number of matching sRNAs was generally increased in the SD-CMVΔsatR-infected sample compared to the uninfected sample ( Table 2 and Fig . 6 ) . At a low-stringency E-value ( le−2 ) , there was no significant difference in the number of sRNAs aligning to the three CMV satRNAs versus the three control sequences . However , using greater E-value stringencies ( i . e . le−3 , le−4 and le−5 ) to improve the alignment quality led to a higher frequency of sRNAs aligning to the Y-Sat genome than the control sequences in both the uninfected and particularly the SD-CMV△satRNA-infected samples ( Table 2 and Fig . 6A , 6B ) . A large proportion of these Y-Sat-matching sRNAs were 24 nt in size ( Table 2 ) . The satCMV110 and SD-satRNA also had more aligning sRNAs in the SD-CMV△satRNA-infected sample than the control sequences when using the higher alignment stringency ( Table 2 ) . In fact , no sRNA aligned to the relevant control sequences when using an E-value of le−5 , with the exception of sRNAs aligning to the SD-CMV control sequence , which is a result of the SD-CMV infection ( Table 2 ) . The 14 unique Y-Sat-matching sRNA sequences aligned with both the 5′ and 3′ regions of the Y-Sat genome ( Fig . 6C ) , suggesting the possible existence of long stretches of Y-Sat-like sequences in the N . tabacum genome . The five SD-satRNA-matching sRNAs identified in the SD-CMVΔsatR-infected sample ( 1e−5 ) showed perfect sequence identity to the SD-satRNA genome , and had a size range of 23–26 nt ( one 23-nt , one 26-nt and three 24-nt; Fig . 6B , the bottom panel; the colour-coded sequences ) . Three of these SD-matching sRNAs also aligned with the nt . 45–74 region of the Y-Sat and satCMV110 genomes ( Fig . 6B , the top and middle panels; the colour-coded sequences ) . Taken together , the sRNA sequencing data indicated the presence of 24-nt sRNAs in the N . tabacum genome with sequence homologies to CMV satRNAs , particularly to Y-Sat , which could account for the 24-nt sRNA signals detected by northern blot ( Fig . 5 ) and explain the DNA methylation of the Y-Sat sequence in the 35S-GUS:Sat transgenes ( Fig . 3 , 4 and S2 Fig . ) . It is noteworthy that the majority of the CMV satRNA-matching sRNAs could not be mapped to the uncompleted N . tabacum genome ( S1 Table ) , indicating that these sRNAs are derived from the unannotated regions of the genome . The CMV satRNA-matching sRNAs were more frequently aligning to certain regions of the satRNA genomes ( Fig . 6 and S4 Fig . ) . Interestingly , these sRNA “hotspots” were relatively conserved among the three different satRNAs ( boxed in Fig . 6 and S4 Fig . ) and corresponded to conserved sequence regions among all CMV satRNA genomes ( S5 Fig . ) . Among the three CMV satRNAs , SD-satRNA showed the most divergent distribution pattern of aligning sRNAs ( Fig . 6A , B ) . Phylogenetic analysis of all published CMV satRNA sequences revealed that SD-satRNA , Y-Sat and satCMV110 are grouped into three distinct clusters , with SD-satRNA being the most ancient among the three ( S6 Fig . ) . In plants , 24-nt siRNAs are usually derived from repetitive DNA regions such as transposable element ( TE ) sequences in the genome [30] . Multiple hybridizing DNA bands were detected in N . tabacum , N . clevelandii , and N . benthamiana plants by Southern blotting using the Y-Sat sequence as a probe ( Fig . 7A ) . The different band patterns among the three Nicotiana species ruled out plasmid DNA contamination in the DNA samples . We also observed differences in the intensity of the hybridization signals , which suggested that Y-Sat-like DNA sequences exist in the Nicotiana species but with different copy numbers . Digestion of N . tabacum DNA with various restriction enzymes confirmed the existence of multiple copies of the Y-Sat-like DNA sequences ( Fig . 7B ) .
In this study we observed that a GUS transgene fused to the CMV Y-Sat sequence was transcriptionally repressed in N . tabacum plants in comparison to a non-fusion GUS transgene . Transcriptional gene silencing of a transgene is usually caused by promoter methylation and characterized by complete or near-complete silencing of the transgene in a subset of transgenic lines [31] , [32] . However , the 35S-GUS:Sat transgenes showed low but consistent levels of GUS expression across the independent transgenic lines . Furthermore , the 35S promoter was not methylated , suggesting that the repression of the 35S-GUS:Sat transgene is not due to conventional TGS . Both McrBC PCR and bisulfite sequencing detected high levels of DNA methylation in the transcribed region of the 35S-GUS:Sat transgene , with the methylation restricted to the Y-Sat sequence . The extent of methylation in the Y-Sat sequence appeared to be inversely correlated to the level of 35S-GUS:Sat transgene expression , suggesting that this methylation may play a direct role in the repression of the transgene . How DNA methylation in the transcribed region represses the transcription of the 35S-GUS:Sat transgene remains to be investigated . However , our finding provides the first example of transgene repression associated with host-induced methylation targeted to a specific sequence in the transcribed region . The Y-Sat-specific DNA methylation is similar to that observed for the Cereal yellow dwarf virus ( CYDV ) satRNA sequence of a GUS:CYDV-satRNA fusion transgene in N . tabacum [22] . The sequence-specific methylation of the CYDV sequence is caused by RdDM directed by sRNAs derived from replicating CYDV satRNA [22] , also implicating RdDM as the cause of Y-Sat-specific methylation . However , methylation of the Y-Sat sequence occurred in the absence of replicating Y-Sat , suggesting that it was directed by host sRNA-induced RdDM . Consistent with host-derived sRNA-induced RdDM being responsible for the methylation of the Y-Sat sequence , sRNAs with sequence homology to Y-Sat were readily detected by both northern blot hybridization and sRNA deep sequencing . Importantly , these sRNAs were primarily 24-nt in size , which are known to direct RdDM . This size distribution is different to that of sRNAs derived from replicating satRNAs , which are dominated by 21-22-nt size classes [19] , ruling out contaminating satRNAs as the source of these sRNAs . In plants , 24-nt sRNAs are derived primarily from repetitive DNA sequences including TE sequences . Indeed , Southern blot analysis detected multiple DNA fragments in the Nicotiana genome with sequence homology to the Y-Sat sequence . Furthermore , the majority of the Y-Sat-matching sRNAs could not be mapped to the published N . tabacum genome sequence , suggesting that they are likely derived from highly repetitive regions of the genome that are usually difficult to assemble during genome sequencing and hence poorly annotated . Consistent with this possibility , a BLASTN search of the published genome sequences of Nicotiana species and two other Solanaceae species Solanum lycopersicum ( tomato ) and S . tuberosum ( potato ) did not yield any long ( >20 bp ) stretches of Y-Sat-like sequences , again implicating unassembled , repetitive regions as the source of the Y-Sat-matching sRNAs . Taken together , our results suggest that the Nicotiana genome contains Y-Sat-like DNA sequences , and that these sequences exist as repetitive DNA giving rise to 24-nt rasiRNA-like sRNAs that induce RdDM against the Y-Sat sequence in the 35S-GUS:Sat transgene . In addition to RdDM , 24-nt siRNAs have previously been shown to be capable of directing RNA degradation [33] . It would be interesting to examine if these host-derived 24-nt siRNAs play a role in the trilateral host-CMV-satRNA interaction by targeting satRNAs affecting satRNA accumulation . Our results raise the possibility that CMV satRNAs originate from repetitive DNA elements in the Nicotiana genome . It has previously been speculated that satRNAs could be generated from the host plant under some unique conditions , such as helper virus infection [1] . Our sRNA sequencing showed that Y-Sat-like sRNAs accumulated at a much higher level in CMV-infected than uninfected N . tabacum plants . This implies that the Y-Sat-like repetitive DNA is transcriptionally repressed under normal conditions by DNA methylation , but is activated by CMV infection , possibly via the function of the 2b silencing suppressor that can repress DNA methylation in plants [34] . This would result in increased levels of transcript from repetitive DNA regions , including the Y-Sat-like DNA repeats , which could serve not only as substrate for sRNA production , but also as potential progenitor of CMV satRNAs . This scenario is consistent with the CMV-infected samples containing a much larger proportion of sRNAs that could not be mapped to the published N . tabacum genome than the uninfected samples ( S1 Table ) . Viral replicases or RNA-dependent RNA polymerases have relatively high error rates [35] , so replication of viral RNAs including satRNAs can be accompanied by a high rate of nucleotide mutations . Furthermore , for the host-derived progenitor RNAs to become satRNAs , they may need to undergo sequence changes to gain nucleotide motifs for efficient replication and encapsidation , and to gain stable secondary structures for resistance to nucleases . Thus , satRNAs , originating from the host , are likely to have substantial sequence variations from the original DNA or RNA sequences of the host genome . This would explain why most of the Y-Sat-matching N . tabacum sRNAs did not share perfect sequence identities with the Y-Sat genome . Our results do not rule out the possibility that the Y-Sat-like sequences in the N . tabacum genome was originally acquired from a viral genome like the non-retroviral RNA virus elements recently discovered in both plants and animals [36] , [37] . However , the fact that CMV satRNAs lack the ability to self-replicate and possess no sequence homology with their helper viruses , together with the previous observation of de novo emergence of satRNAs during serial passaging plants with CMV infection under controlled environmental conditions [12] , favours the view that the satRNAs originate from the sequence of the Nicotiana genome . Our sRNA sequencing data indicated that different CMV satRNAs have different levels of sequence homology to the N . tabacum sRNAs . One possible explanation for this is that the different satRNAs originated from different Nicotiana species , or from different copies of repetitive DNA in one Nicotiana species , that have nucleotide variations . Another possibility is that these satRNAs originated from the Nicotiana genome at different times , with the more ancient ones having greater sequence divergence from the host genome than the more recent ones . This possibility is supported by the phylogenetic analysis of CMV satRNAs , showing the SD-satRNA to be more ancient than Y-Sat , which coincides with N . tabacum sRNAs matching better to Y-Sat than SD-satRNA . In conclusion , by studying the abnormally repressed expression pattern of the 35S-GUS:Sat transgenes in Nicotiana plants , we have generated evidence indicating that CMV satRNAs have originated from repetitive DNA regions in the Nicotiana genome . Our study suggests that a small RNA sequence-based approach can be used to find the origin of satRNAs .
The 35S-GUS-Ocs cassette , prepared using the pART7 plasmid [38] , was previously described [39] . The cassette was excised using NotI and inserted into the NotI site of pART27 that contains a NPTII kanamycin resistance gene as the selectable marker for plant transformation [38] , resulting in the 35S-GUS construct . For the 35S-GUS:sSat and 35S-GUS:asGUS constructs , the CMV 369-nt Y-Sat sequence was assembled using four long overlapping oligonucleotides ( Y-Sat2 , 3 , 4 and 5; S2 Table ) , then PCR-amplified using Y-Sat1 and Y-Sat6 that contained a HindIII restriction site , and cloned into pGEMT-Easy vector ( Promega ) . The full-length Y-Sat fragment was then excised with HindIII and inserted into the HindIII site between the GUS and Ocs sequence in the 35S-GUS-Ocs cassette in either the sense or antisense orientation . The resulting 35S-GUS:sSat-Ocs and 35S-GUS:asSat-Ocs cassettes were then excised with NotI and inserted into pART27 at the NotI site , forming the 35S-GUS:sSat and 35S-GUS:asSat constructs , respectively . For transformation of N . tabacum Wisconsin 38 ( W38 ) , the constructs were introduced into Agrobacterium tumefaciens LBA4404 via triparental mating . For Agrobacterium infiltration assay , the constructs were transformed into A . tumefaciens GV3101 . Transformation of N . tabacum W38 was performed as previously described [10] using 50 mg/L kanamycin as the selective agent plus 150 mg/L timentin to inhibit Agrobacterium growth . Transformed plants with established roots were transferred to soil and grown at 25°C under natural light . Agro-infiltration of N . benthamiana leaves was carried out essentially as described previously [40] with minor modifications . Basically , A . tumefaciens strains containing respective plant expression constructs , including a green florescent protein ( GFP ) construct as a visual marker and a P19 construct as the RNA silencing suppressor [41] , were grown overnight at 28°C in Luria-Bertani medium ( LB ) containing appropriate antibiotics . After centrifugation , Agrobacterium cells were re-suspended in buffer containing 10 mM MgCl2 and 150 µM acetosyringone , to a final optical density at 600 nm ( OD600 ) of 1 . 0 . Agrobacterium cell suspension containing either the 35S-GUS or the 35S-GUS:sSat construct was mixed with the GFP and P19 cell suspensions at a 1∶1∶1 ratio , incubated at room temperature for ∼3 h , and then infiltrated into expanded N . benthamiana leaves using a flat-pointed syringe . For protein and RNA isolation , agro-infiltrated leaf sections at 4 or 5 days post agroinfiltration ( dpa ) were visualized under a blue light torch ( NightSea™ , DFP-1™ , for exciting green light emission ) , excised with a pair of scissors and immediately frozen in liquid nitrogen . Genomic DNA used for Southern blot hybridization and bisulfite conversion was isolated using the CTAB method described by Draper and Scott [42] . Restriction digestion of DNA ( ∼20 µg ) , purification , agarose gel electrophoresis and Southern blot hybridization were essentially as previously described [39] . A HindIII fragment containing the full-length Y-Sat sequence was excised from the pGEM-T Easy clone described above , and used for preparing a 32P-labelled probe using the Megaprime DNA Labelling System ( Amersham Biosciences ) . Hybridized membranes were washed with 1×SSC first at room temperature for 20 min , then at 50°C for 20 min , and finally at 60–65°C for 30 min . The blots were visualized using a phosphorimager ( FLA-5000 , Fuji Photo Film ) . RNA used for small RNA northern blot hybridization was isolated from agro-infiltrated N . benthamiana leaf tissues or transgenic N . tabacum leaves using Trizol Reagent ( Invitrogen ) . For northern blot detection of small RNAs from N . tabacum flowers , total RNAs was isolated using Trizol reagent , high-molecular-weight RNA removed by precipitation with 5% polyethylenglycol 8000 ( PEG 8000 ) and 0 . 5 M NaCl on ice for 20 min , and small RNAs recovered from the supernatant by precipitation with 3 volumes of ethanol at −20°C for 1 hr . RNA used for northern blot analysis of GUS or GUS:Sat fusion mRNA was isolated using the hot-phenol extraction method [43] . sRNA northern blot hybridization was performed as described using 32P-labelled in-vitro antisense transcript of full-length Y-Sat or GUS coding sequence that were fragmented with Na2CO3 treatment [10] . mRNA northern blot hybridization was carried out as previously described [39] using the same full-length antisense GUS RNA ( unfragmented ) as probe . GUS activities were determined using the kinetic MUG ( 4-methylumbelliferyl-β- glucuronide ) assay according to Chen et al . [39] . GUS activities for Fig . 1B was calculated according to Chen at al . [39] , with the Y-axis as pmol MU per min per 5 µg protein . GUS activities for the remaining figures were presented as slope value per 5 µg protein [44] . Nuclei were isolated from approximately 4 g of fresh N . tabacum leaves and used for nuclear run-on assay according to the procedure described in Meng and Lemaux [45] . The full-length GUS gene , the elongation factor 1α ( EF1α ) gene ( used as internal reference ) , and the Fusarium oxysporum FKS1 gene ( used as negative control ) sequences , were amplified by PCR using the following primers ( primer sequences are shown in S2 Table ) : M13-F and M13-R ( from a pGEM-GUS plasmid ) , EF1α-F and EF1α-R , and FKS1-F and FKS1-R , respectively . The PCR product was separated in 1% agarose gel , denatured , neutralized and then blotted onto Hybond N+ membrane using 20× SSC following the manufacturer's instruction . The membrane was hybridized with the nuclear run-on transcript according to Meng and Lemaux [45] and the hybridizing signals were visualized using a phosphorimager ( FLA-5000 , Fuji Photo Film ) . Genomic DNA ( ∼2 µg ) from transgenic 35S-GUS:sSat N . tabacum plants , was mixed with 1×NEB buffer 2 , 0 . 1 µg/µl BSA , 1 mM GTP in 94 µl volume , which was divided in two equal 47 µl aliquots . To one aliquot 3 µl McrBC enzyme ( NEB , 10 units/µl ) was added , to the other 3 µl of H2O . Both were incubated at 37°C overnight and then diluted with H2O to 100 µl . Four µl was used for each PCR reaction , which was performed using the following cycles: 1 cycle of 95°C for 3 min , 33 cycles of 95°C for 30 sec , 56°C for 45 sec , and 72°C for 90 sec , followed by one cycle of 72°C for 10 min . Sequences of the McrBC PCR primers are listed in S2 Table . Real-time PCR was performed using the Rotor-Gene 6000 ( Corbett Life Science , San Francisco , USA ) real-time rotary analyser using SYBR Green reagent and Platinum Taq polymerase ( Invitrogen ) in four technical replicates for each sample . Bisulfite conversion of transgenic N . tabacum genomic DNA ( ∼4 µg ) was performed as previously described [33] . The bisulfite-treated DNA was purified using Qiagen PCR Purification kit . Primer design ( sequences shown in S2 Table ) , nested PCR and direct sequencing of PCR products were as previously described [44] . Total RNA was isolated from wild-type Nt-Xanthi plants and SD-CMVΔSat-infected plants using hot-phenol extraction and small RNAs were prepared as described previously [46] . Small RNA library construction and Illumina sequencing was performed by BGI ( http://www . genomics . cn/en/index ) . For data analysis , adapter and low-quality sequences were removed and cleaned reads used for length distribution and subsequent analysis . Overlapping sequences between different libraries were identified using Perl script ( http://www . perl . org/ ) . All clean reads were mapped to the uncompleted Nt-Xanthi genome ( ftp . sgn . cornel . edu ) using SOAP ( http://soap . genomics . org . cn/ ) , and all perfectly mapped sequences were used to determine the mapping ratio in different samples . All clean reads matching to satRNA and control sequences was examined using BLASTN ( http://blast . ncbi . nlm . nih . gov/Blast . cgi ) with different E-values ( 1e−2 , 1e−3 , 1e−4 and 1e−5 ) to determine different degrees of similarity . ClustalX ( http://www . clustal . org/ ) was used for sequence alignments and phylogenetic analysis of satRNA genomes .
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Satellite RNAs ( satRNAs ) are small RNA pathogens in plants that depend on associated viruses for replication and spread . While much is known about the replication and pathogenicity of satRNAs , their origin remains a mystery . We report evidence for a host genome origin of the Cucumber mosaic virus ( CMV ) satRNA . We show that only the CMV Y-satRNA ( Y-Sat ) sequence region of a fusion transgene was methylated in Nicotiana tabacum , indicating that the Y-Sat sequence is subject to 24-nt small RNA ( sRNA ) -directed DNA methylation . 24-nt sRNAs as well as multiple genomic DNA fragments , with sequence homology to Y-Sat , were detected in Nicotiana plants , suggesting that the Nicotiana genome contains Y-Sat-like repetitive DNA sequences , a genomic feature associated with 24-nt sRNAs . Our results suggest that CMV satRNAs have originated from repetitive DNA in the Nicotiana plant genome , and highlight the possibility that small RNA sequences can be used to identify the origin of other satRNAs .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology",
"and",
"life",
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2015
|
Nicotiana Small RNA Sequences Support a Host Genome Origin of Cucumber Mosaic Virus Satellite RNA
|
The bacterial flagellar type III export apparatus , which is required for flagellar assembly beyond the cell membranes , consists of a transmembrane export gate complex and a cytoplasmic ATPase complex . FlhA , FlhB , FliP , FliQ , and FliR form the gate complex inside the basal body MS ring , although FliO is required for efficient export gate formation in Salmonella enterica . However , it remains unknown how they form the gate complex . Here we report that FliP forms a homohexameric ring with a diameter of 10 nm . Alanine substitutions of conserved Phe-137 , Phe-150 , and Glu-178 residues in the periplasmic domain of FliP ( FliPP ) inhibited FliP6 ring formation , suppressing flagellar protein export . FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring . The crystal structure of FliPP derived from Thermotoga maritia , and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150 , Arg-152 , Ser-156 , and Pro-158 are responsible for the FliP–FliO interactions . Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level , suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure . Copurification assays revealed that FlhA , FlhB , FliQ , and FliR are associated with the FliO/FliP complex . We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold , followed by FliQ , FliR , and FlhB and finally FlhA during MS ring formation .
The bacterial flagellum is supramolecular motility machinery consisting of basal body rings and an axial structure consisting of the rod , the hook , the hook-filament junction , the filament , and the filament cap . Flagellar axial proteins are translocated across the cytoplasmic membrane by a type III protein export apparatus and assemble at the distal end of the growing structure . The export apparatus consists of an export gate complex formed by 5 highly conserved transmembrane proteins ( FlhA , FlhB , FliP , FliQ , and FliR ) and a cytoplasmic ATPase complex consisting of FliH , FliI , and FliJ [1–4] . These flagellar proteins are evolutionarily related to the components of the type III secretion system ( T3SS ) of pathogenic bacteria , also known as the injectisome [5] . The transmembrane protein , FliO , which is not conserved in flagellar and virulence-associated T3SS family , is required for efficient assembly of the export gate complex in S . enterica ( hereafter referred to as Salmonella ) but is not essential for flagellar protein export [6–8] . The flagellar type III export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane to drive protein export [2 , 3] . Recently , it has been shown that ATP hydrolysis by the FliI ATPase and the following rapid protein translocation by the export gate complex are both linked to efficient proton translocation through the gate , suggesting that the export apparatus acts as a proton/protein antiporter to couple the proton flow through the gate with protein export [9] . Interestingly , the structure of the cytoplasmic ATPase complex looks similar to those of F- and V-type rotary ATPases [10–12] . The export gate complex is located inside the basal body MS ring formed by a transmembrane protein , FliF [13 , 14] . FlhA forms a homononamer [8 , 13] and acts as an energy transducer along with the cytoplasmic ATPase complex [15–19] . The C-terminal cytoplasmic domains of FlhA and FlhB form a docking platform for the ATPase complex , flagellar type III export chaperones , and export substrates [20–22] and coordinate flagellar protein export with assembly [23–26] . Genetic analyses have suggested possible interactions of the N-terminal transmembrane domain of FlhA ( FlhATM ) with FliF [27] , FliR [28] and FlhB [29] . Since a FlhB–FliR fusion protein is partially functional in Salmonella , FlhB presumably associates with FliR in a 1-to-1 fashion [30] . FliP and FliR are incorporated into the basal body at the earliest stage of MS ring formation [31 , 32] . The transmembrane export gate complex of the Salmonella SPI-1 T3SS is composed of SpaP ( FliP homologue ) , SpaQ ( FliQ homologue ) , SpaR ( FliR homologue ) , SpaS ( FlhB homologue ) , and InvA ( FlhA homologue ) in a 5:1:1:1:9 stoichiometry [33] . Recently , it has been shown that 5 copies of SpaP and 1 copy of SpaR form a donut-shaped structure with a diameter of about 8 nm [34] . Since the assembly of the export apparatus begins with SpaP , SpaQ , and SpaR , followed by the assembly of SpaS and finally of InvA in the Salmonella SPI-1 T3SS [34 , 35] , the assembly of the flagellar export gate complex is postulated to occur in a way similar to the Salmonella SPI-1 T3SS [8] . FliP is a 25-kDa transmembrane protein that has a cleavable N-terminal signal peptide , 4 transmembrane ( TM ) helices , and a relatively large periplasmic domain ( FliPP ) between TM-2 and TM-3 ( S1 Fig ) [36] . The number of FliP molecules has been estimated to be 4 to 5 per basal body in Salmonella [32] . FliPP of T . maritia ( Tm-FliPP ) forms a homotetramer in solution [37] , raising the possibility that Salmonella FliP ( St-FliP ) forms an oligomer through interactions between FliPP domains . To study the oligomeric structure of FliP , we purified St-FliP from the membrane fraction by solubilizing it with 1% n-dodecyl β-D-maltoside ( DDM ) and analyzed it by electron microscopy ( EM ) and image analysis . We show that FliP forms a homohexameric ring with a diameter of about 10 nm . We also determined the structure of Tm-FliPP at 2 . 4 Å resolution and carried out structure-based photo-crosslinking experiments . We will discuss the assembly mechanism of the transmembrane export gate complex .
To study the oligomeric state of mature form of FliP , we expressed , solubilized , and purified St-FliP . A hexahistidine tag ( LHHHHHH ) was inserted between Gln-22 and Leu-23 of St-FliP ( His-St-FliP ) for rapid and efficient purification ( S1 Fig ) . The membrane fraction of Salmonella cells expressing His-St-FliP was solubilized by 1% DDM , and His-St-FliP was purified by Ni affinity chromatography ( Fig 1A ) , followed by size exclusion chromatography ( SEC ) with a Superdex 200 10/300 column ( Fig 1B , first row ) . The SEC elution profile of His-St-FliP showed 2 distinct peaks ( S2A Fig ) . Many ring-shaped structures were observed by EM of negatively stained particles in the earlier peak fraction ( S2B Fig , Peak 2 ) but not in the later one ( S2B Fig , Peak 4 ) . An apparent molecular mass of the FliP ring structure was estimated to be about 200 kDa by SEC ( S2A Fig ) . Since the deduced molecular weight of His-St-FliP is approximately 25 kDa , the FliP ring structure presumably contains several copies of FliP together with a DDM micelle covering the transmembrane helices of FliP . To estimate the stoichiometry of the FliP ring more precisely , we carried out 2D classification EM image analysis of negatively stained FliP ring particles , followed by autocorrelation analysis for the rotational symmetry ( Fig 2 and S3 Fig ) . The St-FliP rings exhibiting clear blob features were mostly hexameric with a diameter of about 10 nm ( Fig 2 and S3A , S3B and S3C Fig ) . Autocorrelation analysis also showed that 5 , 333 of the 11 , 736 FliP ring particles analyzed were assigned to the 6-fold rotational symmetry and the rest , which did not show proper ring-shaped structures , were assigned to 5-fold or other rotational symmetries ( S3D and S3E Fig ) , suggesting either that the ring structure is flexible or that they could be side views or incomplete partial rings . Previous genetic analyses of a Salmonella ∆fliO mutant have suggested possible interactions between FliO and FliP [6 , 7] . To clarify this , we coexpressed FliO with His-FliP and purified them by Ni affinity chromatography and finally by SEC . In agreement with a previous report [6] , FliO was expressed as 2 forms: FliOL and FliOS ( Fig 1A ) . Both forms copurified with His-FliP from a SEC column ( Fig 1B , second row ) . EM observation of the FliO/FliP complex revealed that 2 to 3 FliP6 rings are connected to each other through an interaction between FliP and FliO ( Fig 2 and S2B and S4 Figs ) . Since the inner diameter of the M ring of the flagellar basal body is about 20 nm [3] and is too small to accommodate such multiring complexes of FliO and FliP , it is likely that only 1 FliP6 ring exists in the final structure of the export gate complex . To test this , we investigated whether overexpression of St-FliP restores motility of a Salmonella ∆fliO mutant . To monitor the expression level of St-FliP , we inserted a HA tag between Gln-22 and Leu-23 of St-FliP ( HA-St-FliP ) . The motility of the ∆fliO mutant overexpressing HA-St-FliP was essentially the same as that of the ∆fliO mutant transformed with a pTrc99A-based plasmid encoding FliO ( S5 Fig ) . This indicates that FliO is not essential for flagellar protein export . We next tested whether FliO itself forms an oligomer . We found that the FliP6 ring dissociates from FliO during storage of the purified FliO/FliP complexes at 4°C ( S6A Fig ) . Thus , we ran purified FliO/His-FliP complex samples on a Ni– nitrilotriacetic acid ( NTA ) column to remove His-FliP6 rings and the FliO/His-FliP complex , followed by SEC to purify FliO ( S6B Fig ) . EM observation and image analysis showed that FliO forms a 5-nm ring structure with 3 flexible clamp-like structures ( Fig 2 and S2 and S4 Figs ) . These observations led us to conclude that the FliO ring complex is not incorporated into the MS ring . The FliP ( R143H ) mutation , which is located in St-FliPP , can bypass the FliO defect to some extent [6 , 7] , raising the possibility that St-FliPP is required for FliP6 ring formation . To test this , we selected relatively well-conserved residues of FliP , Pro-115 , Glu-125 , Phe-137 , Phe-150 , Leu-170 , Phe-172 , Ala-173 , Ser-177 , Glu-178 , Leu-179 , Ala-182 , and Phe-183 ( S1B Fig ) ; replaced each residue with alanine , except for Ala-173 and Ala-182 , which we replaced with serine; and then analyzed the motility of mutant strains in soft agar ( Fig 1C , upper panel ) . These substitutions did not significantly affect the steady cellular level of FliP as judged by immunoblotting with monoclonal HA-tag antibody ( Fig 1C , lower panel ) . HA-St-FliP fully restored the motility of a ∆fliP mutant . The L170A mutant variant complemented the ∆fliP mutant to the wild-type level . The P115A , E125A , P172A , A173S , S177A , L179A , A182S , and F183A mutant variants restored the motility to a considerable degree , although not to the wild-type level . The F137A and F150A mutant variants complemented the ∆fliP mutant to some degree , and the E178A mutant variant did not at all . In agreement with these results , the F137A and F150A mutations in FliP significantly reduced the secretion levels of the hook-capping protein FlgD , the hook protein FlgE , and the hook-filament junction proteins FlgK and FlgL , and the E178A substitution inhibited the export of these flagellar proteins ( Fig 1D ) . These results indicate that highly conserved Phe-137 , Phe-150 , and Glu-178 residues of FliPP are critical for the protein export activity . To investigate whether the F137A , F150A , and E178A mutations affect the FliP–FliO interaction , we carried out copurification assays by Ni-NTA affinity chromatography . FliO coeluted with His-FliP ( F137A ) , His-FliP ( F150A ) , and His-FliP ( E178A ) from a Ni-NTA column ( Fig 1A , Output ) , indicating that they retain the ability to bind to FliO . To test whether these FliP mutations inhibit FliP6 ring formation , we ran FliO/His-FliP ( F137A ) , FliO/His-FliP ( F1507A ) , and FliO/His-FliP ( E178A ) complexes on a SEC column and then analyzed the pooled fractions by EM . His-FliP ( F137A ) , His-FliP ( F150A ) , and His-FliP ( E178A ) dissociated from the FliO complex during SEC and eluted at the same position as peak 4 of wild-type FliP ( Fig 1B and S2A Fig ) , indicating that these mutations reduced the binding affinity of FliP for FliO . The FliO ring structures were seen in their peak 3 fractions , but neither FliP ( F137A ) , FliP ( F150A ) , nor FliP ( E178A ) formed the homohexamer ring ( S2B Fig , Peak 4 ) . These results suggest that Phe-137 , Phe-150 , and Glu-178 in FliPP contribute to the FliP–FliP interactions in the 6-fold rotational symmetry ring as well as the FliO–FliP interaction . To clarify the role of FliPP in FliP6 ring formation , we determined the crystal structure of FliPP . Although no St-FliPP crystal was obtained , the Tm-FliPP crystals were grown [37] , and its structure was solved at 2 . 4 Å resolution . Tm-FliPP formed a homotetramer in the crystal ( Mol A , Mol B , Mol C , and Mol D ) related by pseudo D2 symmetry ( Protein Data Bank [PDB] ID: 5H72 ) ( Fig 3A ) . There are 2 tetramers in the asymmetric unit , and their structures are essentially identical . The 8 Tm-FliPP molecules in the asymmetric unit show no significant structural difference ( root mean square distances for Cα atoms are less than 0 . 46 Å for the 8 molecules ) . Tm-FliPP monomer consists of 3 α-helices: α1 , α2 and α3 ( Fig 3B ) . The N-terminal 13 residues are invisible in the electron density map presumably because of their conformational flexibility . Therefore , the atomic model of Tm-FliPP contains residues from Thr-122 to Lys-188 . Since each subunit of the Tm-FliPP tetramer is related by D2 symmetry , we studied 3 possible intermolecular interactions: between Mol A and Mol B ( Mol C and Mol D ) , between Mol A and Mol C ( Mol B and Mol D ) , and between Mol A and Mol D ( Mol B and Mol C ) ( Fig 3A ) . The A–B interaction is hydrophobic , and Tyr-124 , Phe-128 , Met-154 , Leu-155 , Pro-176 , and Leu-180 are involved in this interaction ( S7A , S7C , S7D and S7E Fig ) . The A–C interaction contains both hydrophilic and hydrophobic nature , and Met-127 , Arg-130 , Val-131 , Arg-134 , Phe-138 , Glu-142 , Glu-182 , Val-185 , Ala-186 , and Phe-187 are responsible ( S7B , S7F , S7G and S7H Fig ) . Arg-134 forms a salt bridge with Glu-142 and Glu-182 . Ala-186 and Phe-187 make hydrophobic interactions with Met-127 , Val-131 , and the side chain arm of Arg-130 . There is no direct contact between Mol A and Mol D . Since sedimentation equilibrium analytical ultracentrifugation measurements have revealed that Tm-FliPP forms a homotetramer in solution [37] , we conclude that the tetramer structure observed in the crystal appears to be equivalent to that in solution . Although the Tm-FliPP tetramer in the crystal is inconsistent with the St-FliP6 ring structure , it is possible that the dimer units seen in the tetramer are responsible for the hexameric ring formation of St-FliP if the hexamer is a trimer-of-dimer structure . Two distinct dimers are present in the Tm-FliPP crystal: A–B dimer and A–C dimer ( Fig 3A and S7 Fig ) . Although the sequence identity between Tm-FliPP and St-FliPP is only about 30% ( Fig 3C ) , we constructed a homology model of St-FliPP based on the Tm-FliPP tetramer structure ( Fig 3D and 3E ) . The interface residues are not well conserved , but the properties of the interface of St-FliPP are similar to those of Tm-FliPP ( S8 Fig ) . The A–B interface of St-FliPP is hydrophobic , and Met-123 , Leu-127 , Leu-149 , Phe-150 , and Pro-172 form the hydrophobic surface ( S8A , S8B and S8C Fig ) . The A–C interface of St-FliPP shows an elongated shape with both hydrophilic and hydrophobic properties ( S8D , S8E and S8F Fig ) . Arg-140 and Gln-141 form a hydrogen-bonding network with those in the other molecule . Phe-137 is in contact with Tyr-174 and the side chain arm of Glu-178 . Considering the hydrophobic nature of the A–B interface , the A–B dimer is more likely to be a dimer unit of the FliP6 ring structure , although the area of the A–B interface is smaller than that of the A–C interface . The C-termini of the A–B dimer can be connected to the periplasmic end of TM-3 without any steric hindrances with the cytoplasmic membrane , suggesting that the A–B dimer contributes to trimer-of-dimer ring formation ( Fig 3F ) . Because the N-terminal 13 residues are invisible , it is possible that both of the 2 N-termini of the dimer can connect to the periplasmic ends of TM-2 helices . In contrast , if the C-termini of the A–C dimer are directly connected to TM-3 , the hydrophilic surface of the dimer core region would be buried in the cytoplasmic membrane ( S7H and S8F Figs ) , which is unlikely . To investigate which dimer form is actually present in the FliP6 ring structure , we carried out structure-based photo-crosslinking experiments . We introduced an amber mutation at positions of 123 , 124 , 127 , 137 , 150 , 152 , 156 , or 158 of St-FliP to incorporate p-benzoyl-phenylalanine ( pBPA ) , which is a photoreactive phenylalanine . Since Ser-14 and Thr-15 in TM-1 of SpaP—which correspond to Leu-51 and Thr-52 in TM-1 of St-FliP , respectively—provide strong SpaP-SpaP photo-crosslinked products [34] , we also introduced an amber mutation at positions of 51 or 52 . We introduced 2 plasmids into the Escherichia coli BL21 ( DE3 ) strain , 1 encoding FliO , FLAG-tagged FliP ( FliP-FLAG ) with an amber mutation , FliQ , and FliR and the other encoding the amber suppressor tyrosyl tRNA and the engineered tyrosyl-tRNA synthetase to incorporate pBPA at the positions of amber codons . We used wild-type FliP-FLAG as a negative control . As expected , UV irradiation of pBPA at positions of 51 or 52 led to the formation of a photo-crosslinked FliP homodimer ( Fig 4A , indicated by a red dot ) , indicating that TM-1 of FliP is responsible for the FliP–FliP interaction in a way similar to the SpaP–SpaP interaction . Both FliP ( F150pBPA ) -FLAG and FliP ( S156pBPA ) -FLAG also reproducibly gave a photo-crosslinked FliP homodimer , whereas the others did not ( Fig 4A , indicated by a red dot ) . This photo-crosslinked product was also observed when only FliO and FliP-FLAG with an amber mutation were expressed in the presence of pBPA ( Fig 4B ) . These results indicate that both FliP-TM1 and FliPP are involved in the FliP–FliP interactions in the hexameric ring structure . Phe-150 and Ser-156 are located at the A–B interface , whereas Phe-137 is located at the A–C interface ( Fig 3D and 3E and S8B and S8E Fig ) . Since we found that both Phe-137 and Phe-150 are required for FliP6 ring formation , protein export , and motility ( Fig 1 ) , the A–B dimer unit seen in the Tm-FliPP crystal structure is likely to exist in the St-FliP6 ring structure , and it is likely that Phe-137 contributes to its trimer-of-dimer formation . UV irradiating pBPA at positions of Phe-150 , Arg-152 , Ser-156 , or Pro-158 produced a 30-kDa crosslinked band , suggesting the presence of a FliP-FliO crosslinked product ( indicated by blue dot ) . This band was also present when only FliO and FliP were expressed ( Fig 4B ) . To confirm the FliO–FliP interaction , we labeled FliO and FliP ( R152pBPA ) with a 3 x FLAG tag and a 3 x HA tag , respectively . FliO-FLAG formed a crosslinked band with FliP ( R152pBPA ) -HA but not with wild-type FliP-HA , proving the presence of the FliO–FliPP interaction ( Fig 4C ) . These results indicate that Phe-150 , Arg-152 , Ser-156 , and Pro-158 of FliPP are in relatively close proximity to FliO ( Fig 4D ) . This is in agreement with our finding that the F150A mutation reduced the binding affinity of FliP for FliO ( Fig 1 ) . Interestingly , the UV irradiation of pBPA at a position of 52 also produced a 30-KDa crosslinked product in the absence of FliQ and FliR ( Fig 4B ) but not in their presence ( Fig 4A ) , indicating that the TM-1 helix of FliP is in close proximity to FliO when FliQ and FliR are absent . The intensity of the FliO-FliP crosslinked band formed by the introduction of pBPA at positions of Phe-150 , Ser-156 , or Pro-158 was weaker in the presence of FliQ and FliR ( Fig 4A ) than in their absence ( Fig 4B ) , whereas the intensity of the FliP-FliP crosslinked band at positions of Phe-150 or Ser-156 was somehow stronger in the presence of FliQ and FliR ( Fig 4A ) than in their absence ( Fig 4B ) . Therefore , we suggest that FliO appears to facilitate oligomerization of FliP and maintain its stability until FliQ and FliR assemble into the FliP6 ring and that the binding of FliQ and FliR to FliP probably induces conformational rearrangements of the FliP ring in the FliO complex . To analyze the interactions of the FliP6 ring with other export gate proteins , we constructed plasmids coexpressing His-FliP with FliR-FLAG , with HA-FliQ and FliR-FLAG , with FliO and FliR-FLAG , with FliO , HA-FliQ , and FliR-FLAG , or with FlhA , FlhB , FliO , HA-FliQ , and FliR-FLAG . These tags did not affect the export function of export gate proteins considerably . To simplify the examination of their interactions , we expressed these proteins from a single pTrc99A-based plasmid in the Salmonella SJW1368 strain , in which no flagellar genes are expressed because of loss of the master regulator complex , FlhD4FlhC2 [1] . The membrane fractions of Salmonella cells coexpressing His-FliP with other export gate proteins were solubilized by 1% DDM , and then the proteins were purified by Ni affinity chromatography , followed by FLAG affinity chromatography ( S9 Fig ) and finally SEC ( Fig 5A ) . FliR-FLAG and FlhB copurified with His-FliP and FliO , whereas neither HA-FliQ nor FlhA did ( Fig 5A and S9A Fig ) . It has been shown that SpaR of the Salmonella SPI-1 T3SS directly binds to the SpaP5 ring [34] . Therefore , we investigated if FliR directly binds to FliP . When only FliR-FLAG was coexpressed with His-FliP , FliR coeluted with the FliP6 ring from a SEC column ( S9B Fig ) , indicating that FliR tightly associates with the FliP6 ring structure . We found that FliQ and FlhA were easily dissociated from the FlhB/FliO/FliP/FliR complex . Therefore , we investigated whether the MS ring , which is made of 26 copies of a single transmembrane protein FliF , stabilizes the structure of the entire export gate complex . Since a C ring protein FliG is required for efficient MS ring formation in the cytoplasmic membrane [8] , we attached a His tag to the C-terminus of FliG for efficient and rapid purification of the MS ring . To carry out copurification assay , we constructed a pTrc99A-based plasmid encoding 8 flagellar proteins: FlhA , FlhB , FliF , FliG-His , FliO , FliP , HA-FliQ , and FliR-FLAG ( S10A Fig ) . Immunoblotting revealed that they were expressed in the Salmonella SJW1368 strain ( S10B Fig ) . The membranes were solubilized by 1% DDM , and then the proteins were purified by Ni affinity chromatography . Only FlhA and FliF copurified with His-FliG from DDM-solubilized membranes of the cells expressing FlhA , FlhB , FliF , FliG-His , FliO , FliP , HA-FliQ , and FliR-FLAG ( Fig 5A ) , indicating that the FliO/FliP/FliR-FLAG/FlhB complex and HA-FliQ dissociate from the FlhA/FliF/FliG complex . The FlhA/FliF/FliG complex was further purified by SEC , and then the main peak fraction containing FlhA , FliF , and FliG was analyzed by EM with negative staining . Many MS rings were observed in the pooled fractions ( Fig 5B ) , indicating that FlhA associates with the MS ring . It has been shown by in vivo photo-crosslinking experiments that SpaQ interacts with SpaP and SpaR in the final assembled export gate complex . However , an assembly intermediate complex isolated from DDM-solubilized membranes contains only SpaP and SpaR , which may be due to loss of SpaQ in response to DDM extraction [33–35] . FliQ is an essential export component of the flagellar type III export apparatus [38] . FlhA requires FliQ for efficient assembly of the FlhA ring structure inside the MS ring [8] , raising the possibility that DDM affects interactions of FlhA and FliQ with other export gate proteins . To test this , we solubilized the membrane fractions of Salmonella cells expressing FlhA , FlhB , FliO , His-FliP , HA-FliQ , and FliR-FLAG by 1% lauryl maltose neopentyl glycol ( LMNG ) instead of DDM and purified it by Ni affinity chromatography , followed by SEC with a Superdex 200 10/300 column ( Fig 6 ) . The SEC elution profile of the FlhA/FlhB/FliO/FliP/FliQ/FliR complex showed 2 distinct peaks ( Fig 6A ) . The first peak ( 10 . 3 ml , Fig 6A ) mainly contained FliO , FlhB , and FlhA along with a much smaller amount of His-FliP ( Fig 6B ) . Since FlhB copurified with His-FliP , FliO , and FliR-FLAG upon membrane solubilization by DDM ( Fig 5A and S8 Fig ) , we suggest that LMNG weakens the interactions of FliO and FlhB with FliP and FliR . The second peak ( 12 . 6 ml , Fig 6A ) mainly contained FliP , FliQ , FliR , and FlhA ( Fig 6B ) , indicating that FliQ and FlhA bind to the FliP/FliR complex , although some of the FlhA molecules are dissociated from the complex along with FliO and FlhB .
The export gate complex is composed of 5 highly conserved transmembrane proteins—namely , FlhA , FlhB , FliP , FliQ , and FliR—although the transmembrane protein FliO is required for efficient assembly of the export gate in Salmonella [6 , 7] . FliP and FliR are postulated to form a core structure for the assembly of other export gate proteins [31 , 32] . Recently , it has been reported that SpaP of the Salmonella SPI-1 T3SS forms a homopentamer [34] . In contrast to SpaP , we showed that St-FliP forms a homohexamer with a diameter of about 10 nm ( Fig 2 ) . The F137A , F150A , and E178A substitutions in FliPP interfered with FliP6 ring formation ( S2 Fig ) and reduced the export function considerably ( Fig 1D ) . Therefore , we suggest that the FliP6 ring is a functional unit in the export gate complex and that Phe-137 , Phe-150 , and Glu-178 are responsible for FliP6 ring formation . Thus , it seems that the core structure of the flagellar export gate complex is somewhat different from that of the T3SS of pathogenic bacteria . However , 3 , 780 of the 11 , 736 FliP ring particles analyzed were assigned to the 5-fold rotational symmetry ( S3E Fig ) , raising the possibility that FliP forms a homopentamer in a way similar to the SpaP5 ring . We have solved the crystal structure of the Tm-FliPP tetramer and have built 2 St-FliPP dimer models on the basis of the Tm-FliPP structure ( Fig 3 ) . We have also mapped 3 functionally important residues—namely , Phe-137 , Phe-150 , and Glu-178 —onto the St-FliPP model . Although the A–B and A–C dimers are found in the crystal , the A–B dimer seems to be the dimer unit of the ring structure , as supported by photo-crosslinking experiments ( Fig 4 ) . However , since Phe-137 and Glu-178 , which are involved in A–C dimer formation ( Fig 3E ) and are located on the bottom surface close to the rectangular corner of the A–B dimer ( Fig 3F ) , are required for the ring formation and export function ( Fig 1 and S2 Fig ) , they are likely to be involved in the ring formation by connecting the dimers . The FliP ( R143H ) and FliP ( F190L ) mutations , which are located in FliPP and TM-3 of FliP , respectively , improve motility of the ∆fliO mutant to some extent [6 , 7] . This suggests the presence of FliO–FliP interaction . Here , we provided direct evidence that FliP binds to FliO ( Figs 1 and 4 ) . Negatively stained EM analysis revealed that FliO forms a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring ( Fig 2 and S4 Fig ) . Photo-crosslinking experiments revealed direct interactions of FliO with FliP-TM1 and FliPP ( Fig 4 ) . Overexpression of FliP restored motility of the ∆fliO mutant to the wild-type level ( S5 Fig ) , suggesting that the FliO ring complex does not exist in the final structure of the export gate complex . In agreement with this , FliO homologues are absent in nonflagellar T3SSs [5] . Therefore , we propose that the FliO ring complex acts as a scaffold to catalyze FliP6 ring formation and that the interactions of FliO with FliP may induce structural rearrangements of the FliPP dimer to facilitate FliP6 ring formation . Because Arg-143 is located on the rectangular corner surface of the A–B dimer and near Phe-137 and Glu-178 ( Fig 3F ) , we suggest that the R143H and F190L mutations in FliP increase the probability of FliP6 ring formation in the absence of FliO . Since the virulence-associated T3SS apparatus does not have the FliO homologue , we assume that FliP homologues may have a self-scaffolding function to facilitate their own ring formation . The export gate complex of the SPI1-T3SS contains 5 SpaP molecules , 1 SpaQ , 1 SpaR , 1 SpaS and 9 InvA subunits [33–35] . SpaQ , SpaR , and SpaS assemble onto the SpaP pentamer and closely interact with each other [34] . Here we showed that FliR and FlhB copurified with the FliO/FliP ring complex when isolated from DDM-solubilized membrane of Salmonella cells expressing FlhA , FlhB , FliO , FliP , FliQ , and FliR ( Fig 5 and S9 Fig ) . Relative band intensities of FliP , FliR , and FlhB in the FlhB/FliO/FliP/FliR complex allowed us to roughly estimate that the complex contains 6 copies of FliP , 2 copies of FliR , and 2 copies of FlhB . This is in good agreement with 2 sets of previous experimental data that FlhB forms a homodimer in the basal body [39] and that FliR and FlhB associate with each other in a 1 to 1 fashion [30] . When we used LMNG as a detergent instead of DDM , both FlhA and FliQ coeluted with the FlhB/FliO/FliP/FliR complex from a Ni-NTA column ( Fig 6B , the lane marked L ) , indicating that they bind to the FlhB/FliO/FliP/FliR complex . In contrast to the complex solubilized by DDM , FliO and FlhB dissociated from the complex during SEC . However , FlhA and FliQ were associated with the FliP/FliR complex , although some of the FlhA molecules were dissociated from the complex along with FliO and FlhB ( Fig 6B ) . Taken all together , we suggest that FlhA , FlhB , FliQ , and FliR assemble onto the FliP6 ring in complex with FliO to form the export gate complex . When FliF , FliO , FliP , FliQ , FliR , FlhA , and FlhB were coexpressed with His-FliG ( S10 Fig ) , only FlhA copurified with the FliF-FliG ring complex from the DDM-solubilized membrane ( Fig 5 ) , indicating that FlhA directly associates with the MS ring . This is in agreement with a previous report that a Salmonella fliF ( ∆174-175 ) mutant gives rise to extragenic suppressor mutations in FlhATM [27] . It has been shown that FlhA forms a homononamer inside the MS ring [8 , 13 , 14] and that the assembly of FlhA to the MS ring is required for FliO , FliP , FliQ , and FliR [8] . Since we found that some FlhA molecules associate with the FliP/FliQ/FliR complex ( Fig 6B ) , we propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold , followed by the assembly of FliQ , FliR , and FlhB and finally of 9 FlhA molecules during MS ring formation in the cytoplasmic membrane ( Fig 7 ) . In summary , we have presented direct evidence that FliP forms a homohexamer with the help of the FliO complex and that FliPP–FliPP and FliPP–FliO interactions are required for efficient FliP6 ring formation . Our most important findings are that FliP6 ring formation is essential for flagellar type III protein export ( Fig 1 ) . Since there are many structural and functional similarities between the flagellar and T3SS proteins , the periplasmic domain of FliP homologues of the T3SSs could be a good target for inhibitors specific for bacterial infection .
Bacterial strains and plasmids used in this study are listed in S1 Table . DNA manipulations , site-directed mutagenesis , and DNA sequencing were carried out as described previously [40] . L-broth ( LB ) and soft tryptone agar plates were used as described before [38 , 41] . The 2×YT medium contained 1 . 6% ( w/v ) Bacto-tryptone , 1 . 0% ( w/v ) Bacto-yeast extract , and 0 . 5% ( w/v ) NaCl . For expression and purification of FliP , Salmonella SJW1368 cells harboring pKY069 were grown in 2×YT medium containing 100 μg ml-1 ampicillin at 30°C until the cell density had reached an OD600 of about 0 . 4–1 . 0 and then were incubated at 16°C for another 24 h . Cells were harvested by centrifugation ( 6 , 400 g , 10 min , 4°C ) and stored at −80°C . The cells were thawed , resuspended in 20 mM Tris-HCl , pH 8 . 0 , 3 mM EDTA , and disrupted by sonication . The cell lysates were centrifuged ( 20 , 000 g , 15 min , 4°C ) to remove cell debris . The supernatants were ultracentrifuged ( 110 , 000 g , 1 h , 4°C ) . The harvested membranes were stored at −80°C . The membranes were solubilized in 50 mM Tris-HCl , pH 8 . 0 , 300 mM NaCl , 5% glycerol , 20 mM imidazole , and 1% DDM at 4°C for 30 min and ultracentrifuged ( 110 , 000 g , 30 min , 4°C ) to remove the insoluble membrane fractions . Solubilized membranes were loaded onto a Ni-NTA agarose column ( GIAGEN ) and washed extensively with 50 mM Tris-HCl , pH 8 . 0 , 300 mM NaCl , 5% glycerol , 20 mM imidazole , and 0 . 1% DDM . Proteins were eluted with a 50–400 mM imidazole gradient . Fractions containing His-FliP were concentrated and further purified by SEC with a Superdex 200 10/300 column ( GE Healthcare ) equilibrated with 20 mM Tris-HCl , pH 8 . 0 , 300 mM NaCl , 2 mM EDTA , 5% glycerol , and 0 . 1% DDM . For purification of the FliO/His-FliP complex , the membrane fractions were prepared from the SJW1368 cells carrying pKY070 or its mutant variant plasmids in a way similar to His-FliP . After solubilization with 1% DDM , the FliO/His-FliP complex and its mutant variants were purified by Ni-NTA chromatography , followed by SEC with a Superdex 200 10/300 column equilibrated with 20 mM Tris-HCl , pH 8 . 0 , 300 mM NaCl , 2 mM EDTA , 5% glycerol , and 0 . 1% DDM . Samples were applied to carbon-coated copper grids and negatively stained with 1 . 0% ( w/v ) uranyl acetate . Micrographs were recorded at a magnification of ×50 , 000 with a JEM-1011 transmission electron microscope ( JEOL , Tokyo , Japan ) operated at 100 kV . To carry out 2D class averaging of the FliP ring structure and the FliO/FliP complex , 11 , 736 and 1 , 961 particle images , respectively , were boxed out with e2boxer . py [42] , aligned , classified , and averaged using the e2refine2d . py program [42] . To estimate the stoichiometry of the FliP ring , a typical class averaged image was converted from cartesian to polar coordinates , and then the autocorrelation function was calculated . The rotational symmetry was analyzed from Fourier transformation of the autocorrelation function . To carry out 2D class averaging of the FliO structure , 14 , 915 particle images were boxed out with e2boxer . py [42] , aligned , classified , and averaged using the RELION program [43] . Multiple sequence alignment was performed by CLUSTAL-Ω ( http://www . ebi . ac . uk/Tools/msa/clustalo/ ) . Details of the expression , purification , and crystallization of Tm-FliPP have been described previously [37] . X-ray diffraction data were collected at the synchrotron beamline BL41XU in SPring-8 ( Harima , Japan ) with the approval of the Japan Synchrotron Radiation Research Institute ( JASRI ) ( Proposal No . 2013B1305 ) . Details of the X-ray data collection and processing are described previously [37] . Crystals were frozen in liquid nitrogen and mounted in nitrogen gas flow at 100 K . The X-ray diffraction data were collected on an MX225HE CCD detector ( Rayonix ) , were processed with iMOSFLM [44] , and were scaled using SCALA [45] . The statistics of the diffraction data have been described previously [37] . The experimental phase was calculated using the SAD data of the Se-Met derivative with the program Phenix [46] . The atomic model was built with Coot [47] and refined to 2 . 4 Å with Phenix [46] against the native crystal data that showed the best resolution limit . The refinement R factor and the free R factor were converged to 21 . 5% and 26 . 2% , respectively . The Ramachandran plot indicated that 96 . 5% and 3 . 5% residues were located in the most favorable and allowed region , respectively . Structural refinement statistics are summarized in S2 Table . The atomic coordinates have been deposited in PDB under the accession code 5H72 . The structure of St-FliPP was modeled by using SWISS-MODEL [48] . The amino acid sequence from Ile-121 to Phe-183 of St-FliP was used for the target sequence , and the crystal structure of Tm-FliPP was used for a template to construct the homology model . Fresh transformants were inoculated onto soft tryptone agar plates containing 100μg ml-1 ampicillin and 0 . 2% arabinose and incubated at 30°C . At least 7 independent measurements were carried out . Details of sample preparation have been described previously [49] . After SDS-PAGE , immunoblotting with polyclonal anti-FlgD , anti-FlgE , anti-FlgK , or anti-FlgL antibody was carried out as described previously [38] . Detection was done with an ECL immunoblotting detection kit ( GE Healthcare ) . At least 3 independent experiments were carried out . E . coli BL21 ( DE3 ) cells were transformed with a low-copy-number pTACO10-based plasmid [35] and the amber suppressor plasmid pSup-pBpa [50] . The transformed BL21 ( DE3 ) cells were cultured at 37°C in LB containing 10 μg ml-1 chloramphenicol and 25 μg ml-1 kanamycin . Cultures were supplemented with 500 μM rhamnose to induce the expression of FliO/FliP-FLAG/FliQ/FliR , FliO/FliP-FLAG , or FliO-FLAG/FliP-HA from the pTACO10-based plasmid . Additionally , the cultures were supplemented with pBPA to a final concentration of 1 mM and afterwards incubated for 5 . 5 h . Two OD units of bacterial cells were harvested and washed once with 1 ml cold PBS ( 8 g of NaCl , 0 . 2 g of KCl , 3 . 63 g of Na2HPO4 12H2O , 0 . 24 g of KH2PO4 , pH 7 . 4 per liter ) . Cells were resuspended in 1 ml PBS and transferred into 6-well cell culture dishes for 30 min UV irradiation ( λ = 365 nm ) using a UV transilluminator table ( UVP ) . Two OD units of bacterial lysates of E . coli were resuspended in 750 μl buffer K ( 50 mM triethanolamine , pH 7 . 5 , 250 mM sucrose , 1 mM EDTA , 1 mM MgCl2 , 10 μg/ml DNAse , 2 mg/ml lysozyme , 1:100 protease inhibitor cocktail ) and incubated for 30 min on ice . Samples were bead milled , and beads , unbroken cells , and debris were removed by centrifugation ( 10 , 000 g , 10 min , 4°C ) . Crude membranes contained in the supernatant were precipitated by ultracentrifugation using a Beckman TLA 55 rotor ( 100 , 000 g , 45 min , 4°C ) . Pellets containing crude membranes were frozen until use . For protein detection , samples were subjected to SDS-PAGE using SERVAGel TG PRiME 8%–16% or SERVAGel TG PRiME 12% precast gels , transferred onto a PVDF membrane ( Bio-Rad ) , and probed with M2 anti-FLAG antibody ( Sigma ) . Anti-mouse IgG DyLight 800 ( Thermo Fisher ) was used as a secondary antibody . Scanning of the PVDF membrane and image analysis were performed with a Li-Cor Odyssey system and Image Studio 2 . 1 . 10 ( Li-Cor ) . His-FliP/FliR-FLAG , His-FliP/HA-FliQ/FliR-FALG , FliO/His-FliP/FliR-FLAG , FliO/His-FliP/HA-FliQ/FliR-FLAG , FliO/His-FliP/HA-FliQ/FliR-FLAG/FlhB/FlhA , or FliO/ FliP/HA-FliQ/FliR-FLAG/FlhB/FlhA/FliF/His-FliG was expressed in Salmonella SJW1368 cells harboring a pTrc99A-based plasmid , solubilized by 1% DDM or 1% LMNG , and purified by Ni-NTA chromatography , followed by FLAG affinity chromatography . Proteins were eluted from anti-FLAG affinity gels ( Sigma ) with 100 μg ml-1 of FLAG peptide ( Sigma ) . The His-FliP/FliR-FLAG , FliO/His-FliP/FLAG-FliR , and FliO/His-FliP/FLAG-FliR/FlhB complexes were further purified by SEC .
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The bacterial flagellar type III export gate complex is a membrane-embedded nanomachine responsible for flagellar protein export and exits in a patch of membrane within the central pore of the basal body MS ring . In this work , we investigate how formation of the export gate complex is initiated . The export gate complex is composed of 5 highly conserved transmembrane proteins: FlhA , FlhB , FliP , FliQ , and FliR . Each subunit protein assembles into the gate during MS ring formation in a well-coordinated manner . The transmembrane protein FliO is required for efficient assembly of the export gate complex in S . enterica but is not essential for flagellar protein export . Here we carry out biochemical and structural analyses of FliP and provide direct evidence suggesting that FliP forms a trimer-of-dimer structure with a diameter of 10 nm . The assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold , followed by FliQ , FliR , and FlhB and finally FlhA during MS ring formation . Given the structural and functional similarities between the flagellar and the virulence-factor-delivering injectisome machineries , we propose that the periplasmic domain of FliP homologues of the injectisome could be a good target for novel antibiotics .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
[
"dimers",
"(chemical",
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"immunoblotting",
"enzymology",
"phosphatases",
"membrane",
"proteins",
"negative",
"staining",
"bacterial",
"diseases",
"enterobacteriaceae",
"elution",
"molecular",
"biology",
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"crystallography",
"cellular",
"structures",
"and",
"organelles",
"bacteria",
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"cell",
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"adenosine",
"triphosphatase",
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] |
2017
|
Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex
|
Human infections with subtype H7 avian influenza viruses have been reported as early as 1979 . In 1996 , a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins , resulting in an eight amino acid deletion in the receptor-binding site . The continuous circulation of these viruses in live bird markets , as well as its documented ability to infect humans , raises the question of how these viruses achieve structural stability and functionality . Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 ( NY107 ) , including complexes with an avian receptor analog ( 3′-sialyl-N-acetyllactosamine , 3′SLN ) and two human receptor analogs ( 6′-sialyl-N-acetyllactosamine , 6′SLN; sialyllacto-N-tetraose b , LSTb ) . Structural results suggest a novel mechanism by which residues Arg220 and Arg229 ( H3 numbering ) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs . Glycan microarray results reveal that NY107 maintains an avian-type ( α2-3 ) receptor binding profile , with only moderate binding to human-type ( α2-6 ) receptor . Thus despite its dramatically altered receptor binding site , this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential .
Influenza is an acute respiratory virus that infects up to 20% of the population in the United States , resulting in ∼36 , 000 deaths annually [1] , [2] . The two membrane glycoproteins on the surface of influenza A virus , hemagglutinin ( HA ) , which functions as the receptor binding and membrane fusion glycoprotein in cell entry , and neuraminidase ( NA ) , which functions as the receptor destroying enzyme in virus release , form the basis for defining subtypes [3] . To date , 16 HA ( H1–H16 ) and 9 NA ( N1–N9 ) have been identified in avian species [4] , while in the last century , only three subtypes , H1N1 in 1918 and 2009 , H2N2 in 1957 , and H3N2 in 1968 [5] , [6] , [7] , have successfully adapted to humans . Hemagglutinin binds to sialic acid ( SA ) glycans present on host cell surfaces . The receptors on epithelial cells of the human upper respiratory tract are mainly α2-6-linked SA moieties [8] . Since avian influenza viruses predominately bind α2-3-linked SA , and human influenza viruses preferentially bind to α2-6-linked SA , human infection by avian influenza viruses is rare [9] . However , since 1997 a growing number of human cases of avian influenza infection have been reported [10] , including H5N1 , H7N2 , H7N3 , H7N7 , and H9N2 strains [11] . Although the current situation with the pandemic H1N1 influenza virus dominates public health efforts , the prospect of a novel pandemic emerging from these isolated cases continues to be a major public health threat around the world . Early cases of human infection by H7 influenza viruses are reported as far back as 1979 [12] , [13] . Since 2002 , multiple outbreaks and human infections of H7 subtype viruses; within both Eurasian and North American lineages have been reported . In the Netherlands in 2003 , a highly pathogenic avian influenza ( HPAI ) H7N7 outbreak resulted in more than 80 cases of human infections , including one fatality [14] , [15] . In New York in 2003 , a single case of human respiratory infection of H7N2 was reported [16] and in British Columbia in 2004 , an H7N3 virus caused two cases of conjunctivitis [17] , [18] . More recently in 2007 , the United Kingdom reported several cases of low pathogenic avian influenza ( LPAI ) H7N2 virus infections that caused influenza-like illness and conjunctivitis [19] . Since 1996 , H7 viruses of the North American lineage have been circulating in regional live bird markets [20] , containing a 24-nucleotide deletion resulting in an eight amino acid deletion in the receptor-binding site ( RBS ) of HA ( Figure S1 ) . The recent human infections with H7 in North America have raised public health concerns as to how these viruses adapt to such a dramatic structural change while remaining one of the predominant circulating viral strains . A recent study of H7 viruses isolated from previous outbreaks revealed efficient replication in both mouse and ferret animal models [21] . In particular , ferret studies with A/New York/107/2003 ( NY107 ) , an H7N2 virus isolated from a man in New York , not only showed efficient replication in the upper respiratory tract of the ferret but also the capacity for intra-species transmission by direct contact [21] , [22] . Interestingly , both an increased preference for α2-6 and decreased preference for α2-3-linked sialosides of this virus compared to the other avian influenza viruses was shown by previous glycan microarray analysis but less so by a competitive solid-phase binding assay [22] , [23] . Here we report a detailed molecular analysis of the RBS of the HA from North American lineage H7N2 virus , NY107 , including glycan microarray analyses and structural analyses of the HA in complex with an avian receptor analog ( 3′-Sialyl-N-acetyllactosamine , 3′SLN ) and two human receptor analogs ( 6′-Sialyl-N-acetyllactosamine , 6′SLN; Sialyllacto-N-tetraose b , LSTb ) . These results provide important insight into the interaction of H7 HAs with both avian and human hosts .
By using x-ray crystallography , the structure of H7 HA from the NY107 virus was determined to 2 . 6 Å resolution ( Table 1 ) . In addition , we also report three H7 HA receptor complex structures , with avian receptor analog ( 3′SLN ) to 2 . 7 Å resolution and with human receptor analogs ( 6′SLN and LSTb ) to 3 . 0 Å and 2 . 6 Å resolution , respectively ( Table 1 ) . The overall structure of NY107 is similar to other reported HA structures with a globular head containing the RBS and vestigial esterase domain , and a membrane proximal domain with its distinctive , central helical stalk and HA1/HA2 cleavage site ( Figure 1A ) . Although five asparagine-linked glycosylation sites are predicted in the NY107 HA monomer , interpretable electron density was observed at only two sites , Asn38 in HA1 and Asn82 in HA2 ( all residue numbers are based on H3 numbering ) . At these sites , only one or two N-acetyl glucosamines could be interpreted . During viral replication , HA is synthesized as a single chain precursor ( HA0 ) and cleaved by specific host proteases into the infectious HA1/HA2 form . In baculovirus expression systems , highly pathogenic HAs , with a polybasic cleavage site , are expressed as an HA1/HA2 form [24] , whereas HAs with monobasic cleavage sites ( single Arg ) from low pathogenic viruses are expressed as the HA0 form [25] . NY107 is regarded as a low pathogenic virus , and as expected , was produced in the HA0 form ( Figure S2 ) . However , subsequent digestion with thrombin protease to remove the His-tag resulted in cleavage to a profile on SDS-PAGE comparable to that of an HA1/HA2 form ( Figure S2 ) . A comparison of the NY107 cleavage site with the consensus cleavage pattern in the MEROPS database ( http://merops . sanger . ac . uk ) suggests it to be a possible thrombin cleavage site . Based on their molecular phylogenies , HAs are divided into two groups and five clades: group 1 includes H8 , H9 , and H12; H1 , H2 , H5 , and H6; H11 , H13 and H16; group 2 includes H3 , H4 , and H14; H7 , H10 and H15 [26] . Among all available HA structures , we selected ten representative HAs from both avian and human subtypes for structural analysis . As expected , NY107 HA is structurally very similar to the Avian-H7 in all comparisons and closely related to H3 , the other group 2 members used in the analyses ( Tables S1 and S2 ) . The RBS is at the membrane distal end of each HA monomer and its specificity for sialic acid and the nature of its linkage to a vicinal galactose residue is a major determinant of host range-restriction . The consensus RBS for all current HAs is composed of three major structural elements: a 190-helix ( residues 188–194 ) , a 220-loop ( residues 221–228 ) , and a 130-loop ( residues 134–138 ) . In addition , highly conserved residues ( Tyr98 , Trp153 , His183 , and Tyr195 ) form the base of the pocket . Although the NY107 RBS is similar to other subtypes ( H1 , H2 , H3 , H5 , and H9 ) , a previously observed specific feature of H7 HAs , is also observed in the NY107 150-loop region: two residues inserted at position 158 result in this loop protruding more than 6Å towards the binding site compared to other subtype HAs ( Figure 1B and Table S2 ) [27] . More interestingly , the eight amino acid deletion , only found in the North American lineage H7s , from position 221 to 228 ( Figure S1 ) , resulted in a complete loss of the 220-loop ( Figure 1B ) . Sequence alignment shows that Arg220 and Arg229 are conserved in all influenza A HA subtypes ( Figure S1 ) , but structural alignment of NY107 HA shows Arg220 occupying the Gly228 position , and the much shorter loop turns at residue Pro217 ( Figure 1C ) . The Cα distance between NY107 Arg220 and its homolog in the Av-H7 structure ( PDB: 1TI8 ) [27] is 5 . 8Å , and they point in opposite directions ( Figure 1C ) . The side chain direction of Av-H7 Arg220 is almost parallel with the beta sheet after Arg229 , whereas the NY107 Arg220 points downward to the binding pocket . The Cα position of Arg229 in both H7 structures remains the same , except the side chain in the NY107 swings away by about 5 . 9Å ( Figure 1C ) and could help to stabilize this region by forming a hydrogen bond to the mainchain carbonyl of Gln210 in the neighboring monomer . In the absence of the 220-loop in NY107 HA , upon glycan binding the long side chain of Arg220 compensates for its loss and is displaced 4Å upward to form hydrogen bonds with receptor analogs inside the binding pocket ( Figure 1D ) . Previously , mutations in the HA receptor binding domains of H1N1 ( Glu190Asp/Gly225Asp ) and H2N2/H3N2 ( Gln226Leu and Gly228Ser ) subtypes were responsible for adaptation of these viruses to pandemic strains [24] , [28] , [29] , [30] . Due to missing residues 221–228 in the NY107 HA RBS , neither mechanism for adaptation is possible . Thus , in order to look more closely at the role of the missing loop and its effect on receptor specificity , we first subjected the recombinant HA ( recHA ) to glycan microarray analyses and compared it to a reverse genetics-derived NY107 virus , and a co-circulating Eurasian virus and recHA , A/Netherlands/219/2003 ( NL219 ) , that has the consensus avian sequence in the 220-loop and it also infected a human [15] . Glycan microarray analysis of recombinant NY107 ( Figure 2A and Table 2 ) revealed a highly restricted binding profile with strong binding to only α2-3 sulfated ( #4–8 ) , α2-3 branched ( #9–11 ) and mixed α2-3/α2-6 branched sialosides ( #60–64 ) as well as to the long linear sialyl di- and tri-lactosamines ( #22 , 24 ) . Weak binding was also observed ( above background ) to other α2-3 glycans on the array . The recombinant NY107 also revealed a strict glycan binding preference to only one α2-6 glycan , the internal structure , Galβ1-3 ( Neu5Acα2-6 ) GlcNAcβ1-3Galβ1-4Glc ( #58; LSTb ) ( Figure 2A ) , a glycan highlighted in a previous study [22] . The virus with higher valency and avidity revealed stronger binding to all α2-3 groups , in addition to the branched di-sialyl α2-6 biantennary structures ( #46–48 ) as well the LSTb ( #58 ) ( Figure 2B and Table 2 ) . In contrast , the NL219 recHA ( Figure 2C and Table 2 ) bound well to only the avian α2-3 containing sialyl-glycans ( sulfated , branched , linear and fucosylated ) . Its corresponding virus also reflected this specificity although it also revealed strong binding to α2-3 N-glycolylneuraminic acid ( Neu5Gc ) containing glycans ( #66–70 ) ( Figure 2D and Table 2 ) . To further assess the effect of the missing 220 loop on HA structural stability and receptor specificity it was essential to evaluate these functions on the ancestral HA containing the full length 220-loop . To this end , we engineered an HA with an avian H7 consensus ( PQVNGQSG ) 220-loop re-introduced ( NY107-220ins ) into the NY107 HA and recovered this virus by reverse genetics . Compared to the NY107 virus ( Figure 2A ) glycan microarray analyses of the resulting NY107-220ins virus ( Figure 3A and Table 2 ) revealed a decrease in binding to branched ( #9–11 ) and linear ( #12–27 ) α2-3 sialosides and a loss of binding to the branched di-sialyl α2-6 biantennary structures ( #46–48 ) , LSTb ( #58 ) as well as the mixed α2-3/α2-6 branched sialosides ( #60–64 ) . In addition , sequence analysis of the NY107-220ins HA revealed the presence of quasispecies in the second position of the inserted loop , P ( Q/K ) VNGQSG , suggesting that re-introduction of the loop alone is not tolerated and does not create an avian-type binding profile . Thus other amino acid substitutions in the HA might have co-evolved with the deletion of the 220 loop to help stabilize the RBS/HA to maintain functionality . When viruses containing this 220-loop deletion emerged in North America in the mid 90's , four additional amino acid substitutions , Gly114Arg , Asp119Gly , Gly186Glu and Gly205Arg , in the HA1 as well as an Asp19Asn in the HA2 chain were also introduced to most of the circulating isolates . Of these , Gly186Glu and Gly205Arg in the HA1 are close to the RBS , at the monomer interface , and could potentially modulate its structure and/or function . NY107 viruses with a restored consensus 220-loop and a single Glu186Gly ( NY107-ins-186 ) or Arg205Gly ( NY107-ins-205 ) substitution as well as the Glu186Gly/Arg205Gly double substitution ( NY107-ins-186/205 ) were derived by reverse genetics and evaluated . Glycan microarray analysis for the three resulting viruses revealed similar glycan binding profiles with increased binding to α2-3 sialosides , including mixed α2-3/α2-6 branched sialosides ( #60–64 ) , α2-3 Neu5Gc ( #66–70 ) , but limited binding to the α2 , 6 sialosides ( Figures 3B , 3C , 3D ) , resulting in a binding profile virtually identical to that of the NL219 virus and other avian influenza viruses ( Figure 2D ) [30] . Sequence analysis of the three reverse genetics derived viruses revealed no mutations/quasispecies in the HAs of either the NY107-ins-186 or the NY107-ins-186/205 virus stocks , indicative of replication fitness . For the NY107-ins-205 virus however , a Glu186Gly substitution emerged in the HA after only two passages in eggs following recovery from DNA transfection , indicating the importance of the co-variant position 186 with respect to HA functionality/glycan specificity . Altogether , the data indicates that the H7 subtype avian influenza viruses that were circulating in aquatic birds and poultry in North America before 1996 exhibited a classic avian α2-3 sialoside binding preference . In order for the 220-loop deletion to be tolerated , concurrent Gly186Glu and Gly205Arg substitutions in the vicinity of RBS of HA emerged to achieve a restricted α2-3 binding profile and only a moderate/limited increase in binding to branched di-sialyl α2-6 biantennary structures ( #46–48 ) as well the α2 , 6 internal sialoside , LSTb ( #58 ) . To understand from a structural perspective how NY107 interacts with host receptors , we solved the structure of NY107 in complex with an avian and two human receptor analogs . For the avian receptor analog , 3′SLN , the electron density maps revealed well-ordered features for the Sia-1 , Gal-2 , and GlcNAc-3 in the NY107 HA complex structure ( Figure 4A ) . Structural comparison of NY107 HA binding to other , H1 , H2 , H3 , H5 , and H9 subtypes ( Figure S2A ) revealed that 3′SLN binding to NY107 resembled binding of the other published HAs . Indeed , the terminal Sia-1 moiety is positioned almost identically in all structures , and forms the majority of hydrogen bonds and contacts with residues in the RBS ( Figure 4A and Table S3 ) . Published avian HA structures with an intact 220-loop form very close interactions with Gal-2 of 3′SLN via residue Gln226 which is important in receptor specificity and host adaptation . For example , in the avian H7/3′SLN HA structure it interacts with Gal-2 O4 [31] . In the NY107 HA structure , although Gln226 is absent and no other residue occupies the same space as Gln226 ( Figure 1B ) , Arg220 does forms a hydrogen bond between Arg220 NH2 and Gal-2 O4 ( Figure 4A ) . Interestingly , although there was interpretable density for the GlcNAc-3 ( Figure 4A and Figure S4B ) , no hydrogen bonding was apparent between the HA and the GlcNAc-3 , which is consistent with other reported structures [32] . Thus , for binding to avian receptors , the trans conformation of α2-3 linkages is essential and perhaps only the first two saccharides are required . Indeed , due to the absence of 220-loop in the NY107 HA structure , the “aperture” of the RBS formed by 220-loop and 130-loop in regular HAs is increased by ∼10 Å , so that the branched , internal , and perhaps more complicated glycans might be accommodated more efficiently . In the NY107/6′SLN complex , only Sia-1 and Gal-2 are ordered ( Figure 4B ) . The Sia-1 remains in the same position as previously analyzed glycan/HA complexes from H1 , H2 , H3 , H5 , and H9 ( Figure S3B ) , whereas the Av-H7 complex structure with Sialyllacto-N-tetraose c ( LSTc ) did not reveal any density for the Sia-1 in the receptor binding site [31] . The Gal-2 position varies significantly among different subtypes . Compared to the human-adapted H1 HA [32] , Gal-2 in the NY107 HA is 3Å higher , and thus is further from the protein ( Figure S3B ) . In NY107 , the Gal-2 only forms an intramolecular , saccharide-saccharide interaction with Sia-1 . The poor electron density map and fewer interactions with protein residues suggest that the cis conformation of α2-6 linkages in 6′SLN trisaccharides show a reduced binding affinity with NY107 . Glycan array results with NY107 revealed a strong binding signal for the internal α2-6 sialoside , LSTb . To further investigate this interaction , we solved the structure of the NY107/LSTb complex . The final model contained Sia-1 , NAG-2 , Gal-3 , and Gal-5 in the RBS . Although glycan microarray data indicated NY107 to have a specific affinity for LSTb , few interactions were apparent from the crystal structure . Sia-1 still forms multiple hydrogen bonds with residues in the RBS ( Table S3 & Figure 4C ) . The branched Gal-5 interacts with Ser137 , to help stabilize the LSTb binding . However , Arg220 and Lys193 , the two residues showing close binding with 3′SLN , did not form any hydrogen bonds with LSTb . In the structure , Gal-5 also interacts with a crystal packing symmetry mate and thus the flexibility of whole LSTb may be restricted . In solution , with more freedom , the LSTb should be able to tilt closer to the RBS , and thus Glc-4 may have more interactions with the 190-helix than seen in the crystal structure .
Human infections by avian influenza viruses , including H7 subtypes , continue to pose a major public health threat . Although the species barrier prevents avian influenza viruses from widespread infection of the human population , the molecular determinants of efficient interspecies transmission and pathogenicity are still poorly understood . The viral coat protein HA however , is perhaps a critical molecule since previous pandemic viruses modified their receptor specificity and overcame the interspecies barrier to spread in the human population . Although HA structures alone and in complex with receptor analogs provide considerable insight into receptor binding , it is clear that HAs from different species and subtypes have significant structural variation . Indeed , low-pathogenic H7N2 avian influenza viruses with an 8 amino acid deletion within its RBS started to circulate in live-bird markets in the northeast United States in 1996 . Despite what one would consider a debilitating mutation , these viruses have been reported as the predominant isolate [33] . Whether such a deletion contributed to their evolutionary success and how are an important questions , especially in light of NY107's ability to produce respiratory illness in humans [16] , as well as its reported increased affinity for human-type receptors and ability for contact transmission in ferrets [21] . To try to help answer these questions , we have analyzed the molecular structures of NY107 and its complexes with receptor analogs to explain receptor specificity at the molecular level . The crystal structures of NY107 and its complexes with both avian and human receptor analogs describe a mechanism as to how an influenza virus might adapt by dramatically altering its RBS , and still be functional . Arg220 of the HA1 chain of NY107 compensates for the loss of the 220-loop , by forming hydrogen bonds with Gal-2 from the avian analog ( binding was not observed in either of the structures complexes with the human analogs ) . However , in the LSTb complex , branched Gal-5 forms extra interactions with the 130-loop , thus improving the binding preference for this particular glycan . Consistent with the structural evidence , glycan microarray analyses of NY107 revealed a strong binding preference for the branched α2-6 sialoside , LSTb . Except for the absence of the 220-loop , other key residues within the RBS are conserved in NY107 and thus , direct interactions with sialic acid are maintained . The 220-loop is recognized as one of the three crucial structural elements in the RBS . Aside from the North American lineage H7N2 viruses , which have been circulating with a deletion ( 221–228 ) in this loop , there has been one other report describing a seven amino acid deletion ( 224–230 ) in a laboratory generated H3N2 escape mutant which was reported to have a slightly increased affinity for α2-3-linked glycans by hemagglutination assay [34] . Meanwhile , the equivalent region in the hemagglutinin-esterase-fusion ( HEF ) protein of influenza C virus reveals a rearrangement resulting in a truncated 260-loop in its RBS ( Figure S5 ) [35] . However , without structural data with appropriate receptor analogs , it is not possible to compare the role of these loop variants in receptor binding to the H7 HA structure described here . When compared to NL219 , another co-circulating H7 avian virus HA ( Figure 2C and D ) , overall binding to α2-3-linked glycans was markedly reduced , while increased binding to α2-6-linked receptors was only marginal . However , these results focus attention on only 2 sub-classes of human-type receptors that may be important for infection ( and transmission in ferrets ) . The NY107 virus interaction with biantennary glycans ( Figure 2B ) , although weak ( not seen in Figure 2A with recHA ) , is a possible route for virus entry as biantennary structures are common on tissues , i . e . glycan profiling data from human lung tissue on the Consortium for Functional Glycomics ( CFG ) web site . In addition , the internal sialoside , LSTb , was observed in both virus and recHA microarray data , suggesting this type of glycan has good affinity for this HA . The significance of this is unknown since LSTb has only been described in human milk [36] . Interestingly , NY107 and NL219 virus receptor binding and specificity has been addressed previously using glycan microarray analysis that reported a significantly increased preference for α2-6 and decreased preference for α2-3-linked sialosides [22] . In addition , the same viruses were also included in a recent study from Gambaryan et al . using a competitive solid-phase binding assay [23] . Our findings confirm and extend the receptor binding specificity reported by these authors in that they reported both viruses binding to sulfated sialylglycans with a lactosamine ( Galβ1-4GlcNAc core and reported only a moderate binding affinity for α2-6-sialyllactosamine , the human-type receptor analog used in their assay . The 220-loop is an integral feature of the receptor binding site , and thus one would predict that such a deletion might have compromised this strain to be deleted from the population of circulating viruses . However , this was not the case [33] and its existence appears to be in part due to the additional mutations at positions 186 and 205 . Restoration of the loop with either or both residues mutated back to the pre-1994 consensus sequence resulted in a classic avian influenza virus binding profile . The emergence of the Glu186Gly mutation in the HA of the NY107-ins-205 mutant after only two passages of the rescued virus in eggs , also indicates the importance of these positions for HA functionality/glycan specificity . Analysis of the structural data reveals that positions 186 and 205 are on opposite sides of a monomer but are both close to the 220-loop deletion region in the trimeric form . The Glu at position 186 is close to Arg220 and may interact with Arg220 when binding avian receptors . Position 205 in the neighboring monomer may be important in trimer stability and maintaining RBS functionality . If one models the pre-1996 220-loop restored into the NY107 structure , Arg205 , Glu186 and the loop all clash , thus explaining the Glu186Gly mutation that emerged in the NY107-ins-205 virus HA after limited egg passage . The NY107 RBS with its more restricted α2-3 glycan binding preference and weak/moderate increase in α2-6 binding may have given the virus a selective advantage to be maintained in poultry at live bird markets and supplying farms . Certain terrestrial birds , such as quails and chickens , have recently been shown to present both human and avian types of receptors in the trachea and intestine [37] , [38] , [39] . Although it is not known what specific glycans are presented in these animals , it is conceivable that a virus with mixed specificity might have a distinct advantage over avian viruses that have specific avian receptor requirements , particularly in bird markets where multiple species coalesce . Previous results with H7N2 , H9N2 and H5N1 viruses all highlight the fact that an increase in α2-6-binding preference is not sufficient for efficient transmission of avian influenza viruses to humans [22] , [40] , [41] . Although it remains to be seen whether prolonged circulation of viruses in terrestrial birds , such as domestic chickens , can provide a possible route for viruses to adapt for efficient human infection [11] , continued surveillance of influenza viruses from avian and other animal reservoirs is urgently needed to define their zoonotic potential .
Based on H3 numbering [42] , cDNA corresponding to residues 11–329 ( HA1 ) and 1–176 ( HA2 ) of the ectodomain of the hemagglutinin ( HA ) from A/New York/107/2003 ( H7N2; Genbank:ACC55270 ) and A/Netherlands/219/2003 ( H7N7; Genebank: AAR02640 ) was cloned into the baculovirus transfer vector , pAcGP67-A ( BD Biosciences ) , incorporating a C-terminal thrombin cleavage site , a “foldon” sequence [43] and a His-tag at the extreme C-terminus of the construct to enable protein purification [25] , [44] . Transfection and virus amplification were carried out according to the baculovirus expression system manual ( BD Biosciences Pharmingen ) . Soluble NY107 was recovered from the cell supernatant by metal affinity chromatography using Ni-NTA resin ( Qiagen Inc . ) . Fractions containing NY107 were pooled and dialyzed against 10 mM Tris-HCl , 50 mM NaCl , pH 8 . 0 , then subjected to ion-exchange chromatography ( IEX ) using a Mono-Q HR 10/10 column ( GE Healthcare ) . IEX purified NY107 was subjected to thrombin digest ( 3 units/mg protein; overnight at 4°C ) and purified by gel filtration chromatography using a Superdex-200 16/60 column ( GE Healthcare ) and 50 mM Tris-HCl , 100 mM NaCl , pH 8 . 0 as running buffer . Protein eluting as a trimer was buffer exchanged into 10 mM Tris-HCl , 50 mM NaCl , pH 8 . 0 and concentrated to 14 . 5 mg/ml for crystallization trials . At this stage , the protein sample still contained the additional plasmid-encoded residues at both the N ( ADPG ) and C terminus ( SGRLVPR ) . Initial crystallization trials were set up using a Topaz Free Interface Diffusion ( FID ) Crystallizer system ( Fluidigm Corporation , San Francisco , CA ) . Crystals were observed in several conditions containing PEG 3350 or PEG 4000 . Following optimization , diffraction quality crystals for NY107 were obtained at room temperature using a modified method for microbath under oil [45] , by mixing the protein with reservoir solution containing 20% PEG 3350 , 0 . 2 M magnesium chloride at pH 7 . 2 . For receptor analog complexes , crystals were soaked for 3 hours in the crystallization buffer containing 10 mM 3′SLN or 6′SLN ( V-labs Inc . , Covington , LA ) , or overnight in 10mM LSTb ( Sigma , St . Louis , MO ) . All crystals were flash-cooled at 100K using 20% glycerol as the cryo-protectant . Datasets were collected at Advanced Photon Source ( APS ) beamlines 22 ID and BM at 100K . Data were processed with the DENZO-SACLEPACK suite [46] . Statistics for data collection are presented in Table 1 . The structure of NY107 was determined by molecular replacement with Phaser [47] using the structure of the avian H7 ( Av-H7 ) from A/turkey/Italy/2002 , pdb:1TI8 ( HA1 , 78% identity; HA2 , 90% identity ) as the searching model . One HA trimer occupies the asymmetric unit with an estimated solvent content of 58% based on a Matthews' coefficient ( Vm ) of 2 . 9 Å3/Da . Rigid body refinement of the trimer led to an overall R/Rfree of 28 . 6%/37 . 4% . The model was then “mutated” to the correct sequence and rebuilt by Coot [48] , then the protein structures were refined with REFMAC [49] using TLS refinement [50] . The final models were assessed using MolProbity [51] . The three complex structures were refined and evaluated using the same strategy . All statistics for data processing and refinement are presented in Table 1 . Electron density maps ( 2fo-fc ) were generated in Refmac [49] while simulated annealing omit maps were generated by sa-omit-map , a part of the Crystallography and NMR System ( CNS ) software [52] . Wild type and mutant viruses of NY107 ( H7N2 ) and A/Netherland/219/2003 ( H7N7 ) were generated from plasmids by a reverse genetics approach [53] . To generate viruses with amino acid insertion or substitution in the HA , mutations were introduced into plasmid DNA with an overlap extension PCR approach [54] . Viruses derived by plasmid transfection of HK293 cells were propagated in eggs . The genomes of resulting virus stocks were sequenced to detect the emergence of possible variants during amplification . Glycan microarray printing and recHA analyses have been described previously [24] , [30] , [44] , [55] ( see Table 2 for glycans used for analyses in these experiments ) . Virus were analyzed on the microarray as described previously [30] . The atomic coordinates and structure factors of NY107 are available from the RCSB PDB under accession codes 3M5G for the unliganded NY107 , 3M5H for the NY107 with 3′-SLN and 3M5I and 3M5J for NY107 with 6′SLN and LSTb , respectively . A/New York/107/03 ( H7N2 ) , Genbank: ACC55270; A/Netherlands/219/03 ( H7N7 ) , Genbank: AAR02640; A/Hong Kong/1-9/68 ( H3N2 ) , 2HMG; A/Duck/Ukraine/1/63 ( H3N8 ) , PDB: 1MQL; A/South Carolina/1/18 ( H1N1 ) , PDB: 1RD8; A/Puerto Rico/8/34 ( H1N1 ) , PDB: 1RU7; A/Swine/Iowa/15/30 ( H1N1 ) , PDB: 1RUY; A/Singapore/1/1957 ( H2N2 ) , PDB: 2WRC; A/Viet Nam/1203/04 ( H5N1 ) , PDB: 2FK0; A/Duck/Singapore/3/97 ( H5N3 ) , PDB: 1JSM; A/Swine/Hong Kong/9/98 ( H9N2 ) , PDB: 1JSD; A/Turkey/Italy/8000/02 ( H7N3 ) , PDB: 1TI8; C/Johannesburg/1/66 , 1FLC .
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Influenza virus adaptation to different hosts usually results in a switch in receptor specificity of the viral surface coat protein , hemagglutinin . Indeed , the hemagglutinin subtypes from the last two human influenza pandemics of the 20th Century ( H2 in 1957 and H3 1968 ) both adapted successfully to human-type receptor specificity through only two amino acid mutations in the receptor binding pocket ( Glutamine226→Leucine and Glycine228→Serine ) . The recent human infections reported with other avian subtypes such as H5 , H7 and H9 have raised public health concerns and focused efforts on identifying potential subtypes from which a future pandemic strain may emerge . Since 1996 , H7 viruses of the North American lineage have been circulating in regional live bird markets , containing an eight amino acid deletion in the receptor-binding site of HA . Here we report a detailed structural analysis of the receptor binding site of a hemagglutinin from the North American lineage of H7N2 viruses , in complex with avian and human receptor analogs , to understand how these viruses have adapted to such a dramatic structural change in the binding site while remaining one of the predominant circulating viral strains .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/viral",
"infections",
"biochemistry/biomacromolecule-ligand",
"interactions",
"infectious",
"diseases/respiratory",
"infections"
] |
2010
|
Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site
|
Asparagine synthetase ( AS ) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source . There are two distinct types of AS , asparagine synthetase A ( AS-A ) , known as strictly ammonia-dependent , and asparagine synthetase B ( AS-B ) , which can use either ammonia or glutamine . The absence of AS-A in humans , and its presence in trypanosomes , suggested AS-A as a potential drug target that deserved further investigation . We report the presence of functional AS-A in Trypanosoma cruzi ( TcAS-A ) and Trypanosoma brucei ( TbAS-A ) : the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP , ammonia and Mg2+ . TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor , but surprisingly , can also use glutamine , a characteristic so far never described for any AS-A . TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite . However , growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine . As expected , mice infections with induced and non-induced T . brucei RNAi clones were similar to those from wild-type parasites . However , when induced T . brucei RNAi clones were injected in mice undergoing asparaginase treatment , which depletes blood asparagine , the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites . Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting , but is unlikely to be suitable as a drug target .
Asparagine is a naturally occurring non-essential amino acid found in many proteins . Due to its high nitrogen/carbon ratio , asparagine is likely to be linked to nitrogen homeostasis and protein biosynthesis [1] . AS is the protein involved in asparagine biosynthesis . There are two distinct types of AS , AS-A and AS-B , encoded by asnA and asnB genes , respectively . AS-A encoding genes have been reported in archaea [2] , [3] , prokaryotes [4]–[7] , and in the protozoan parasite Leishmania [8] . The AS-B encoding gene is present in prokaryotes [5] , [9] and also in eukaryotes , including mammalian cells [10] , [11] , yeasts [12] , algae [13] , and higher plants [14] . Both types catalyze the ATP-dependent conversion of aspartate into asparagine . While AS-B can use both ammonia and glutamine ( reaction B ) as amide nitrogen donors [5] , [15]–[20] , Escherichia coli ( E . coli ) AS-A was reported to be dependent strictly on ammonia ( reaction A ) [21] , [22] . AS-A and AS-B share no sequence or structural similarities . Their three-dimensional structures provided important information concerning their distinct catalytic mechanisms [2] , [23]–[25] . AS-A exists as a dimer where each monomer has a core of eight β-strands flanked by α-helices , resembling the catalytic domain of class II aminoacyl-tRNA synthetases such as aspartyl-tRNA synthetase [24] . AS-A synthesizes asparagine in two steps: the β-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP , followed by amidation by a nucleophilic attack with an ammonium ion [2] . The AS-B enzyme also forms a dimer , but each monomer contains two distinct domains , each of which contains a catalytic site . The N-terminal site catalyzes the conversion of glutamine into glutamic acid and ammonia , while aspartate reacts with ATP in the C-terminal site , generating the intermediate β-aspartyl-AMP [26] , [27] . Similarly to other glutamine dependent amidotransferases , ammonia released in the N-terminal domain of the enzyme travels through an intramolecular tunnel connecting the active sites , and reacts with the reactive acyladenylate intermediate to produce asparagine [28] . An open reading frame encoding a putative AS-A is present in the genome of the protozoan parasites , Trypanosoma cruzi ( T . cruzi ) and Trypanosoma brucei ( T . brucei ) [29]–[31] . T . cruzi and T . brucei are transmitted to a mammalian host through an invertebrate vector , and are responsible for Chagas disease and African sleeping sickness , respectively . Disease control is dependent on drug therapy , but treatment options are limited , both by high toxicity and recent emergence of drug resistance [32]–[34] . Vaccines for T . brucei infections are unlikely to be developed not only because of extensive antigenic variation [35] , but also because infections compromise host humoral immune competence [36] . Trypanosome AS-A might be a drug target due to the absence of a homologue in humans [8] . AS-A is important in other microorganisms . For example , asnA is an essential gene in Haemophilus influenzae ( DEG10050178 ) [37] , and is strongly up-regulated in Pasteurella multocida during host infection [38] , and when Klebsiella aerogenes is grown in an amino acid-limited but ammonia rich environment [5] . We therefore undertook biochemical and genetic studies of AS-A in trypanosomes to ascertain its biological role and evaluate its potentiality as drug target .
All experiments involving animals were carried out in accordance with the IBMC . INEB Animal Ethics Committees and the Portuguese National Authorities for Animal Health guidelines , according to the statements on the directive 2010/63/EU of the European Parliament and of the Council . IL , JT and ACS have an accreditation for animal research given by the Portuguese Veterinary Direction ( Ministerial Directive 1005/92 ) . Procyclic and bloodstream forms of Trypanosoma brucei brucei Lister 427 were used . Procyclic forms were grown in MEM-Pros medium supplemented with 7 . 5 µg/ml hemin , 10% fetal calf serum ( FCS ) and 100 IU/mL of penicillin/streptomycin at 27°C , with cell densities between 5×105 cells/ml to 1–2×107 cells/ml . Bloodstream forms were grown in complete HMI-9 medium ( supplemented with 10% FCS and 100 IU/mL of penicillin/streptomycin ) [39] in vented tissue culture flasks; these cultures were diluted when cultures reached the cell density of 2×106/ml and incubated in a humidified atmosphere of 5% CO2 , at 37°C . Bloodstream RNAi cell cultures were supplemented with 7 . 5 µg/ml hygromycin and 0 . 2 µg/ml phleomycin . T . brucei asparagine synthetase A ( TbASA ) and T . cruzi asparagine synthetase ( TcASA ) genes were obtained by performing PCR on genomic DNA from Trypanosoma brucei brucei TREU927 and Trypanosoma cruzi CL Brener Non-Esmeraldo-like . Fragments of the open reading frames of TbASA ( Tb927 . 7 . 1110; chromosome Tb927_07_v4; 28861 to 289067 ) and TcASA ( Tc00 . 1047053503625 . 10; chromosome TcChr29-P; 687159–688206 ) were PCR-amplified using a Taq DNA polymerase with proofreading activity ( Roche ) . The sequences of the primers were as follows: sense primer 5′ - CTAATTACATATGGGCGACGACGGTTATTC - 3′ and antisense primer 5′ - CCCAAGCGAATTCTTACAACAAATTGTGC - 3′ , sense primer 5′ - CAAT TTGCATATGACATCGGGAGATCC - 3′ and antisense primer 5′ - CCCAAGCAAGCTTTCACAGCAAGGG - 3′ , respectively . PCR conditions were as follows: initial denaturation ( 2 min at 94°C ) , 35 cycles of denaturation ( 30 s at 94°C ) , annealing ( 30 s at 45°C ) and elongation ( 2 min at 68°C ) for TbAS-A , and annealing ( 30 s at 50°C ) and elongation ( 2 min at 68°C ) for TcAS-A , and a final extension step ( 10 min at 68°C ) . The PCR products were isolated from a 1% agarose gel , purified by the Qiaex II protocol ( Qiagen ) , and cloned into a pGEM-T Easy vector ( Promega ) and sent to Eurofins MWG ( Germany ) for sequencing . The TbASA and TcASA genes were subcloned into pET28a ( + ) expression vector ( Novagen ) . The recombinant 6-His-tagged proteins were expressed in E . coli BL21DE3 by induction of log-phase cultures with 0 . 5 mM IPTG ( NZYTech ) for 3 h at 37°C ( TcAS-A ) and at 18°C , overnight ( O/N ) ( TbAS-A ) . Bacteria were harvested and resuspended in buffer A [0 . 5 M NaCl ( Sigma ) , 20 mM Tris . HCl ( Sigma ) , pH 7 . 6] . The sample was sonicated and centrifuged to obtain the bacterial crude extract . The recombinant proteins were purified using Ni2+ resin ( ProBond ) and washing and elution with increasing levels ( 25 mM to 1 M ) of imidazole ( Sigma ) . The presence and purity of the recombinant protein in the several fractions was determined by SDS-PAGE and Coomassie staining . Dialysis was performed against PBS [137 mM NaCl ( Sigma ) , 2 . 7 mM KCl ( Sigma ) , 10 mM Na2HPO4 . 2H2O ( Riedel-de Haën ) , 2 mM KH2PO4 ( Riedel-de Haën ) pH 7 . 4] . To generate rat and rabbit polyclonal antibodies against TbAS-A , each animal was first immunized with 150 µg of recombinant TbAS-A protein . After 2 weeks , 4 boosts with 100 µg of recombinant TbAS-A were given weekly . The collected blood samples were centrifuged to obtain the serum . Extracts were obtained in RIPA buffer [ ( 20 mM Tris-HCl ( Sigma ) ( pH 7 . 5 ) , 150 mM NaCl ( Sigma ) , 1 mM Na2EDTA ( Sigma ) , 1 mM EGTA ( Sigma ) , 1% Nonidet P-40 ( Sigma ) , 1% sodium deoxycholate ( Sigma ) , 2 . 5 mM sodium pyrophosphate ( Sigma ) , 1 mM β-glycerophosphate ( Sigma ) , 1 mM Na3VO4 ( Sigma ) ] , with freshly-added complete protease inhibitor cocktail ( Roche Applied Science ) . The total protein amount was quantified using Biorad Commercial Kit ( Reagents A , B and S ) and the samples were then kept at −80°C . For analysis of parasites from mice , trypanosomes were purified from mouse blood using a DE-52 ( Whatman ) column [40] . For Western blotting , 2 µg of recombinant TbAS-A and TcAS-A proteins , 10 µg of total soluble cell extract , or 1×107 parasites , were resolved in SDS/PAGE and transferred on to a nitrocellulose Hy-bond ECL membrane ( Amersham Biosciences ) . The membrane was blocked in 5% ( w/v ) non-fat dried skimmed milk in PBS/0 . 1% Tween-20 ( blocking solution ) , followed by incubation with an anti-His-tag rabbit antibody ( MicroMol-413 ) ( 1∶5000 ) or a combination of an anti-TbAS-A rabbit antibody ( 1∶1000 ) with an anti-aldolase rabbit antibody ( 1∶5000 ) in blocking solution at 4°C O/N , respectively . Blots were washed with PBS/0 . 1% Tween-20 ( 3×15 min ) . Horseradish peroxidase-conjugated goat anti-rabbit IgG ( Amersham ) ( 1∶5000 for 1 h , at room temperature ) in blocking buffer was used as the secondary antibody . The membranes were developed using SuperSignal WestPico Chemiluminescent Substrate ( Pierce ) . ImageJ software ( version 1 . 43u ) was used for protein bands semi-quantification . AS activity was assessed by quantification of asparagine formation [41] . The reactions were performed in a total of 150 µl of enzyme assay mixture in 85 mM Tris-HCl ( Sigma ) containing aspartate ( Sigma ) , ammonia ( Sigma ) , ATP ( Sigma ) and 8 . 4 mM Mg2+ ( Sigma ) . Following incubation for set times at 37°C , enzymatic reactions were terminated by boiling 4 min , and then centrifuged at maximum speed for 30 s . 100 µl of the reaction mixture supernatant was added to 900 µl of ninhydrin 0 . 05% in ethanol . The resulting mixtures were boiled at 100°C for 5 min , then centrifuged for 30 s and maintained on ice . 300 µl of clear supernatant fluids were transferred to 96-well plates , and the absorbance at 340 nm determined [41] . Based on reaction linearity studies , 7 . 5 µg of enzyme and 15 min incubation at 37°C were selected as final conditions . To determine Kms , the concentrations of substrates were varied in the following ranges: 1 . 25–20 mM ( aspartate ) , 0 . 78–50 mM ( ammonia ) and 0 . 62–10 mM ( ATP ) , while the remaining substrates concentrations were in excess ( [aspartate] >20 mM , [ATP] >10 mM , and [ammonia] >50 mM ) . Km for glutamine was determined using a concentration range of 1 . 5625–25 mM , while ATP and aspartate were maintained in excess . Measurements were performed in triplicate , and the initial rate was analyzed to obtain values of Vmax and Km by curve fitting using GraphPad Prism ( 5 . 0 version ) . Using a query based on L-cysteine-S-sulfinic acid inhibitor [42] , the ZINC database was screened using the program ROCS ( version 2 . 3 . 1 ) to find compounds that have good shape similarity ( measured by 3D Tanimoto ) and similar functional group overlap to the query molecule . L-cysteine-S-sulfate ( Sigma; PubChem Substance ID 24892471 ) was used under the following conditions: 2 . 5 mM aspartate , 1 . 25 mM ATP , 12 . 5 mM ammonia , and 8 . 4 mM Mg2+ . The characterization of the mechanism of inhibition consisted in the determination of Km and Vmax for each substrate , in the presence of four inhibitor concentrations ( 0 . 025 , 0 . 050 , 0 . 1 and 0 . 2 mM ) . The following substrate concentration ranges 1 . 25–10 and 1 . 25–50 mM were used for aspartate and ammonia , respectively , while to determine Km for ATP , a range from 0 . 625 to 10 mM ( TbAS-A ) or from 0 . 3125 to 5 mM ( TcAS-A ) was assayed . Ki was determined by “Km app Method”[43] . EcAS-A , TbAS-A and TcAS-A protein alignments were performed using ClustalW [44] . Aline , Version 011208 [45] , was used for editing protein sequence alignments and preparing Fig . 1 . TbAS-A and TcAS-A homology models were obtained with SWISS-MODEL , using EcAS-A crystal structure ( Protein Data Bank ( PDB ) accession code 12AS [24] ) as a template ( percentage of sequence identity of 56% and 57% , respectively ) [46]–[48] . The 3D structures were rendered in PyMOL ( The PyMOL Molecular Graphics System , Version 1 . 3 , Schrödinger , LLC ) . The “stuffer strategy” was used to generate RNAi-mediated AS-A depletion . First , the TbASA fragment ( amplified with a sense oligo with a BglII – SphI linker 5′ - GAGAAGATCTGCATGCGCGACGACGGTTATTCGTCATAC - 3′ , and an antisense oligo with a EcoRI – SalI 5′ – CGGAATTCGTCGACACTCCGTTTTTCGGATTGCGGC – 3′ ) was cloned twice in opposite direction on either sides of a ‘stuffer’ fragment of the pHD1144 vector ( also digested with SphI and SalI ) ( Fig . S1A ) . The resulting [ ( target ) -stuffer- ( reverse-complement target ) ] construct obtained through HindIII and BglII digestion , which generates a stem-loop RNA , was cloned into pHD1145 ( also digested with HindIII and BglII ) ( Fig . S1B ) . The final construct was linearized with NotI and 10 µg of DNA was transfected into 2×107/ml bloodstream form cell line carrying pHD1313 plasmid ( contains two copies of the tet repressor and a phleomycin resistance cassette ) by electroporation using Amaxa Basic Parasite Nucleofector Kit ( Lonza ) . Transcription occurs on induction with tetracycline ( 100 ng/ml ) , hence producing mRNA homologs to the target the gene . Stable individual clones were selected 5 to 7 days after transfection with 7 . 5 µg/ml of hygromycin . To analyse growth , T . brucei RNAi cell line and cells expressing the tet repressor only ( wt ) , were seeded at 2×105 cells/ml of complete HMI9 medium , in the presence and absence of tetracycline . Cell growth was monitored microscopically on a haemocytometer ( Marienfeld ) and the culture diluted back to 2×105 cells/ml daily . The same protocol was repeated in complete HMI9 medium with basal IMDM without asparagine , complete HMI9 medium with basal IMDM without asparagine supplemented with 6 . 7×104 nM of asparagine ( levels found in human plasma [49] ) , and complete HMI9 medium with basal IMDM without asparagine supplemented with 1 . 67×105 nM of asparagine ( levels found in normal medium ) . Wild-type and transgenic bloodstream T . brucei parasites were cultured in the absence of selecting drugs ( hygromycin and phleomycin ) for 24 h , then tetracycline was added . After a further 48 h , parasites were inoculated in mice . For each experiment , 4 groups of BALB/c mice ( 6–8 weeks old , n = 4 ) ( Harlan Laboratories , United Kingdom ) were infected by intraperitoneal injection of 104 T . brucei bloodstream forms . 2 groups were injected with wt strain ( with or without tetracycline ) and the other 2 groups were injected with RNAi cell line ( with or without tetracycline ) . 48 h prior infection the 2 RNAi induced groups were given doxycycline ( treated with 1 mg/ml doxycycline hyclate and 5% sucrose containing water ) . The 2 non-induced groups were given standard water . To evaluate the virulence of RNAi induced parasites in mice with reduced plasmatic levels of asparagine , animals were treated with 50 IU of E . coli L- asparaginase ( ProSpec-Tany TechnoGene ) 48 h before injection and every 48 h . According to the literature , L-asparagine could not be detected in the blood 48 h following an intravenous injection of E . coli L-asparaginase , at a dose of 50 IU/mouse [50] . Mice were monitored every day for general appearance and behaviour . Parasitemia was monitored daily from the fifth day post-infection , using tail-vein blood , in a haematocytometer under a microscope . Animals with a parasitemia greater than 108 parasites/ml were euthanized , as previous studies had established that these levels were consistently lethal within the next 24 h . T . brucei bloodstream forms from log-phase cultures , with or without RNAi , were fixed in μ-Chamber 12 well ( Ibidi ) for 15 min , at room temperature , in PBS containing 3% p-formaldehyde , washed twice with PBS , and then permeabilized in PBS containing 0 . 1% of Triton X-100 . Fixed cells were incubated for 60 min in PBS containing 10% FCS at room temperature ( RT ) , in a humidified atmosphere , then washed twice with PBS/2% FCS . Cells were then incubated with primary rat or rabbit polyclonal antibody against TbAS-A ( 1∶100 and 1∶5000 respectively , both diluted in blocking solution ) overnight at 4°C , followed by two washes with PBS/2% FCS . Subsequently , cells were incubated with Alexa Fluor 647 conjugated goat anti-rat or Alexa Fluor 488 conjugated goat anti-rabbit secondary antibodies ( Molecular probes from Life technologies ) ( 1∶500 diluted in blocking solution ) for 1 h at RT in an humidified atmosphere , then washed twice with PBS . Next , the slides were stained and mounted with Vectashield-DAPI ( Vector Laboratories , Inc . ) . Images were captured using fluorescence microscope AxioImager Z1 and software Axiovision 4 . 7 ( Carl Zeiss , Germany ) . Pseudo-coloring of images was carried out using ImageJ software ( version 1 . 43u ) . In case of TbAS-A immunolocalization , T . brucei wt bloodstream forms cells were co-stained using rat anti-TbAS-A antibody ( 1∶100 diluted in blocking solution ) , rabbit anti-aldolase antibody ( 1∶5000 diluted in blocking solution ) , anti-BiP antibody ( kindly provided by Dr . Jay Bangs , 1∶500 diluted in blocking solution ) , anti-enolase antibody ( kindly provided by Dr . Paul Michels , 1∶5000 diluted in blocking solution ) or anti-GRASP antibody ( kindly provided by Dr . Graham Warren , 1∶200 diluted in blocking solution ) . Alexa Fluor 647 conjugated goat anti-rat ( 1∶500 ) and Alexa Fluor 488 conjugated goat anti-rabbit ( 1∶500 ) were used as secondary antibodies . Staining with MitoTracker Orange ( Invitrogen ) followed by Alexa Fluor 488 conjugated goat anti-rabbit ( 1∶500 ) , as a secondary antibody . The labelling of parasites with MitoTracker was done by adding 250 nM to the cell culture medium ( without FCS ) for 30 minutes , prior to washing , fixing and staining using the protocol described above . Images were captured using the confocal microscope Leica TCS SP5II and LAS 2 . 6 software ( Leica Microsystems , Germany ) . Again , image analysis was done using ImageJ version 1 . 43U software . For each sample condition , 1 . 0×107 bloodstream cells were washed once with cold trypanosome homogenization buffer ( THB ) , containing 25 mM Tris ( Sigma ) , 1 mM EDTA ( Sigma ) and 10% sucrose ( Sigma ) , pH = 7 . 8 . Just before cell lysis , leupeptin ( Sigma ) ( final concentration of 2 µg/ml ) and different digitonin ( Calbiochem ) quantities ( final concentrations of 5 , 12 . 5 , 25 , 50 , 100 , 150 and 200 ug/ml ) were added to 500 µl of cold THB , for cell pellet resuspension . Untreated cells ( 0 µg/ml of digitonin ) and those completely permeabilized ( total release , the result of incubation in 0 . 5% Triton X-100 ) were used as controls . Each sample was incubated 60 min on ice , and then centrifuged at 2000 rpm , 4°C , for 10 min . Supernatants were transferred to new chilled tubes and 500 µl of cold THB was added to each pellet and then mixed . All fractions were analysed through Western blot as described above . T . brucei bloodstream forms were analyzed by flow cytometry for DNA content following RNAi induction . Cells were collected by centrifugation and washed twice with PBS containing 2% FCS . Each 2×106 cells were resuspended in 1 mL of PBS/2% FCS and 3 mL of cold absolute ethanol was added while vortexing . Cells were fixed for 1 hour at 4°C and then washed twice in PBS . 1 mL of staining solution [3 . 8 mM sodium citrate dehydrate ( Sigma ) , 50 µg/mL propidium iodide ( Sigma ) , 0 . 5 µg/µL RNAse A ( Sigma ) in PBS] was added to the cell pellets and vortex . Samples were analysed by FACS ( Becton Dickinson ) after a incubation at 4°C for 30 min . Data was analyzed by FlowJo software ( Ashland , OR ) . One-way ANOVA and two-tailed Student's test were used for statistical analysis . Statistical analysis was performed using GraphPad Prism Software ( version 5 . 0 ) , and p values≤0 . 01 were considered to be statistically significant . Asterisks indicate statistically significant differences ( *** p≤0 . 001 , ** p≤0 . 01 ) .
One open reading frame that code for a putative AS-A is present in the genomes of T . cruzi CL Brener Non-Esmeraldo-like and T . brucei TREU927 ( http://tritrypdb . org ) [29]–[31] . A protein multiple sequence alignment , performed using ClustalW [44] , of AS-A from T . brucei ( Tb927 . 7 . 1110 , NCBI-GeneID:3658321 ) , T . cruzi ( Tc00 . 1047053503625 . 10 , NCBI-GeneID:3534325 ) and E . coli ( NCBI-GeneID:948258 ) is shown in Figure 1 . The amino acid residues known to be involved in the active-site formation in E . coli [24] are highly conserved within the three sequences ( Fig . 1 ) . Protein alignments demonstrated 58% similarity for EcAS-A versus TbAS-A , 60% for EcAS-A versus TcAS-A , and 63% for TbAS-A versus TcAS-A . Like EcAS-A , TbAS-A and TcAS are predicted to be dimeric , as seen from superimposed homology models with the EcAS-A crystal structure [24] ( Fig . 2A ) . The only structurally divergent region ( area marked by dashed rectangle ) ( Fig . 1 , 2B ) , is present in both TbAS-A ( from residues Q232 to S250 ) and TcAS-A ( from residues D232 to S247 ) , but absent in EcAS-A . This region is distant from the enzyme active site and the dimer interface and its functional and structural significance are unknown . The amino acids involved in asparagine binding are all strictly conserved , while in the AMP binding pocket , the majority of the residues are conserved , except for three residues ( Fig . 1 ) . In EcAS-A , E103 ( D106 and E106 in TbAS-A and TcAS-A , respectively ) and L109 ( I112 and T112 in TbAS-A and TcAS-A , respectively ) ( Fig . 1 ) are not directly involved in polar interactions with the nucleotide base , but form part of the outer wall of the binding site [24] . The main chain of L249 in EcAS-A ( V271 and L268 in TbAS-A and TcAS-A , respectively ) is directly involved in hydrogen bonds with ribose from AMP , however the different side chains of leucine and valine do not affect the shape of AMP binding site . TbAS-A and TcAS-A coding sequences were cloned into the bacterial expression vector pET28a . Histidine-tagged fusion proteins were purified under non-denaturing conditions ( Fig . 3A , B ) . As expected , the recombinant proteins were recognized by anti-His Tag monoclonal antibody ( Fig . 3A , B ) . Rabbit polyclonal antibodies produced against recombinant TbAS-A recognized the protein in total extracts from two different parasite developmental stages , bloodstream forms ( mammalian host parasite stage ) and procyclic forms ( insect vector parasite stage ) ( Fig . 3C ) . The capacities of TbAS-A and TcAS-A to produce asparagine from aspartate in the presence of ATP , ammonia and Mg2+ were determined using a specific quantitative colorimetric assay for L-asparagine [41] . The pH optimum was 7 . 6 , with detectable activity from 6 . 0 to 9 . 0 ( data not shown ) . Mg2+ was an essential co-factor for TbAS-A and TcAS-A ( data not shown ) , as previously described for EcAS-A [22] . We included 8 . 4 mM Mg2+ in the final reaction mixture . Lower concentrations ( 2 , 4 and 6 mM ) gave lower activity while increased concentrations ( up to 16 mM ) resulted in no substantial activity improvement ( data not shown ) . TbAS-A and TcAS-A showed similar Kms for aspartate and ammonia ( p>0 . 01 ) , while TcAS-A showed higher Km for ATP than TbAS-A ( p = 0 . 0042 ) ( Table 1 ) . ATP is the substrate required for the generation of the β-aspartyl adenylate intermediate , which reacts with ammonia , releasing asparagine . In its absence , the reaction did not occur ( data not shown ) . To our surprise , both TbAS-A and TcAS-A could also use glutamine as a nitrogen donor ( Table 2 ) . TbAS-A showed higher Km for this nitrogen donor than TcAS-A , however not statistically significant ( p>0 . 01 ) . Both enzymes present higher Km values for ammonia than for glutamine , but these differences were not statistically significant ( p>0 . 01 ) ( Table 1 and 2 ) . TbAS-A had a higher Vmax than TcAS-A , for both ammonia ( p<0 . 0001 ) and glutamine ( p = 0 . 0043 ) dependent-activities ( Table 1 and 2 ) . TbAS-A had similar catalytic rates for both glutamine and ammonia-dependent activities ( p>0 . 01 ) , whereas TcAS-A presented a slightly higher , but not statistically significant , rate for glutamine-dependent activity ( p>0 . 01 ) ( Table 1 and 2 ) . The high conservation of the active sites and the small amino acid differences identified in the homology models do not allow an accurate structural interpretation of the small differences observed . Indeed , these might have been due to slight differences in the proportion of protein that was correctly folded . L-cysteine-S-sulfate , considered a putative AS-A inhibitor from a virtual screening , inhibited both enzymes , with IC50s of 126 and 100 µM for TbAS-A and TcAS-A , respectively ( Fig . 4A ) . For both enzymes , the kinetic characteristics suggested competition with ATP binding ( Fig . 4C , D ) . No changes in the Kms and Vmaxs for aspartate and ammonia were observed ( p>0 . 01 ) ( Fig . 4E , F , G , H ) , suggesting the inhibition is exclusively due to ATP binding interference ( p≤0 . 01 ) . Ki values of 137 and 128 . 9 µM were determined for TbAS-A and TcAS-A , respectively ( Fig . 4B ) . The subcellular localization of TbAS-A was determined by immunofluorescence and digitonin fractionation in bloodstream forms . As expected , induction of RNAi resulted in a decrease in the fluorescence intensity ( Fig . S2A , B , C ) . TbAS-A is in the cytosol , as revealed by colocalization with the cytosolic enzyme enolase [51] ( Fig . 5A ) and no colocalization with aldolase , BiP , GRASP or mitotracker ( Fig . S3 ) , markers for glycosomes [52] , endoplasmic reticulum [53] , Golgi and mitochondria compartments [54] , respectively . Controls performed with rat or rabbit pre-immune sera and secondary antibody alone , showed no detectable signal ( data not shown ) . Digitonin fractionation also resulted in similar profiles for AS-A and enolase ( cytosolic marker ) and no similarity to aldolase ( glycosomes marker ) ( Fig . 5B ) . To study the biological role of AS-A in T . brucei bloodstream forms , cells were stably transfected with an RNA interference plasmid construct . RNAi against asparagine synthetase A was induced in normal medium ( complete HMI-9 ) by adding tetracycline . No difference was observed in cell proliferation between induced and non-induced cells ( Fig . 6A ) , although AS-A protein was reduced to ≈13% of the normal level within 48 hours ( Fig . 6B ) . When , however , the AS-A-depleted cells were grown in HMI-9 medium with only the asparagine from the fetal calf serum , growth was impaired , with an increase in the proportion of cells in G0/G1 ( Fig . 6C and 7B , C ) . Presumably the asparagine from the serum allowed this slower growth . Levels of asparagine usually found in human serum ( 6 . 7×104 nM ) [49] , which are somewhat lower than in normal medium ( 1 . 67×105 nM; IMDM - Iscove's modified Dulbecco's basal medium from Gibco Invitrogen ) , were sufficient to overcome this defect ( Fig . 6E , G ) . In complete HMI-9 medium , with only asparagine from the fetal calf serum , the growth defects of induced RNAi clones are abrogated at day 5 post-induction ( Fig . 6C , 7D ) , and the percentage of cells in GO/G1 and S phases of the cell cycle return to the ones found in non-induced cells ( Fig . 7A ) , suggesting the appearance of RNAi revertants , as is also visible on the Western blot ( Fig . 6D ) . Similar reversion to evade lethal RNAi in trypanosomes has been seen many times before [55] . In the presence of asparagine , low AS-A levels were maintained ( Fig . 6B , F and H ) . Normal T . brucei parasites also showed a statistically significant slower growth under conditions of asparagine limitation ( p≤0 . 01; data not shown ) ( compare Fig . 6C , with A , E , G ) . It is therefore possible that even when the parasite has AS , it also requires external asparagine for optimal in vitro growth . To test whether AS-A is important for parasite infection in a disease model , two groups of mice ( n = 4 ) were inoculated with the parental cell line , and other two groups with RNAi cells . Two mice groups were fed with water containing doxycycline to induce down-regulation of TbAS-A , while the remaining mice were kept as non-induced controls . Within six days of inoculation , all mice from the different groups developed high levels of parasitemia ( Fig . 8A ) , and all had to be euthanized after seven or eight days post-infection ( Fig . 8B ) . The results confirm that the asparagine in mouse blood is sufficient to compensate for the ≈87% downregulation of AS-A ( Fig . 8C , D ) . To assess the contribution of blood L-asparagine in vivo , mice were treated with L-asparaginase [50] . L-asparaginase treatment did not affect growth of normal parasites in mice ( Fig . 9A ) and consequently did not extend animal survival ( Fig . 9B ) . However L-asparaginase treatment in mice infected with TbAS-A RNAi-induced parasites caused a decrease in the parasitemia ( Fig . 9D ) , thus leading to an increase of mice survival ( Fig . 9E ) . Even so , the infection resulted in death . As happened in vitro , RNAi revertants appeared during the course of infection in asparaginase-treated , but not untreated , mice ( Fig . 9F ) . Parasites extracts from wt infected mice were used as controls ( Fig . 9C ) .
In this study we demonstrated that trypanosomes AS-A use both ammonia or glutamine as nitrogen donors for the ATP dependent conversion of aspartate into asparagine . Such hybrid activity was only previously demonstrated for type B enzymes , which prefer glutamine to ammonia [15]–[19] . The small differences in Km of TbAS-A and TcAS-A for ammonia and glutamine ( 1 . 5 and 2 fold , respectively ) are lower than the difference found in most AS-B enzymes , with the exception of the human enzyme , which has similar affinities for both [16]–[20] . Purified E . coli AS-A used only ammonia as the nitrogen source , and results from Klebsiella aerogenes also suggested that AS-A preferentially uses ammonia as substrate [5] , [21] , [22] . The conclusions for AS-A enzymes of these two Gram-negative organisms relied on both biochemical and genetic analysis , but given technical limitations at the time , and the fact that background enzyme activity was seen in the absence of both ammonia and glutamine , some re-examination in bacteria would be worthwhile . Moreover the overall Km values of trypanosomes AS-A for aspartate , are 6 up to 20 fold higher than the ones found in the literature for prokaryotic asparagine synthetase type A [5] , [21] , [22] . Trypanosoma AS-A structures were not yet been solved and our protein homology models are not completely enlightening , nevertheless we can speculate that such differences may result in the fact that parasite enzymes were expressed and purified as recombinant proteins in bacteria and not purified directly from trypanosomes extracts . As a consequence , differences in protein post-transcriptional processing and/or changes in protein conformation cannot be excluded . Our results suggest that bloodstream-form parasites rely on two major sources of asparagine to ensure normal proliferation: uptake from the extracellular medium and biosynthesis by AS-A . Bloodstream form proliferation , either in vitro or in vivo , was only significantly affected when both asparagine sources were compromised . Also consistent with this idea , in the published RNAi screen , a very slight ( possibly insignificant ) growth disadvantage was seen in bloodstream forms depleted of AS-A [56] . In the same way , our in vitro results are corroborated with previous studies , as mammalian cells with low expression of AS are similarly susceptible to asparagine depletion [57]–[59] , and asparaginase isolated from E . coli and Erwinia carotovora act as potent anti-leukemic agents [60] . In the trypanosomes genome there is a second open reading frame ( Tb927 . 3 . 4060 ) coding for a putative AS domain . However this is apparently not a classical AS , despite the presence of a good Pfam AS domain ( PF0073 ) at the C-terminus . A BLASTp search using the T . brucei sequence revealed a variety of proteins of unknown function that aligned not only across the AS domain , but also in the N-terminal region , which contains N-terminal aminohydrolase domains . Best matches originate from extremely diverse eukaryotes including a plant , an alga , a member of the fungi and an amoeba . BLASTp against the Saccharomyces cerevisiae predicted proteome yielded YML096W , and the reciprocal BLASTp on the trypanosome genome indeed gave Tb927 . 3 . 4060 as best match . The function of YML096W is not known , and in a trypanosome RNAi screen no growth defect was seen for Tb927 . 3 . 4060 [56] . The capacity of trypanosomes to grow using asparagine from the extracellular environment , and the lack of growth defect when the levels of AS-A are reduced , show that only a combination therapy using both a TbAS-A inhibitor and an extracellular asparagine depletor ( e . g . L-asparaginase ) or an asparagine transport blocker could inhibit parasite growth . This is not appropriate for African sleeping sickness treatment . A combination that absolutely required simultaneous activities of two different drugs would be wide open to resistance development , and drug combination including an intravenously-introduced enzyme is likely to be both too expensive and logistically inappropriate for treatment of African trypanosomiasis . Moreover , L-asparaginase treatment in cancer results in serious adverse events [61]–[63] . We therefore conclude that AS-A is not a good candidate as a sleeping sickness drug target . Its role in Trypanosoma cruzi , however , remains to be established .
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The amino acid asparagine is important not only for protein biosynthesis , but also for nitrogen homeostasis . Asparagine synthetase catalyzes the synthesis of this amino acid . There are two forms of asparagine synthetase , A and B . The presence of type A in trypanosomes , and its absence in humans , makes this protein a potential drug target . Trypanosomes are responsible for serious parasitic diseases that rely on limited drug therapeutic options for control . In our study we present a functional characterization of trypanosomes asparagine synthetase A . We describe that Trypanosoma brucei and Trypanosoma cruzi type A enzymes are able to use either ammonia or glutamine as a nitrogen donor , within the conversion of aspartate into asparagine . Furthermore , we show that asparagine synthetase A knockdown renders Trypanosoma brucei auxotrophic to asparagine . Overall , this study demonstrates that interfering with asparagine metabolism represents a way to control parasite growth and infectivity .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2013
|
Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine
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IL-23 regulates myriad processes in the innate and adaptive immune systems , and is a critical mediator of the proinflammatory effects exerted by Th17 cells in many diseases . In this study , we investigated whether and how hepatitis B virus ( HBV ) causes liver damage directly through the IL-23 signaling pathway . In biopsied liver tissues from HBV-infected patients , expression of both IL-23 and IL-23R was remarkably elevated . In vivo observations also indicated that the main sources of IL-23 were myeloid dendritic cells ( mDCs ) and macrophages . Analysis of in vitro differentiated immature DCs and macrophages isolated from healthy donors revealed that the HBV surface antigen ( HBsAg ) efficiently induces IL-23 secretion in a mannose receptor ( MR ) -dependent manner . Culture with an endosomal acidification inhibitor and the dynamin inhibitor showed that , upon binding to the MR , the HBsAg is taken up by mDCs and macrophages through an endocytosis mechanism . In contrast , although the HBV core antigen ( HBcAg ) can also stimulate IL-23 secretion from mDCs , the process was MR- and endocytosis-independent . In addition , IL-23 was shown to be indispensible for HBsAg-stimulated differentiation of naïve CD4+ T cells into Th17 cells , which were determined to be the primary source of IL-17 in HBV-infected livers . The cognate receptor , IL-17R , was found to exist on the hepatic stellate cells and mDCs , both of which might represent the potential target cells of IL-17 in hepatitis B disease . These data provide novel insights into a yet unrecognized mechanism of HBV-induced hepatitis , by which increases in IL-23 expression , through an MR/endocytosis-dependent or -independent manner , produce liver damage through the IL-23/IL-17 axis .
Hepatitis B virus ( HBV ) is a noncytopathic , hepatotropic and stealth DNA virus that has been implicated in the etiology of chronic hepatitis B ( CHB ) and HBV-associated acute-on-chronic liver failure ( ACLF ) . HBV-induced hepatic injury is known to be mediated by a variety of immunocytes that play important roles in the development and progression of hepatitis B . Among these host immune cells , the T cells are considered the main effector cells contributing to the pathogenesis of hepatitis B disease . Furthermore , the CD4+ and CD8+ T cell subpopulations have both been shown to play key roles in antiviral defenses , as well as in the hepatocellular damage that accompanies hepatitis B viral infection [1] . CD4+CD25+Foxp3+ regulatory T ( Treg ) cells are a subset of the CD4+ T cells that function as inhibitors of T cell-mediated responses . While this action ameliorates T cell-mediated hepatocellular damage , it also creates an environment favorable to persistent hepatitis viral infection and tumor formation [2] , [3] . More recently , however , it has been reported that HBV-infected patients have a remarkably high amount of another subset of T helper ( Th ) cells , the interleukin ( IL ) -17-secreting Th17 cells , which have been verified as closely associated with the development and severity of liver damage in patients with hepatitis B [4]–[6] and HBV-related liver fibrosis [7] , [8] . However , the detailed mechanisms for the roles of Th17 cells in hepatitis B disease remain to be fully elucidated . It has been demonstrated that , in hepatitis B patients , the Th17 cell-associated liver inflammation is positively associated with IL-23 cytokine level , as described in our previous review [6] . IL-23 , a member of the IL-12 family , has been shown to regulate a myriad of processes in the innate and adaptive immune systems [9] , [10] , including promoting the antigen presentation abilities of antigen presenting cells ( APCs ) and enhancing the effector function of T cells [11] . In our previous experiment , we found that the mRNA expressions of IL-23 were enhanced in the liver tissue of hepatitis B patients [12]; recent study further indicated that IL-23 plays a pathological role through IL-17 production in Concanavalin A ( Con A ) -induced hepatitis [13] . All these data suggest that IL-23 might play an important role in the pathogenesis of hepatitis B . However , the precise source cells of IL-23 , the mechanism by which HBV induces their production of IL-23 , and the concrete functions of IL-23 in hepatitis B patients have to be clarified in order to gain an accurate and useful understanding of the immunological mechanisms underlying HBV infection . IL-23R has been demonstrated to be mainly expressed on Th17 cells ( interleukin-17-producing CD4+ T cells ) and IL-23 is thought to play a pivotal role in the differentiation and maintenance of function of Th17 cells [14] , [15] . Recent research indicated that a remarkable amount of Th17 cells infiltrate the liver tissue in hepatitis B patients and the extent of Th17 infiltration correlated with severity of liver damage [4] , [16] , [17] . IL-17 must bind to its cognate receptor ( IL-17R ) to fulfill its functions , such as inducing the release of other cytokines that lead to proinflammatory processes and neutrophil-mobilization [14] , [18] . In Con A-induced hepatitis [19] , IL-17R was found to be highly expressed on Kupffer cells and the IL-17/IL-17R signaling pathway was characterized as critically involved in the pathogenesis . Zhang et al . reported that IL-17R was uniquely expressed on monocytes and myeloid dendritic cells ( mDCs ) from the peripheral blood mononuclear cell ( PBMC ) population of CHB patients [4] . However , the cell type source of IL-17R in HBV-infected liver tissue remains unknown , and may involve any one or multiple types of the immune cells known to reside in the diseased organ , including lymphocytes , neutrophils , mDCs , or/and plasmacytoid dendritic cells ( pDCs ) [20] , [21] . Herein , we investigated the role of the IL-23/IL-17 axis in HBV-infected humans by using human liver biopsy samples . We found that , upon binding to the mannose receptor ( MR ) on mDCs and macrophages , the hepatitis B surface antigen ( HBsAg ) could be taken up through an endocytosis mechanism , followed by efficient IL-23 secretion from these cells . In contrast to HBsAg , HBV core antigen ( HBcAg ) could also stimulate mDCs and macrophages to produce IL-23 , but in an MR- and endocytosis-independent manner . Clinical observation further demonstrated that IL-23 expressions were significantly up-regulated in hepatitis B patients , and identified mDCs and macrophages as the main sources of IL-23 . In response to the elevated IL-23 level , Th17 cells were found to be stimulated to produce large amounts of IL-17 , which would subsequently contribute to the pathogenesis of hepatitis B disease . We also found that the key Treg cell functional cytokine , IL-10 , and not TGF-β , could markedly inhibit the HBsAg-stimulated IL-23 production from mDCs in vitro . Thus , the findings from this study not only provide novel insights into the mechanisms of the IL-23/IL-17 axis , supporting the pathogenesis of HBV infection , but also identify potential targets of interventional strategies for treating hepatitis B patients through manipulation of the IL-23/IL-17 axis .
IL-23 has been demonstrated to be involved in many pivotal processes of the innate and adaptive immune systems [10] , [11] . To explore the role of IL-23 in pathogenesis of hepatitis B , we first assessed the expression of IL-23 in liver tissues from healthy individuals and patients with CHB or ACLF . Results showed that IL-23 transcripts and protein were strongly expressed in liver tissues of ACLF and CHB patients , as compared to that detected in tissues from healthy controls . Moreover , the liver tissues of ACLF patients exhibited a much higher expression level of IL-23 over that in CHB livers ( Figure 1A and B ) . The high IL-23 expression in the livers of CHB and ACLF patients was further confirmed by in situ immunohistochemistry assay . We found that the frequency of IL-23+ cells was significantly increased in liver tissues of infected patients , as compared to healthy controls , and those cells were mainly located in the portal tract ( Figure 1C ) . The significant difference of IL-23 expression among ACLF , CHB and healthy controls suggests an association between IL-23 and liver damage in HBV infection . To further confirm the source cell type of IL-23 , we performed immunofluorescence staining on liver tissues from patients with hepatitis B . Confocal microscopy of liver tissues showed that IL-23p19 , the specific subunit of IL-23 heterodimers , co-localized with the CD11c mDC marker , but not with the CD303 pDC marker ( Figure 2A and 2B ) . Besides DCs , macrophages are another important innate immune cell and APC type . Therefore , we investigated whether liver macrophages were also responsible for IL-23 production upon HBV infection . Confocal microscopy assay showed that most of the macrophages but not the hepatocytes in hepatitis B patients secrete IL-23 ( Figure 2C and 2D ) . Immunofluorescence assay of liver biopsy samples from healthy controls showed IL-23 staining in less than 50% of mDCs , while those from hepatitis B patients showed staining in over 80% of mDCs ( Figure 2E ) . Similarly , only about 10% of macrophages in the samples from healthy controls were IL-23-positive , while over 60% of macrophages in the hepatitis B samples expressed IL-23 ( Figure 2F ) . These results indicate that the elevated IL-23 expression in livers of hepatitis B patients is mainly derived from APCs , including mDCs and macrophages , and suggests that these cells are likely involved in the immunological responses to HBV . As a functional cytokine , IL-23 functions only if it binds to its cognate receptor , IL-23R , which is found to be mainly expressed by activated T cells [10] . To investigate the potential effects of IL-23 on hepatitis B , we evaluated the expression of IL-23R in HBV infected liver tissues . Results showed that the mRNA and protein expression levels of IL-23R in liver tissue infected with hepatitis B were significantly higher than in tissues from healthy controls ( Figure 3A and 3B ) . Most of the IL-23R+ cells were located in the portal tract , and the frequency of IL-23R+ cells in liver tissue of patients with hepatitis B was remarkably higher than in liver from healthy controls ( Figure 3C and 3D ) . Furthermore , the expression of IL-23R was significantly correlated with IL-23 in liver tissue with hepatitis B ( P<0 . 001 , Figure 3E ) . Since IL-23/IL-23R expression was significantly elevated in livers of patients infected by HBV ( Figures 1–3 ) and IL-23 is essential for the functional maintenance of Th17 cells [15] , we anticipated that IL-17 expression might be enhanced in HBV infected liver tissues , as a result of the elevated IL-23 expression . Indeed , we found that IL-17 expression was significantly elevated in the livers of CHB and ACLF patients , as compared to that in healthy controls ( Figure S1A–C ) . Furthermore , IL-17 expression in liver tissues of ACLF patients was higher than that in CHB patients , which was in agreement with a previous report of elevated Th17 cell frequency in hepatitis B patients [22] . We also observed that the IL-17 expression levels in liver were much higher than that in PBMCs from the same hepatitis B patients ( Figure S1D ) . Further statistical analyses confirmed a significant positive correlation between IL-23/IL-23R and IL-17 expression levels in liver biopsies from the patients with CHB ( P<0 . 001; Figure 4A and 4B ) . To explore the role of IL-23 on IL-17 production in hepatitis B pathogenesis , we investigated which cell type ( s ) adopted IL-23 signals to produce the pathogenic IL-17 by detecting the expression of IL-23R in IL-17+ cells in liver tissue . Confocal microscopy revealed that IL-17 was almost completely co-localized with IL-23R in hepatitis B-infected liver tissues ( Figure 4C ) . Moreover , although not all CD4+ T cells expressed IL-17 , the IL-17 was mainly expressed in CD4+ T cells and only rarely expressed in γδT cells or neutrophils ( Figure S2 ) . When determining the expression of IL-23R in PBMCs of patients with CHB by flow cytometry ( FCM ) analysis , we also found that most IL-23R+ cells were IL-17+CD4+ T cells ( Figure 4D , left panel ) , which indicated that the circulating Th17 cells expressed a high level of IL-23R under the hepatitis B condition . Confocal microscopy results further confirmed the co-expression of IL-17 and IL-23R in PBMCs ( Figure 4D , other panels ) . Together , these results indicate that IL-23 might stimulate CD4+IL-23R+ T ( Th17 ) cells within HBV-infected livers and PBMCs to produce the pathogenic IL-17 . To further address the role of IL-23/IL-17 pathway in pathogenesis of HBV infection , we analyzed the relationship between the expression of IL-23/IL-17 and clinical phenotypes of CHB patients . Results in Figure 5 showed that the expression of IL-23 and IL-17 in livers of CHB patients was significantly correlated with the serum alanine aminotransferase ( ALT ) level and the proinflammatory cytokines , including tumor necrosis factor ( TNF ) -α and IL-8 , but not with the plasma HBV load . Interestingly , we found that there was a remarkable correlation between the expressions of IL-23/IL-17 and the level of serum HBsAg , when the HBsAg concentration was above 104 IU/mL , suggesting that only a high enough level of HBsAg could efficiently induce the production of IL-23/IL-17 . HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation , with HBsAg being predominant . As such , there is substantial opportunity for interaction between HBV antigens and APCs in vivo . Since IL-23 appeared to mainly be derived from APCs and IL-23 expression was closely correlated with plasma HBsAg level ( Figure 5 ) , we investigated whether the main HBV-derived antigen , HBsAg , could directly prime APCs , such as DCs and macrophages , to secrete IL-23 in vitro . In addition , another important HBV antigen , HBcAg , was also investigated in parallel . Results showed that treatment of human monocyte-derived DCs with recombinant HBsAg or HBcAg led to a dose-dependent increase in IL-23 , but had little effect on IL-12 expression , as compared with the medium-only control and the human serum albumin ( HSA ) negative control ( Figure 6A ) . Similar results were obtained in monocyte-derived macrophages upon stimulation by HBsAg or HBcAg ( data not shown ) . As the HSA and HBV antigens were prepared by the same protocol using a mammalian CHO-cell system , it was unlikely that contamination of lipopolysaccharide endotoxin or differential arrays of other Toll-like receptor ( TLR ) ligands would significantly affect the experiment . To further explore the mechanisms of HBsAg-stimulated IL-23 production from mDCs of patients with hepatitis B , patients' sera with high concentrations of HBsAg were added into the culture system . As shown in Figure 6B , the sera from patients with ACLF significantly induced IL-23 production from mDCs , but this effect was sharply reversed by addition of HBsAg antibody ( HBsAb ) prepared from the anti-sera of healthy volunteers who received inoculation of preventive HBsAg vaccine and were HBsAb-positive . Similar results were obtained in vitro with macrophages stimulated by HBsAg or patients' sera ( Figure S3 ) . Since DCs have been shown to uptake HBsAg through their surface mannose receptors [23] , we investigated whether interaction between the glycoprotein HBsAg and the MR is responsible for the production of IL-23 from DCs . To this end , DCs were treated with mannan ( as a competitive inhibitor [23] ) or increasing amounts of neutralizing anti-MR antibodies to assess the effects HBsAg on MR-blocked DCs . As shown in Figure 6C , both mannan and MR-blocking antibody effectively decreased the HBsAg-induced IL-23 production from DCs , suggesting that HBsAg could induce IL-23 expression by binding to and signaling through the MR on DCs . However , IL-23 production from DCs induced by HBcAg was unaffected by mannan or the neutralizing antibody targeting the MR ( Figure 6C ) , indicating that HBcAg stimulated the DCs in a MR-independent manner . Similar results were observed when macrophages were used in this assay , which was expected since macrophages also express MR [24] ( data not shown ) . In our previous studies , we found that the frequencies of both Treg and Th17 cells were increased in hepatitis B patients [12] , [25] . However , Th17 cells might be inhibited by the functional cytokines of Treg cells , such as IL-10 , which has been demonstrated to suppress the production of Th17 cytokines by down-regulating the RORγt expression [26] , [27] . Therefore , in this study , we further investigated whether the critical cytokines of Treg cells ( i . e . IL-10 and TGF-β ) could influence Th17 cells through affecting the IL-23 production from mDCs . Results showed that the exogenous IL-10 protein significantly inhibited the HBsAg-induced IL-23 production by mDCs; however , TGF-β addition did not influence the IL-23 production from HBsAg-stimulated mDCs ( Figure 6D ) . To further dissect the mechanisms responsible for IL-23 production , we investigated whether the IL-23 production from DCs was dependent upon endocytosis or endosome acidification . The HBsAg-stimulated high-level secretion of IL-23 by DCs was found to be significantly blocked in the presence of ammonium chloride , chloroquine or Dynasore , similar to the results obtained with HBsAb blocking ( Figure 7A ) . Moreover , a similar trend was observed in macrophages ( data not shown ) . Immunofluorescence assay showed that the cell surface of both DCs and macrophages bound much of the FITC-labeled anti-MR antibody , indicating the rich expression of MR on these cells ( Figure 7B ( b ) ) . When HBsAgs were incubated with DCs , they appeared to be internalized , as evidenced by the FITC-anti-HBsAg-mAb staining pattern ( Figure 7B ( c ) ) . However , this internalization was dependent on endocytosis and was MR-mediated , and was almost completely blocked by pretreatment with chloroquine or MR-blocking antibody ( Figure 7B ( d–e ) ) . A similar trend was observed with macrophages ( Figure 7C ) . To verify these results , we further examined the co-localization of MR/HBsAg , and HBsAg/endosomal marker LysoTracker by confocal microscopy . We found that HBsAg was almost completely co-localized with MR or LysoTracker ( Figure 7D and 7E ) , indicating that HBsAg was taken up by mDCs through binding to MR and subsequent endocytosis . To confirm whether HBcAg-stimulated IL-23 production from mDCs was an endocytosis-dependent process , mDCs were stimulated by HBcAg alone or in the presence of ammonium chloride , chloroquine , Dynasore , or HBcAg-blocking antibody for 40 hours . Effects on IL-23 production by the mDCs were examined by FACS , and the IL-23 level was found to be significantly decreased by the HBcAg-blocking antibody , but only minimally decreased by ammonium chloride , chloroquine and Dynasore ( Figure S4 ) . These results suggest that HBcAg-stimulated IL-23 production in mDCs occurs through a certain yet unknown pathway , other than endocytosis . We also observed similar results with HBcAg-stimulated macrophages ( data not shown ) . As shown in Figure 7 , IL-23 production by DCs appeared to be significantly blocked by ammonium chloride , chloroquine , and Dynasor . However , the absence of IL-23 production could merely reflect poor viability of DCs since these reagents are toxic under some culture conditions [28]–[30] . To confirm that the DCs remain fully functional in our experimental system , we examined DCs responsiveness to LPS stimulation in the presence of each of these reagents because LPS activates host cells by TLR4 signaling , which does not require endocytosis or endosomal acidification . After 12 hours of stimulation with LPS in the presence of chloroquine , the expression patterns of co-stimulatory molecules , including CD40 , CD80/86 , CD274 and HLA-DR , on DCs were nearly identical to those on DCs stimulated with LPS in the absence of chloroquine ( Figure S5 ) . In addition , similar results were achieved with stimulation in the presence of ammonium chloride or Dynasor ( data not shown ) . The significantly positive correlations that were observed between IL-23/IL-23R and IL-17 expression in patients with hepatitis B led us to investigate whether IL-23 is indispensable for HBV antigen-stimulated IL-17 production . Naïve CD4+ T cells that were co-cultured with HBsAg or HBcAg pretreated DCs produced a significant amount of IL-17 and showed a markedly higher frequency of IL-17+ T cells than controls . Addition of recombinant IL-23 further enhanced these effects , while addition of IL-23-blocking antibody dramatically decreased these effects ( Figure S6 ) . Together , these data suggest that IL-23 derived from HBV antigen-stimulated mDCs can effectively polarize naïve CD4+ T cells toward the IL-17-producing Th17 phenotype . This finding might at least partially reflect the in vivo effects of HBV infection on mDCs and the subsequent effects on Th17 cells . It is well known that cytokines initiate downstream signaling pathway activities by binding to specific receptors expressed on the target cells , thus IL-17 should also bind to its cognate receptor to mediate liver injury during HBV infection . IL-17 receptors are reported to be expressed on several kinds of cells including epithelial cells , fibroblasts , B and T lymphocytes , myelomonocytic cells , and marrow stromal cells [31] . However , the IL-17R expression pattern in hepatitis B liver should be clarified specifically . To this end , we first examined the IL-17R expression in the livers of CHB patients by Western blot assay and in situ immunostaining . Results demonstrated that IL-17R expression was appreciably higher in patients with hepatitis B infection and the expression was mainly in the fibrotic septa ( Figure S7A and B ) . The localization of positively stained cells , and the essential role of HSCs in hepatic fibrosis prompted us to investigate whether the cells observed as IL-17R+ were in fact HSCs . By double-fluorescence staining , we confirmed that the IL-17R was indeed expressed on HSCs ( Figure S7C ) . We further found that a few mDCs also expressed IL-17R ( Figure S7D ) . These results indicate that HSCs and mDCs might be the potential target cells of IL-17 in the liver of hepatitis B patients .
Proper inflammatory response is crucial to the positive outcome of patients infected by hepatitis B virus [32] , [33] . The host IL-23 signaling pathway is critical to the immune response and is the target of manipulation by pathogens seeking to establish infection [34] , [35] . Here , we describe our investigations into the role of intrahepatic IL-23/IL-23R signaling during development of lethal or chronic hepatitis . We found that the IL-23/IL-23R pathway was strongly activated in liver tissues from HBV infected human patients , as compared with those from healthy controls . Moreover , we determined that in these patients IL-23 was principally derived from the intrahepatic myeloid DC population and macrophages . When stimulated by HBsAg and HBcAg in vitro , monocyte-derived DCs and macrophages produced large amounts of IL-23 . The effect for HBsAg was dependent on the availability and function of the surface MR , while the mechanisms underlying the HBcAg-induced effect remain unclear . After binding to MRs , HBsAg entered the cells via endocytosis , which subsequently induced the polarization of Th17 cells from naïve CD4+ T cells and IL-17 production . In addition , IL-17R expression was mainly detected on HSCs and a few mDCs in liver tissue , suggesting that these cells may be potential targets of IL-17 in hepatitis B patients . IL-23 is a heterodimeric cytokine composed of an IL-12 shared p40 subunit and a unique p19 subunit [9] , [10] , and it is primarily produced by APCs upon encounter with pathogen associated molecular patterns ( widely known as PAMPs ) [10] , [36] . In HBV infection patients , we found that IL-23 expression was largely elevated , as compared to that detected in the healthy controls , which agreed with the previous observations reported by Xia et al . in hepatitis B patients [5] . In the current study , the concentration of HBsAg in serum was found to be positively correlated with IL-23 expression for patients with hepatitis B . Confocal fluorescence microscopy further confirmed that mDCs and macrophages , and not pDCs or hepatocytes , were the main source cell types for IL-23 production . Surprisingly , Xia et al . had demonstrated that the hepatocytes were the main cell types for IL-23 production and that the HBx antigen acted as the active transcription factor for the IL-23 gene in liver cells [5] . These apparently incongruous findings may simply reflect the different experimental methods used in each study; for example , we used a fluorescence confocal microscopy strategy that may more directly show the source cells than the routine immunohistochemical analysis used by Xia et al . Furthermore , Xia et al . used an HBx-overexpressing plasmid-transformed liver cell line that may not reflect the real intrahepatic condition in hepatitis B patients as closely as the freshly isolated human cells or human liver tissues used in our study . It has been shown that the frequencies of both Treg and Th17 cells are higher in hepatitis B patients than in healthy controls [12] , [25] . In addition , Treg cells have also been shown to inhibit the function of Th17 cells by secreting key inhibitory cytokines , such as IL-10 , which suppress the function of Th17 cells by negatively regulating the production of Th17-related proinflammatory cytokines by down-regulating RORγt expression in PBMCs [26] , [27] . Therefore , in this study , we further investigated whether IL-10 and another key cytokine of Treg cells , TGF-β , could exert negative regulatory effects on Th17 cells by suppressing the production of IL-23 in mDCs , which is known to be a critical step in the differentiation and maintenance of Th17 cells [14] , [15] . In an in vitro system , we found that the exogenous IL-10 protein significantly inhibited the HBsAg-induced IL-23 production by mDCs; however , TGF-β addition did not influence the HBsAg-stimulated IL-23 production from mDCs . Recent studies have shown that IL-10 levels in hepatitis B patients are significantly higher than in non-infected individuals [37] , [38]; nevertheless , no significant differences were observed between chronic hepatitis B patients and healthy controls for the levels of TGF-β [38] . Notwithstanding the increased IL-10 level in hepatitis B patients , we observed high-level IL-23 expression and Th17 frequency in these patients , suggesting that other mechanisms likely exist to support Th17 function by antagonizing the suppressive effects of IL-10 during HBV infection in vivo . The primary pathogenic mechanism of HBV involves direct infection of hepatocytes . However , it is possible that HBV and its antigens may also be recognized and taken up by host immune cells , especially DCs and macrophages , which then provoke the anti-HBV immune responses . It is known that DCs can recognize HBV antigens by at least three different mechanisms [39] . First , HBV DNA binding to TLR9 on DCs activates the NF-κB signaling pathway that results in secretion of type I IFN and inflammatory cytokines , and induces DC maturation and the adaptive immune response [39] , [40] . Second , unlike HBc/HBeAg which seem to be recognized only by the B cells [41] , HBsAg appears to be able to be recognized by APCs , such DCs . While several putative binding factors have been described for this interaction , including asialoglycoprotein receptor and mannose-binding lectin , their exact role in HBV attachment to and uptake by DCs remains unclear [42] . In one of the more recent studies , the MR on intrahepatic DCs was shown to functionally interact with HBsAg [43] . Considering that macrophages also express MRs [24] , it is probable that these cells will be able to uptake HBsAg in a similar manner . In this study , we observed that blocking MR with MR-neutralizing antibody or blocking HBsAg with antibody obtained from healthy volunteers , who had received inoculation of preventive HBsAg vaccine and were HBsAb-positive , significantly suppressed HBsAg ( HBsAg protein or ACLF serum ) -induced IL-23 production by mDCs and macrophages . We further verified that the MR-mediated IL-23 production from mDCs and macrophages was dependent on an endocytosis mechanism because such stimulations were significantly decrease in the presence of inhibitors of endosomal acidification , such as ammonium chloride and chloroquine , or of the cell permeable dynamin inhibitor Dynasore that blocks cell endocytosis . Immunofluorescence assay further clearly showed that the cell surface of both DCs and macrophages express rich MR protein . HBsAgs could be efficiently internalized by mDCs and macrophages in an endocytosis and such internalization was MR-mediated because the internalization was almost completely blocked by pretreatment with chloroquine or MR-blocking antibody . Furthermore , we found that HBsAg almost completely co-localized with MR or the endosomal marker LysoTracker . This finding indicates that MR on DCs and macrophages not only acts as an endocytic receptor of HBsAg but also mediates an intracellular signaling response that is triggered upon encounter with HBV-related antigens [44] . Studies of fungal pathogens have shown that MR can induce production of a number of cytokines , including IL-8 , IL-10 , IL-17 , TNF-α and MCP-1 , as well as inhibit that of others , such as IL-12 and TNF-α [45] , [46] . However , how the downstream molecular signaling mechanisms of MR have not yet been defined because the MR cytoplasmic tail is devoid of any classical signaling motifs [45] . Accordingly , it has been theorized that MR requires functional interactions with other receptors , such as TLR2 and FcRγ , to trigger any signaling cascades [45] , [46] . As such , the role of MR in host immune response to a pathogenic infection represents an additional level of complexity to studies attempting to understand the cellular activation process . Moreover , we found that not only synthetically engineered HBsAg protein but also the ACLF patients' sera containing large amounts of HBsAg were able to directly induce mDCs and macrophages to secrete IL-23 protein in a dose-dependant manner in vitro . All these effects were reversed upon exposure to the antibody obtained from healthy volunteers who had received inoculation of the preventive HBsAg vaccine and were HBsAb-positive . These data demonstrated the specific effects of HBsAg on mDCs and macrophages , and , more importantly , provided clinical evidence of HBsAg function as a stimulator of IL-23 production from host cells . Although no definitive receptors for HBcAg have been reported to date , according to our data , HBcAg can stimulate DCs and macrophages to produce IL-23 in a MR-independent manner . The underlying mechanisms remain undefined , but may include: 1 ) binding to currently unknown HBcAg receptor ( s ) and triggering a downstream signaling pathway; 2 ) phagocytosis by phagocytes such as mDCs and macrophages , and thereby initiating intracellular signal transmission . Future studies should investigate these hypotheses . In addition , our results in this study , along with the observations from Zhang et al . [4] , verified that both Th17 cells and IL-17 are increased and correlated with the clinical phenotypes of hepatitis B patients . However , in order to understand the functions of the IL-23/IL-23R axis related to liver damage caused by HBV infection , the interplay between elevated IL-23/IL-23R and IL-17 in hepatitis B patients has to be clarified . It was found that mice deficient in IL-23p19 were resistant to experimental allergic encephalitis , and failed to produce IL-17 [47] . Meanwhile , Aggarwal et al . found that IL-23 was able to stimulate secretion of IL-17 from memory T cells in vitro [48] . It has also been demonstrated that IL-23 signaling is required for the terminal differentiation of activated T cells into functional effector Th17 cells in mice [15] . In humans , the factors involved in Th17 cell development and regulation are more complicated; however , among the potential factors , the critical role of IL-23 for the function of Th17 cells has also been observed in most studies ( reviewed in reference [49] ) . In the present study , we found that IL-23R in HBV-infected liver tissue was mainly expressed on IL-17+ cells , indicating the in situ IL-17 production being dependent upon the IL-23/IL-23R axis . Meanwhile , in the co-culture experiments , IL-23 derived from mDCs and macrophages was able to stimulate the secretion of IL-17 from CD4+ T cells in the presence of HBV antigen; however , IL-23-blocking antibody significantly decreased the production of IL-17 . Thus , these results firstly demonstrated that the IL-23/IL-23R axis was indispensable for the elevated IL-17 production that occurs in HBV infection cases . However , we cannot exclude potential roles for other factors , such as IL-1β and IL-6 in Th17 cell function because we found that blocking the IL-23/IL-23R axis did not diminish all of the IL-17 expression or completely eliminate Th17 cells from the system . IL-17 is considered the principal mediator of Th17 cell functions . In several experimental disease models , IL-17 binding to the IL-17 receptor on target cells has been shown to induce the expression and secretion of proinflammatory factors , such as IL-6 and IL-8 , which in turn mediate the inflammatory response [31] , [50] . Zhang et al . reported that IL-17R was uniquely expressed on peripheral monocytes and mDCs in CHB patients [4] . In vitro analysis in that study also revealed that IL-17 can activate mDCs and monocytes and enhance their capacity to produce proinflammatory cytokines . However , the authors did not detect IL-17R expression on the mDCs and monocytes from the liver tissue of CHB patients . This negative finding might simply be due to the technical difficulty associated with detecting IL-17R positive cells by using a chromogenic immunohistochemisty method . Even though we used the much more sensitive immunofluorescence method , we could only detect IL-17R expression on a few mDCs from the liver tissue of CHB patients . Nevertheless , we detected a substantial amount of IL-17R positive cells in the liver tissue , mainly within the fibrotic septa area , and these cells were subsequently identified as HSCs . We further verified that a few mDCs in the liver tissue of hepatitis B patients could also express IL-17R , suggesting that both HSCs and mDCs might be target cells of IL-17 and involved in the pathogenesis of hepatitis B disease . In conclusion , the findings from this study demonstrate that the IL-23/IL-23R axis is highly activated and closely correlated with Th17 cells , liver damage and clinical phenotypes of hepatitis B patients . The HBsAg binds to the MRs on mDCs and macrophages , enters cells by endocytosis , and finally efficiently stimulates those mDCs and macrophages to produce IL-23 . However , IL-10 , and not TGF-β , was able to decrease IL-23 production from mDCs in an in vitro system . In contrast , HBcAg could stimulate mDCs to secrete IL-23 via a yet unknown mechanism that functions in an MR- and endocytosis-independent manner . IL-23 induction of naïve CD4+ T cells to produce IL-17 facilitates the process by which this cytokine would be secreted and then bind to its cognate receptor on liver HSCs and liver mDCs , thereby contributing to the pathogenesis of hepatitis B disease . Collectively , the data presented in this study not only provide further insights into the mechanisms underlying HBV pathogenesis , but also suggest potential intervening targets for patient treatment .
A total of 166 HBV-infected patients , including 108 patients with CHB and 58 patients with ACLF , were enrolled in this study along with 62 healthy volunteers . CHB and ACLF were diagnosed according to the described criteria [4] , [20] . Exclusion criteria were: positive test results for HCV , HDV or human immunodeficiency virus; the presence of any concomitant illness; detection of serological markers of autoimmune disease . All included patients were hospitalized and followed-up in the Department of Infectious Diseases , Southwest Hospital , Third Military Medical University , China from December 2007 to December 2011 . The clinical characteristics of these patients and healthy controls are shown in Table 1 . Written , informed consent was obtained from all subjects prior to participation , and this study was approved by the ethics committee of the Third Military Medical University , Chongqing , China . IL-17 , IL-23 , IL-8 , TNF-α , IL-6 and IL-1β levels were determined by use of the Ready-SET-Go ELISA kit ( eBioscience , San Diego , CA , USA ) , following the manufacturer's instructions . PBMCs were isolated from 10 mL of heparinized blood by Ficoll-Hypaque density gradient centrifugation . Separation of monocytes and naïve CD4+T cells was carried out with immunomagnetic beads ( MiltenyiBiotec , Bergisch Gladbach , Germany ) . DCs were prepared by treating the monocyte population with GM-CSF ( 500 U/mL ) and recombinant human IL-4 for five days , and then treated with HBsAg , mannan , ammonium chloride , chloroquine , Dynasore ( Sigma-Aldrich , St . Louis , MO , USA ) , HBsAg-blocking antibody , or neutralizing anti-MR antibody ( clone 19 . 2 , BD ) . The recombinant HBsAg and HSA were prepared from Chinese Hamster Ovary ( CHO ) cells as previously described [51] . The purity of both HBsAg and HSA reached 99% , as determined by high performance liquid chromatography . HBsAg-blocking antibody ( 95% purity ) was provided by Dr . Qing Mao ( Department of Infectious Diseases , Southwestern Hospital , Third Military Medical University , Chongqing , PR China ) . Co-culture of mDCs and naïve CD4+ T cells ( 1 . 5∶1 ) was performed for seven days , and then the expression of IL-17 was detected by FCM or ELISA . The following antibodies used in FCM analysis were from eBioscience: Allophycocyanin-eFluor 780–conjugated anti-CD3; FITC-conjugated anti-CD8; PerCP-conjugated anti-IL-17A; PE-conjugated IL-23R . PBMCs were cultured with 50 ng/mL phorbol 12-myristate 13-acetate ( PMA ) and 0 . 5 µg/mL calcium ionophore III ( both from Sigma–Aldrich ) , in the presence of GolgiStop ( BD Biosciences Pharmingen , San Jose , CA , USA ) . Cells were then stained with surface markers , fixed and permeabilized with Cytofix/Cytoperm solution ( BD Biosciences Pharmingen ) , and finally stained with anti-IL-17A . Labeled cells were detected on a FACSAriaI digital cell sorter ( BD Biosciences Pharmingen ) and data were analyzed by FlowJo v . 5 . 7 . 2 ( TreeStar , Inc . , Ashland , OR , USA ) . PBMCs and liver tissues were dissolved in TRIzol reagent ( Invitrogen , Carlsbad , CA , USA ) and total RNA was extracted and subjected to RT-PCR using the PrimeScript RT reagents kit ( TaKaRa , Shiga , Japan ) according to the manufacturer's directions . qPCR was performed using the SYBR Premix Ex Taq polymerase ( TaKaRa ) . Gene-specific primers for human IL-17 , IL-23 , IL-23R , TNF-α and IL-8 were designed as shown in Table S1 . Fluorescence signals were measured after 40 PCR cycles , and all samples were normalized to GAPDH RNA content . All samples were run in duplicate . Total protein was extracted from liver tissue using T-PER Tissue Protein Extraction Reagent ( Pierce , Rockford , IL , USA ) and quantified with BCA Protein Assay ( Pierce ) . The proteins were separated by 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride ( PVDF ) membranes for immunoblotting . The membranes were incubated with goat anti-human IL-17 or IL-17R ( R&D Systems , Wiesbaden , Germany ) , mouse anti-human IL-23p19 ( BioLegend , San Diego , CA , USA ) or goat anti-human IL-23R ( Abcam , San Francisco , CA , USA ) for 1 h at room temperature . After incubation with peroxidase-conjugated rabbit anti-goat IgG , or rat anti-mouse IgG for 1 h at room temperature , specific protein bands on the membranes were visualized by the enhanced chemiluminescence method ( Amersham , Piscataway , NJ , USA ) according to the manufacturer's instructions . Liver tissues were embedded in paraffin using the standard protocol . Immunohistochemistry for human IL-17/IL-17R and IL-23/IL-23R was performed as previously described [52] with minor modifications . Briefly , masked antigens were retrieved by microwaving for 20 min in citrate buffer , pH 6 . 1 . After cooling , non-specific binding was blocked by incubation with 5% bovine serum albumin ( BSA ) in phosphate-buffered saline ( PBS ) for 30 min . Then , incubation was carried out with the primary antibodies to IL-17 ( Santa Cruz Biotechnology , Santa Cruz , CA , USA ) , IL-23 ( BioLegend ) , IL-23R ( Abcam ) and IL-17RA ( goat anti-human IL-17RA polyclonal IgG; R&D Systems ) for 48 h at 4°C in a humidified chamber; after which the slides were incubated with the secondary , biotin-conjugated antibody , followed by sequential incubation with horseradish peroxidase-streptavidin and the peroxidase substrate 3′-diaminobenzidine ( DAB ) . Slides were then counterstained with hematoxylin . For the double-staining of anti-IL-17 plus anti-CD4 , anti-γδTCR , anti-MPO , or anti-IL-23R , anti-IL-23p19 plus anti-CD11c , anti-CD303 , CD68 , or anti-hepatocyte and anti-IL-17RA plus anti-α-SMA , anti-IL-17 , anti-IL-23p19 antibody , the first antibody ( 1∶100 ) was applied and slides incubated for 48 h at 4°C in a humidified chamber . The second antibody anti-human CD4 , δTCR , MPO , IL-23R , CD11c , CD303 , CD68 , Hepatocyte or α-SMA ( 1∶100 in PBS containing 5% BSA ) was then applied for overnight at 4°C . TRITC- , FITC- or CY5-labeled anti-goat IgG ( 1∶500 in PBS ) and Dylight488-labeled anti-mouse or anti-rabbit IgG ( diluted 1∶200; Zhongshan Goldenbridge Biotechnology , Beijing , China ) were applied for 60 min at 37°C . Nuclear counterstaining was performed by incubating slides for 5 min with diaminidophenylindol ( DAPI , 1∶100 in PBS; Invitrogen ) . Normal mouse , goat or rabbit IgG was used as a negative control . Images were obtained by the digital confocal laser scanning system MRC-600 ( Bio-Rad , Hercules , CA , USA ) . The commercial-source human primary HSCs isolated from healthy human liver ( ScienCell , San Diego , CA , USA ) or the freshly prepared primary HSCs from liver tissue of ACLF patients who received surgical liver transplantation [53] were resuspended in HSC medium ( DMEM+10% fetal bovine serum ( FBS; Invitrogen ) +1% penicillin/streptomycin ) and plated on a 24 well non-tissue culture-treated plate . After overnight incubation , fresh culture solution with 5 µg/mL of IL-17R-blocking antibody or 10 ng/mL of rhIL-17 was added and the HSCs were cultured for an additional 48 h . The supernatants were subjected to ELISA to measure the levels of secreted cytokines IL-6 , IL-8 , TNF-α , and IL-1β . The virological assay was performed as previously described [54] . Comparisons between various groups were performed using the Mann-Whitney U test . Results are expressed as the mean ± standard error of the mean ( SEM ) , unless noted otherwise . Correlations between variables were evaluated using the Spearman's rank correlation test . For all tests , two-sided P<0 . 05 was considered significant .
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While it is known that IL-23 plays a pivotal role in maintenance of the Th17 phenotype and their production of the IL-17 cytokine , the mechanisms by which HBV induces particular immune cell types to produce IL-23 remain unknown . In the study of human hepatitis B described herein , we demonstrated that IL-23 is principally derived from the liver myeloid dendritic cells ( mDCs ) and macrophages . In vitro assay showed that mDCs produce large amounts of IL-23 upon stimulation with HBV surface antigen ( HBsAg ) through the mannose receptor ( MR ) and an endocytosis mechanism . In contrast , although the HBV core antigen ( HBcAg ) was also capable of stimulating IL-23 secretion from mDCs , the process occurs in an MR- and endocytosis-independent manner . IL-23 was also shown to efficiently stimulate the differentiation of naïve CD4+ T cells into Th17 cells in the presence of HBsAg or HBcAg; furthermore , the Th17 cells were shown to be the primary source of IL-17 . The results also indicated that both hepatic satellite cells and mDCs might be the potential target cells of IL-17 in hepatitis B disease . Therefore , our study not only provides further insights into the mechanisms underlying HBV pathogenesis , but also suggests the potential intervening targets for patient treatment .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"medicine",
"infectious",
"diseases",
"hepatitis",
"b",
"hepatitis",
"viral",
"diseases",
"gastrointestinal",
"infections"
] |
2013
|
Hepatitis B Virus Induces IL-23 Production in Antigen Presenting Cells and Causes Liver Damage via the IL-23/IL-17 Axis
|
A central question in cognitive neuroscience regards the means by which options are compared and decisions are resolved during value-guided choice . It is clear that several component processes are needed; these include identifying options , a value-based comparison , and implementation of actions to execute the decision . What is less clear is the temporal precedence and functional organisation of these component processes in the brain . Competing models of decision making have proposed that value comparison may occur in the space of alternative actions , or in the space of abstract goods . We hypothesized that the signals observed might in fact depend upon the framing of the decision . We recorded magnetoencephalographic data from humans performing value-guided choices in which two closely related trial types were interleaved . In the first trial type , each option was revealed separately , potentially causing subjects to estimate each action's value as it was revealed and perform comparison in action-space . In the second trial type , both options were presented simultaneously , potentially leading to comparison in abstract goods-space prior to commitment to a specific action . Distinct activity patterns ( in distinct brain regions ) on the two trial types demonstrated that the observed frame of reference used for decision making indeed differed , despite the information presented being formally identical , between the two trial types . This provides a potential reconciliation of conflicting accounts of value-guided choice .
Accounts of how the brain supports value-guided decision-making have been characterised as lying along a continuous spectrum [1] . At one end of the spectrum , it is argued that decisions are a serial process , in which stimuli are first perceived , then assigned values and fed to a subsequent decision stage where comparison takes place [2] , [3] . Evidence in favour of such a view comes from comparing the relative prevalence and timing of pre- and post-decision variables encoded during economic choice [4] , [5] . At the other end of the spectrum , decisions are framed as a parallel process , in which valuation , decision formation and action selection proceed simultaneously [6] , [7] . Such a hypothesis is supported by the representation of potential responses in motor regions prior to decision termination [8]–[11] , from probing the motor system behaviourally during the evolution of a decision [12] , [13] , and by comparing the relative timing of motor preparation responses in free- and forced-choice decisions [14] . The diversity of accounts is perhaps a symptom of value correlates being isolated in many different brain regions [15] – such as medial prefrontal [16]–[21] , parietal [22]–[24] , and motoric [25] , [26] structures – and also of the diverse frames of reference in which these value correlates have been found . For example , one prominent serial model of decision making proposes that value comparison occurs in the frame of reference of abstract goods , prior to the representation of choice [27] . This would most likely occur in regions such as orbitofrontal and ventromedial prefrontal cortex , where goods-space value correlates have been isolated [5] , [18] , [28] . By contrast , a prominent parallel model suggests that comparison may take place in the frame of reference of actions needed to obtain a certain outcome [7] . This comparison may occur in structures such as motor and premotor cortex , in which value-related neural signals tied to specific actions can be found [8] , [14] , [24] , [29] . It remains unclear whether decision processes occur serially or in parallel , and whether decision formation is principally resolved in action- or goods-space . It is possible that each account may be partially true , or that decisions are reached via a consensus between different systems [30] . One further reconciliation between the accounts might propose that the mechanism of decision formation might be task-dependent – that is , the frame of reference in which value-related signals are observed might depend upon both the framing of the decision and the way the data are analysed . Whilst both systems may still operate in parallel , the sensitivity to detect signals in a particular frame of reference might be strongly influenced by the task used – and so the differences between the tasks used across different studies might explain why goods-space signals are observed in some studies , and action-space signals in others . To test this hypothesis , we designed a task in which subjects faced two different , interleaved types of trial . Each trial type comprised formally identical decisions , but had information presented in a subtly different fashion . In the first type of trial ( ‘comparison’ trial , as reported in [5] ) , both options were presented simultaneously and subjects were free to respond at any time . Importantly , such a trial can be solved in several different ways . Decision formation could be carried out in the frame of reference of action values , tied to both left and right options , presumably in late motoric structures . Alternatively , it could occur in the frame of reference of abstract goods , presumably in frontal structures such as orbitofrontal or ventromedial prefrontal cortex ( VMPFC ) before undergoing a goods-to-action transformation . Although it is noteworthy that items are rarely encountered exactly simultaneously in nature , it is also true that such paradigms have been the norm in many studies of value guided choice [4] , [14] , [16]–[18] , [28] , [31] , [32] . We hypothesised , based on signals observed in these tasks [4] , [27] , [28] , that value-related signals might be observed in a goods frame of reference in this condition , and also that these goods value signals might be localised in structures such as VMPFC . In the second type of trial ( ‘sequential’ trial ) , each option was presented sequentially , with a delay between the presentation of the first and second option , and a further delay before subjects executed their response . There were thus two differences between this trial type and the ‘comparison’ trial: first , its sequential nature , and second , the additional delays before a response was allowed . Again , such a trial could either be solved in an action-space or a goods-space frame of reference . However , both sequential presentation of options and the imposition of a delay prior to response have previously been used in tasks where action value signals have been recorded [8] , [10] , [26] , [33] . Thus , we hypothesised that these two manipulations , although subtle , might push subjects towards a strategy of integrating information across probability and magnitude on each action as it is presented , and contribute towards the representation of a subjective value of making that action . This would suggest that the decision process could take place in the frame of reference of integrated action values [3] , [17] , [34] , or that decision formation might occur coincidentally with the planning of the action necessary to execute the choice [7] . Either of these possibilities would lead to value signals in an action-space frame of reference as the decision was being made , and these signals might be predicted to occur in later , motoric structures .
18 subjects completed 324 trials of each type , pseudorandomly interleaved , whilst undergoing magnetoencephalography ( MEG ) . In ‘comparison’ trials , both options were presented simultaneously , until response . In ‘sequential’ trials , each option was presented sequentially , with a delay before a response was allowed ( figure 1A ) . Subjects were not instructed to perform the task differently in each condition , except that in the sequential trials , they had to wait until the end of the delay period before they could respond ( see Materials and Methods ) . We first compared subject choice behaviour in comparison and sequential trials . We used logistic regression to test the influence of each option's reward probability and magnitude on subjects' choices during each type of trial ( figure 1B ) . Reward probability , reward magnitude and their interaction each had a highly significant influence on subject choices ( one-sample T-test on regression coefficients , all T ( 17 ) >4 . 6 , all p<0 . 0005 ) , but importantly there was no significant difference in these influences between the two types of trial ( paired T-test on regression coefficients between trial types , all |T ( 17 ) |<1 . 52 , all p>0 . 14 ) . There was a slight bias towards choosing the second presented option on sequential trials ( T ( 17 ) = 2 . 90 , p<0 . 01 ) , but no such bias towards choosing the left or right option on comparison trials ( T ( 17 ) = −0 . 17 , p = 0 . 87 ) . We also fit models from Prospect theory [35] , [36] to describe subject choice behaviour on both types of trial ( figure 1C–E ) . We fit a three-parameter model ( α to describe curvature in subjective reward magnitude weighting , γ to describe non-linearities in subjective probability weighting , and β to describe stochasticity in choice behaviour ) using maximum likelihood estimation . There was a strong correlation across subjects between α on sequential and comparison trials ( figure 1A; R = 0 . 84 , p<0 . 0001 ) , and similarly for β ( figure 1D; R = 0 . 75 , p<0 . 0005 ) , although no such correlation for γ ( figure 1C; R = 0 . 11 , p = 0 . 67 ) . ( This difference is potentially explained by the differing variances associated with the different parameters ( coefficients of variation: α , 0 . 51; β , 0 . 70; γ , 0 . 17 ) , which may imply that cross-subject variance in γ is primarily driven by noise in parameter fitting , rather than true variability in the population . ) Importantly , there was no significant difference between fitted parameters on the two trial types , except for a trend towards α being larger in comparison trials ( paired T-test , α: T ( 17 ) = 2 . 08 , p = 0 . 052; β: T ( 17 ) = 1 . 58 , p = 0 . 13; γ: T ( 17 ) = −0 . 956 , p = 0 . 35 ) . In summary , behavioural results indicated that , even if subjects were to have adopted a different strategy in solving the two types of trial , their resultant choice behaviour was very similar in sequential and comparison trials . In both trial types , subjects chose left and right options with left and right thumbpresses respectively , allowing us to investigate decision formation in the frame of reference of actions by interrogating the timecourse of lateralised responses in motor cortices . We first investigated lateralised responses in sequential trials . We localised motor cortex by performing a contrast of right minus left planned responses , 500–1000 ms after the presentation of the second option ( figure 2A ) . In the beta band ( 13–30 Hz ) , there was a greater degree of desynchronisation in the hemisphere contralateral to the planned movement ( i . e . left hemisphere desynchonisation was greater on trials where a rightward movement was planned ( peak T ( 17 ) = −5 . 59 ( Montreal Neurological Institute ( MNI ) coordinates = −36 , −34 , 54 mm ) , T ( 17 ) = −5 . 86 ( MNI = −50 , −34 , −54 ) whole-brain family-wise error corrected p<0 . 05 ) ) , and a lesser degree of desynchronisation in the hemisphere ipsilateral to the movement ( i . e . right hemisphere desynchronisation was lesser on trials where a rightward movement was planned ( peak T ( 17 ) = 4 . 63 ( MNI = 56 , 0 , 34 mm ) ) . This pattern of pre-movement beta desynchronisation is as would be expected from many previous studies of response selection [37]–[40] . Having localised this beta desynchronisation during movement preparation , we then investigated the temporal evolution of value correlates in the same region . In all analyses , we included the eventual categorical choice as a coregressor , to test whether signals were better predicted by value or by choice . At the time of option 1 presentation , beta desynchronisation ( in the hemisphere contralateral to the side option 1 was presented on ) was found to correlate with the value of this option; the higher the value , the more negative the beta power ( figure 2B ) . As shown in figure 2B/E , this signal first emerged approximately 500 ms after stimulus presentation ( significant cluster delineated by black line in figure 2B , tested via a cluster-based permutation test that corrects for multiple comparisons across time and frequency ( see Materials and Methods ) ) . It remained in the region throughout the delay period ( in which the option was removed and replaced with a central word ‘OR’ ) ( peak T ( 17 ) = −3 . 83 , t = 1975 ms post-stimulus presentation , 14 Hz; cluster-corrected p<0 . 05 , permutation test ) . The negative coefficient of the value correlate ( shown in figures 2B and 2E ) reflects increased desynchronisation [26] , [37]–[40] in the beta band at the time of option 1 . At the time of option 2 presentation , there was a negative correlate of the difference in value between the option contralateral to the hemisphere and the option ipsilateral to the hemisphere ( figure 2C ) , with a significant cluster centred around 400 ms post-stimulus presentation; the greater the value difference between contralateral and ipsilateral options , the more negative the beta power ( peak T ( 17 ) = −3 . 91 , t = 325 ms post-stimulus , 23 Hz; cluster-corrected p<0 . 05 ) . Such a signal is a value difference signal , but importantly it is tied to the frame of reference of a specific action ( contralateral vs . ipsilateral movement ) , rather than the frame of reference of which option will be chosen on the current trial . ( It is notable that part of this signal may be driven by the value of option 1 , which is known prior to option 2 presentation – and so the effect of value difference may arise much earlier than when analysed time-locked to option 1 presentation . Indeed , when split into the separate subcomponents , option 1 influenced beta desynchronisation earlier than option 2 ( see figure S1 ) ) . Using the regressor for categorical choice , we also identified a signal reflecting the categorical commitment to a rightward or leftward action ( figure 2D ) in the same region of interest , with beta desynchronisation being more negative when choices ( button presses ) were made to the side contralateral to the hemisphere than to the side ipsilateral to the hemisphere . Such a finding is unsurprising , as the region of interest was selected on the basis of differential beta desycnchronisation on left vs . right buttonpresses . However , the critical test is the timing of this categorical decision signal ( figure 2D ) relative to the action value signals ( figure 2B/C ) . When the first option was presented ( figure 2E ) , beta descynchronisation was explained by the value of the contralateral option ( blue line ) over and above any possible variance that could be attributed to the eventual choice that the subject would make ( red line ) . This was because both value and choice regressors were included in the same multiple regression model , and whereas value correlates were significantly different from zero , choice correlates were not . From figure 2F , we see that in a similar multiple regression model , the categorical decision signal emerged prior to the time at which subjects were allowed to make their response , but after the value difference signal ( peak T ( 17 ) = −7 . 36 , t = 775 ms post-stimulus , 18 Hz ) . This suggests a transition from initially representing action value difference , to subsequently representing categorical choice . We formally compared the relative timing of these two signals by comparing the time of the peak T-statistic in each subject for the two signals ( figure 3 ) ; this confirmed that the value-related signal preceded the categorical decision signal ( paired T ( 17 ) = 2 . 14 , p<0 . 05 ) . The relative timing of these value-related and categorical choice signals may reflect two possibilities . It may suggest that in sequential trials , late motoric structures directly support the comparison of values tied to specific actions . Alternatively , it may be that an evolving decision process taking place in other cortical structures is continually biasing action preparation or planning in motor cortex . In either case , it is clear that value correlates are present in motor cortex before a categorical decision has been reached . We next investigated whether similar value signals could be seen prior to the representation of choice in comparison trials . Again , we found that 500–1000 ms after the decision was presented , there was a differential response for right versus left buttonpresses , with less beta band desynchronisation in the right hemisphere for rightward than for leftward movements ( figure 4A; peak T ( 17 ) = 11 . 42 , MNI = 28 , −14 , 54 , voxelwise whole brain corrected p<1*10−5 ) . When searching for a correlate of the value of the options contralateral versus ipsilateral to the hemisphere , we timelocked to the response rather than the stimulus , as in this condition responses occurred at varying latencies rather than a fixed delay – and so , because reaction times correlate negatively with value difference [5] , beta desynchronisation that was in fact associated with responses made at different latencies would give rise to spurious correlations with value . Using this analysis , we found that there was no correlate of the difference in value between the option contralateral and the option ipsilateral to the hemisphere in the beta band , nor indeed in any frequency band from 1–40 Hz ( figure 4B ) . By contrast , consistent with figure 4A , there was still a strong correlate of the categorical choice , with beta desynchronisation being more negative when choices were made to the contralateral side than to the ipsilateral side ( figure 4C ) , peaking near the time of the response ( peak T ( 17 ) = −5 . 46 , t = 175 ms post-response , 26 Hz ) . Thus , on comparison trials , in contrast to the sequential trials , there was a categorical representation of choice but no lateralised representation of action value prior to the formation of the decision . On both sequential and comparison trials , we also found a similar set of signals emerged if we examined activity in lateral pre-motor , rather than primary motor , cortex ( figure 5 ) . The absence of an action-value signal in comparison trials is a negative result , and so might be interpreted as a consequence of insufficient statistical power . To demonstrate that this was not the case , we used a formal interaction test ( described below ) and found a significant difference in action-value signals between the two conditions . We then searched for response-locked correlates of value on comparison trials in ventromedial prefrontal cortex ( VMPFC ) , a region we have previously identified as playing an important role in value comparison on these trials [5] , [16] . We analysed data from the same region of VMPFC identified in our previous study [5] , in which we found that ( stimulus-locked ) there was a temporal evolution from a representation of overall value to value difference in low frequencies ( 2–10 Hz ) . The location of this region of interest ( MNI = 6 , 28 , −6 mm ) also lies within a cluster of activations identified in a recent meta-analysis of human functional MRI studies of value-guided choice [19] . We hypothesised , based on signals observed in other studies of this region , that it would not encode value in the frame of reference of actions , but of choice [5] , [16] , [18] , [32] – which might be the result of a comparison occurring in ‘goods space’ [27] , [28] . Based on our previous work [5] , we also hypothesised that this region might particularly encode value on ‘harder’ trials , in which probability and magnitude advocate opposing choices , but not on ‘nobrainer’ trials , in which both probability and magnitude were both larger on the same side than on the other . Critically , we note that these harder trials are precisely those on which a comparison of attribute differences might be necessary to resolve the decision . On harder comparison trials , there was a positive correlate of the difference in value between chosen and unchosen options in the beta band approximately 750 ms prior to the response ( figure 6A; peak T ( 17 ) = 4 . 05; t = 975 ms pre-response; F = 10 Hz ) . This value difference signal is in a different frame of reference to that isolated in primary motor cortex: it is not tied to the frame of reference of one or other specific action , but instead to the choice that is to be made . Importantly , when split into its subcomponents , this ‘goods-value’ signal contained both a positive correlate of the value of the chosen option and a negative correlate of the value of the unchosen option ( figure 6D ) . Again , we formally compared the relative timing of this value-related signal in VMPFC to that of the categorical choice signal in motor cortex , by extracting the peak T-statistic for each signal in each subject ( figure 7 ) ; we found that the VMPFC value related signal preceded the categorical motor signal ( paired T ( 17 ) = 2 . 25 , p<0 . 05 ) . In contrast , on ‘nobrainer’ comparison trials , we found no significant correlation in VMPFC with the value difference between the chosen option minus the value of the unchosen option , nor of the subcomponents of this signal ( figure 6B/E ) . However , this finding was complemented by signal in the posterior superior parietal lobule ( pSPL ) , a region isolated in our previous study as showing similar dynamics to VMPFC in lower frequency bands ( 2–10 Hz ) , but across both harder and nobrainer trials [5] . In the beta band ( 13–30 Hz ) , pSPL showed a synchronisation that correlated positively with chosen-unchosen value across both harder ( figure 8A/D ) and nobrainer ( figure 8B/E ) trials , consistent with our previous study . Finally , both VMPFC and pSPL also showed no correlation of chosen-unchosen value ( or the separated subcomponents ) on harder sequential trials ( figure 6C/F; figure 8C/F ) , or nobrainer sequential trials . This finding is particularly important , as it suggests that when values are represented in ‘action space’ as a choice is being made ( as was the case in sequential trials ) , there was no longer a detectable ‘goods space’ comparison in these regions . As before , we note that this is a negative result , and so we test it by comparing the strength of goods-value signals in each trial type formally below . We were also unable to detect any action value signal or categorical choice signals ( equivalent to those observed in motoric structures above ) in VMPFC or pSPL ( figures S2 and S3 ) . Finally , we formally compared the effect of value in VMPFC and motor cortex across the two trial types . In each subject , we extracted the peak T-statistic for the effect of value difference between the options in the ipsilateral and contralateral hemispheres from motor cortex , and the effect of value difference between chosen and unchosen options in VMPFC . We restricted our analysis to the beta band ( 13–30 Hz ) , focussing on the period from 2nd stimulus onset up to 1 s post-stimulus on sequential trials , and from 1 s pre-response until response time on comparison trials . As the same frequency range was examined in each region/condition , and the regions of interest were isolated via orthogonal contrasts , this analysis was protected against circular inference [41] . A repeated measures ANOVA with independent variables of brain region ( VMPFC/motor cortex ) and trial type ( comparison/sequential ) revealed a significant interaction between these two variables on the peak effect of value ( F1 , 17 = 7 . 29 , p<0 . 02 ) . Post-hoc T-tests revealed that there was a significantly greater effect of chosen-unchosen value in VMPFC on comparison trials than on sequential trials ( paired T ( 17 ) = 2 . 42 , p<0 . 05 ) , and a slightly greater effect of ipsilateral-contralateral value in motor cortex on sequential trials than on comparison trials ( paired T ( 17 ) = 1 . 83 , p<0 . 05 one-tailed ) .
Conflicting accounts of value-guided choice have proposed that decision formation is either supported principally by comparing the value of alternative goods , or by the comparing the value of alternative actions . In the present study , we isolated evidence in support of both accounts , but in two distinct types of trial – one ( comparison trials ) in which goods-space value comparison signals were more readily apparent , and another ( sequential trials ) in which action value-space signals were found . These findings therefore present a possible reconciliation of the two accounts – that the brain adaptively adopts the strategy most appropriate to the current context . The hypothesis that different tasks may be solved in different frames of reference may help to resolve apparently discrepant findings from previous studies in the literature . In one set of studies examining single unit activity during an economic choice task , Padoa-Schioppa and colleagues have identified dissociations between activity in orbitofrontal cortex ( OFC ) and anterior cingulate cortex ( ACC ) . In this task , OFC neurons encode both pre- and post-decision variables , but not in the frame of reference of actions [28] . By contrast , ACC neurons encode solely post-decision variables , and are modulated by movement direction [4] . This has led to the hypothesis that in this task , items ( here quantities of fruit juice ) are compared in an abstract ‘goods space’ in OFC/VMPFC , before undergoing a goods-to-action transformation in ACC in order to implement the required action to obtain that item [27] . This hypothesis gains support from the presence of post-decision ( chosen value ) signals in VMPFC in a task in which a stimulus value-based comparison is made , but the action needed to implement the decision is not yet known [18] . On the other hand , it appears that when subjects are presented with tasks that can only depend upon learnt action values rather than stimulus values , then the structure critical for value-guided choice may change , with lesions to ACC and not OFC affecting behaviour [31] , [42] . In even simpler forced-choice trials , on a task that does not require integration of information across multiple dimensions , there appears to be a temporal evolution from the initial coding of option values to the subsequent coding of action-related signals within relatively late , motoric structures , such as the supplementary eye fields [43] . In experiments where multiple possible actions are presented and held in working memory prior to a decision cue , enhanced representations of these actions can be seen prior to the decision in premotor cortex [8] . Similarly , when a free choice is made between alternative arm movements , evidence for a competitive decision mechanism ( in the frame of reference of actions ) is found in the parietal reach region [44] . Thus , in tasks where decisions in stimulus space or goods space are favoured , then neural correlates of the decision process is found in a stimulus- or goods-related frame of reference , whereas in tasks more closely tied to the comparison of different actions , correlates of the decision process appear in an action frame of reference . This observation unifies apparently discrepant findings as to the precise locus of decision-making processes in the brain . On the other hand , the differing signals across the two trial types may not be a reflection of different neural mechanisms of choice being used in each context , but instead differential sensitivity to one or other mechanism in our analysis . For instance , it is possible that action value signals are present in motor cortices in all trials , but without a delay period they become too transient to be detected . Similarly , it is possible that the relatively weak sensitivity of MEG to deep anterior structures such as VMPFC [45] means that on sequential trials , any value comparison process that takes place over a space of several seconds is too weak to be detected . Future studies may address these questions by direct invasive recording from these structures , across different conditions . Our findings from the sequential trials suggested one of two possibilities . One interpretation is that these trials were solved using a comparison of action values , as demonstrated by the transformation from a lateralised action value signal into a categorical choice signal in motor cortex beta band oscillations . An alternative account is that this signal is better interpreted as a ( graded ) motor planning signal , but there was a continual updating of this plan as a consequence of value comparison taking place elsewhere . Here , it is perhaps telling that many signals that have been interpreted as the intention to move by one set of researchers [46] have been related to decision-related signals by others [22] . In either case , these results have additional implications for our understanding of the role of motor cortex beta band oscillations in action selection . Whereas early accounts of these oscillations suggested that they might reflect an ‘idling rhythm’ [47] , more recent suggestions have proposed that beta desynchronisations may reflect a change in the current sensorimotor set or status quo [38] , or an increased likelihood of generating a novel voluntary action [39] , [40] . Such proposals align with a role for decreases in beta band activity during response preparation , an idea corroborated by recent findings that lateralised beta reflects the accumulation of evidence for a leftward or rightward response during perceptual discrimination [9] . By contrast , a recent study has highlighted that lateralised beta band desynchronisation reflects the evidence for a particular response , rather than response preparation per se , whilst integrating evidence to make a decision [26] . The current findings on ‘sequential’ trials suggest a similar role for beta desynchronisation , as evidenced by the correlation with action value above and beyond any correlation with the categorical response that is going to be effected on a given trial ( figure 2 ) . On comparison trials , we found that in the beta band , value comparison signals emerged in VMPFC ( figure 6 ) that preceded categorical choice signals in primary motor cortex . Critically , we found that such signals were present on trials in which magnitude and probability advocated opposing choices ( ‘harder’ trials ) , but not on trials in which they both advocated the same choice ( ‘nobrainer’ trials ) . Such trials are those on which conflict between the two attributes comes into play , and attention must be guided to the attribute that is most salient for determining the current decision . Notably , this was not the case in the posterior superior parietal lobule ( figure 8 ) , in which goods value difference signals were present on both ‘harder’ and ‘nobrainer’ trials . This replicates findings in lower frequency ranges ( 2–10 Hz ) from the same dataset [5] , and may reflect an important difference between VMPFC and parietal cortex when considering value-guided choices with multiple attributes . One further noteworthy difference between the signals observed in comparison and sequential trials is the relative timing of value difference and categorical choice signals in the two trial types . In sequential trials ( figure 3 ) , there was a median latency difference of approximately 200 ms between the peak of ( action ) value difference signal in motor cortex , and the peak of the categorical choice signal in the same region . By contrast , in comparison trials ( figure 7 ) , there was a median latency difference of around 500 ms between the peak of the ( goods ) value difference signal in VMPFC , and the peak of the categorical choice signal in motor cortex . Such differences would be expected if it were assumed that there is a temporal cost for translating signals in goods space into action space , and for conveying the results of computations from one brain region to another . It is important to note that there are two differences between the comparison and sequential trials – both the imposition of a delay prior to the response , and the sequential vs . simultaneous presentation of options . These differences were selected as they captured some of the key differences between previous paradigms in which goods and action value signals had been observed in previous tasks . It is , of course , completely reasonable that investigators have designed paradigms more like our ‘comparison’ trial type [4] , [14] , [16]–[18] , or like our ‘sequential’ trial type [4] , [27] , [28] – importantly , however , the signals they observe may lead them to different conclusions about the neural mechanisms of value-guided choice . Future work will be needed to refine precisely which of these two manipulations is most critical for pushing signals towards being found in one space or another . It is noteworthy , for instance , that in some experiments where options have been presented simultaneously but a delay is still imposed , goods-space value signals can still be isolated ( albeit using different measures of neural activity ) [16] , [48] . In previous fMRI studies of sequential choice [10] , VMPFC has been found to encode a goods value signal at the time of option presentation . At first sight , this appears discrepant with the absence of a goods value signal in VMPFC on our ‘sequential’ trials . Whilst a beta-gamma desynchronisation in VMPFC appeared to carry some information about the value of option 1 , this did not reach statistical significance ( figure S2 ) . It is important , however , to consider the differences between what computational processes are likely to be visible to fMRI and MEG recordings . We have previously demonstrated that the MEG signal during goods value comparison can be modelled by the dynamics of competition in an excitation-inhibition network ( EIN ) [5] . This suggests valuation signals visible to MEG reflect trial-to-trial variability in this dynamic , competitive process . By contrast , the relationship between EIN activity and the BOLD fMRI signal is more complex , but it is related not only to local processing , but also to afferent input to a brain region [49] . One potential reconciliation of these findings is therefore that a goods value ‘afferent input’ signal is always present in VMPFC , and so can be seen in VMPFC fMRI signal , even when comparison can be found to take place in later , motoric structures [10] . By contrast , in situations when VMPFC supports comparison of options in goods space , this local processing is witnessed in both MEG dynamics [5] and also in fMRI value difference signals [16] , [32] , [50] . In summary , we have here presented evidence that when performing two formally identical decision tasks , the temporal evolution of value-related and choice signals differs depending upon how the information is revealed to subjects . If the value of each action is revealed separately , decision signals appear in an action-based frame of reference , reflected by beta desynchronisations in motor cortex . If both options are presented simultaneously , and subjects have to integrate across dimensions to form their decision , decision signals appear in an abstract frame of reference ( chosen value minus unchosen value ) , reflected in beta synchronisations in VMPFC .
18 subjects ( age range 21–33 , 10 male , 8 female , recruited from the University of Oxford ) repeatedly chose between two risky prospects , comprising differing reward magnitudes ( represented by bar width ) and probabilities ( represented numerically ) , in order to obtain monetary reward ( figure 1A ) . The probabilities of winning on each option were independent; thus , on any given trial , both , neither or either option ( s ) might yield reward . Stimuli were drawn such that reward magnitude and probability were never identical across the two options; subjects therefore needed to integrate across stimulus dimensions to make optimal choices . On some trials , however , both probability and magnitude were larger on one side than the other , a decision we classify as a ‘no brainer’ . On comparison trials , decisions were presented onscreen until a response was made . On sequential trials , one option was presented for 800–1200 ms jittered , followed by a 200–400 ms jittered delay , then the second option for 800–1200 ms jittered; subjects could respond only after removal of the second option . Stimuli were presented on either side of a fixation point; subjects selected the left option with a left-thumb button press , and the right option with a right-thumb buttonpress . The difference between the two conditions was explained to the subjects in the instruction sheet thus: ‘For half of the decisions you have to make , you will see the screen as shown above ( in figure 1A ) . In these trials , simply respond as soon as you feel that you have made your decision . For the other half of the decisions you have to make , you will see the two gambles one after the other , and then be presented with a screen displaying only a question mark . In these trials , you must wait for the question mark to appear before responding . ’ On choosing a rewarded option , a ‘winnings bar’ displayed at the bottom of the screen increased in magnitude in proportion to the width of the chosen option . When this winnings bar reached a gold target on the far right of the screen , £2 was added to subjects' earnings , and the winnings bar reset itself to its original size . Total typical earnings for the task ranged from £26 to £34 . All subjects provided informed consent in accordance with local ethical guidelines . Subjective utility functions were derived from Prospect Theory [35] , and were of the following form:where ro and po are the reward magnitude and probability of gaining reward , respectively , on outcome o . The subjective expected value of outcome o was calculated as:The probability of choosing each option was then calculated using a softmax choice rule:where n is the number of options ( 2 for this study ) and τ is a temperature parameter that determines the stochasticity of action selection . Values of α , γ , and 1/τ ( inverse temperature , denoted by β in results section ) were fit by maximizing the likelihood of each subject's choices in the experiment , using non-linear fitting routines in MATLAB ( The Mathworks , Natick , MA ) , separately for sequential and comparison trials . As in [5] , we found that Bayesian Information Criteria ( BIC ) favoured Prospect theory over a simpler model that used objective probability and magnitude to compute expected value . A comparison between fitted parameter values in sequential and comparison trials is shown in figure 1C–E . The fitted values were used to calculate subjective expected values , which have been found to provide a better fit to neural data in value-guided decision tasks [51] , [52] , to use as trialwise regressors in analysis of MEG data . We used logistic regression to investigate the influence of p1-p2 ( probability difference between option 1 and 2 ) , r1-r2 ( reward magnitude difference ) and EV1-EV2 ( objective expected value difference ) on the probability of choosing option 1 ( see figure 1B ) . This was performed separately for each trial type . We normalised each explanatory variable before entry into the logistic regression ( to ensure that parameter estimates were comparable across the different variables ) , and included a constant term to model any bias towards choosing one option over the other . For each explanatory variable and each trial type , we then performed a one-sample T-test across subjects' parameter estimates , to infer which variables had a significant effect on choice behaviour . We also performed a paired T-test between parameter estimates for sequential and comparison trials for each explanatory variable , to infer whether any variables had a greater or lesser influence on behavior between the two trial types . MEG data were sampled at 1000 Hz on a 306-channel VectorView system ( Elekta Neuromag , Helsinki , Finland ) , with one magnetometer and two orthogonal planar gradiometers at each of 102 locations distributed in a hemispherical helmet across the scalp , in a magnetically shielded room . A band-pass filter of 0 . 03–330 Hz was applied during acquisition . Head position was monitored at the beginning of each run , and at twenty-minute intervals during each run , using four head position indicator ( HPI ) coils attached to the scalp . Data were acquired in two or three runs , with pauses between blocks to save data acquired . HPI coil locations , headpoints from across the scalp , and 3 anatomical fiducial locations ( nasion , left and right pre-auricular points ) were digitized using a Polhemus Isotrak II prior to data acquisition . Simultaneous 60-channel electroencephalography data was acquired using a MEG-compatible EEG cap ( ANT Neuro , Enschede , Netherlands ) , but is not discussed here . Vertical electrooculogram ( EOG ) and electrocardiogram were also measured to detect eye blinks and heartbeat , respectively . Stimuli were presented on a screen situated 1 . 5 meters away from the subject , inside the magnetically shielded room; stimuli were displayed via projector ( refresh rate 60 Hz ) situated outside the room . Stimulus presentation and timing was controlled using Presentation software ( Neurobehavioral Systems , Albany , CA ) . Magnetic resonance imaging ( MRI ) data for forward model generation were acquired using an magnetization-prepared rapid gradient echo ( MP-RAGE ) sequence on a Siemens 3T TRIO scanner , with voxel resolution 1×1×1 mm3 on a 176×192×192 grid , echo time = 4 . 53 ms , inversion time = 900 ms , recovery time = 2200 ms . External noise was removed from MEG data using the signal space separation method [53] , and adjustments in head position across runs ( detected using HPI ) were compensated for using MaxMove software , both implemented in MaxFilter version 2 . 1 ( Elekta Neuromag , Helsinki , Finland ) . Continuous data were down-sampled to 200 Hz and low-pass filtered at 40 Hz , before conversion to SPM8 format ( http://www . fil . ion . ucl . ac . uk/spm ) . Eye blinks were detected from the EOG channel ( EOG data was bandpass filtered at 1–15 Hz; local maxima lying more than 3 standard deviations from the mean were considered blinks ) . Detected eye blinks were used to generate an average eye blink timecourse , on which principle components analysis was run to obtain spatial topographies describing the average eye blink; these were regressed out of the continuous data ( as per [54] , without inclusion of brain source vectors as co-regressors; see http://www . fil . ion . ucl . ac . uk/~lhunt ( ‘Resources’ tab ) for an SPM-based tutorial ) . Data were epoched with respect to stimulus onset ( −1000 to 2000 ms around stimulus , with −200 to 0 ms pre-stimulus baseline ) , and button press ( −2000 to 1000 ms around response , again with −200 to 0 ms pre-stimulus baseline ) . Artifactual epochs and bad channels were detected and rejected via visual inspection , using FieldTrip visual artifact rejection routines [55] . All MRI processing and forward modelling was performed using SPM8 . MRI images were segmented and spatially normalized to an MNI template brain in Talairach space; the inverse of this normalization was used to warp a cortical mesh derived from the MNI template to each subject's MRI space [56] . Digitized scalp locations were registered to head model meshes using an iterative closest point algorithm , to affine register sensor locations to model meshes [56] . Forward models were generated based on a single shell using superposition of basis functions which will approximately correspond to the plane tangential to the MEG sensor array [57] . The forward models are implemented in FieldTrip's forwinv toolbox [55] . Source reconstruction was carried out using linearly constrained minimum variance ( LCMV ) beamforming [58] adapted for use on Elekta Neuromag data by using variance normalization between ( magnetometer and planar gradiometer ) sensor types , and dimensionality reduction to 64 spatial principal components [59] . This was used to reconstruct data to a grid across MNI space , sampled with a grid step of 7 mm . Full details of the beamforming approach used are given in [5] . The sensor covariance matrix was estimated separately for stimulus-locked and response-locked data using data pass band-filtered between 1 and 40 Hz , and 0% regularization . In a preliminary whole-brain analysis , we looked for areas with greater beta power ( 13–30 Hz ) on trials where the right button was pressed than on those where the left button was pressed , 500 ms–1000 ms after the last stimulus was presented ( i . e . after option 2 was presented in sequential trials; after both options were presented in comparison trials ) . We performed this contrast at each of the beamformed voxels to produce a whole brain image , sampling the brain with a 7 mm gridstep . We then performed a one-sample T-test across subjects to produce the T-statistic images shown in figure 2A/3A ( upsampled to 2 mm isotropic for display purposes ) . Inference was performed using a threshold of p<0 . 05 corrected voxelwise under assumptions of Gaussian Random Field theory . We then beamformed data to the peaks from this analysis , and to a VMPFC peak identified in a previous paper [5] , to perform time-frequency regression in order to test for correlates of value in these areas . We used multiple regression to estimate the contribution of the value of each option and the response made to power in each frequency band at each timepoint through the decision . In the sequential trials , at the time of option 1 presentation ( figure 2B/E ) , we included the value of this option as the regressor ( and searched in contralateral M1 for responses ) . The full regression model at each timepoint and frequency band therefore consisted of three terms – a constant ( β0 in regression model below ) , the effect of the value of option 1 ( β1 below ) , and a categorical term reflecting which option was chosen ( β2 ) . At the time of option 2 presentation ( figure 2C/F ) , we included the ( action-space ) value difference between contralateral and ipsilateral options ( and calculated the differential response in contralateral minus ipsilateral M1 ) . The full regression model consisted of four terms – a constant ( β0 ) , the value of contralateral ( β1 ) and ipsilateral ( β2 ) options , and a categorical term reflecting which option was chosen ( β3 ) . The effect of action value difference was estimated by performing a contrast of parameter estimates for β1 and β2 . In the comparison trials , we performed the same action-space analysis in M1 ( figure 4B ) ; and a goods-space analysis in VMPFC , in which we included the value difference between chosen and unchosen trials ( figure 6A/B/D/E ) , separately for harder and nobrainer trials . Again the full regression model consisted of four terms – a constant ( β0 ) , the value of chosen ( β1 ) and unchosen ( β2 ) options , and a categorical term reflecting which option was chosen ( β3 ) . The effect of goods-space value difference was estimated by performing a contrast of parameter estimates for β1 and β2 . We also performed the same analysis for harder sequential trials ( figure 6C/F ) . Importantly , in all regressions , the inclusion of the final decision regressor as a covariate allowed us to isolate the variance that could be explained by value independent of choice . Value regressors were normalized prior to regression , so they occupied a similar range of values across subjects . At each trial , the source-reconstructed data d ( ri ) was decomposed into 40 time-frequency bins linearly spaced between 1 and 40 Hz , by convolving the data with Morlet wavelets ( Morlet factor 5 ) [60] . This yielded , at each trial tr , frequency f , and timepoint t , an instantaneous estimate of the power at that frequency . Linear regression was then used to estimate the contribution of the n explanatory variables ( EV ) to this estimated power:where ε is the residual from the regression . The parameter estimates β1…n , normalized by their variances , were submitted to a group-level one-sample T-test to test for significant effects of each explanatory variable . For statistical inference on the effects of overall value and value difference on region of interest data , we performed a cluster-based permutation test at the group level . The logic of this permuation test is identical to that used in non-parametric statistical inference of cluster sizes in functional MRI and other MRI based analyses [61] . We generated 5000 randomly permuted T-statistics for each timepoint and frequency bin , by randomly sign-flipping the group design matrix 5000 times . We then thresholded each permutation's time-frequency decomposed T-statistic map at a threshold of T ( 17 ) >2 . 0 , and measured the maximum size of any cluster passing this threshold in the map , to build a null distribution of cluster sizes . We then compared the size of clusters from the true T-statistic map to those from the null distribution . We report clusters at a significance level of p<0 . 05 , corrected for multiple comparisons across time and frequency .
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There are several competing theories of how the primate brain supports the ability to choose between different opportunities to obtain rewards – such as food , shelter , or more abstract goods ( e . g . money ) . These theories suggest that the comparison of different options is either fundamentally dependent upon regions in prefrontal cortex ( in which representations of abstract goods are often found ) , or upon motoric areas such as pre-motor and motor cortices ( in which representations of specific actions are found ) . Evidence has been provided in support of both theories , derived largely from studies using different behavioural tasks . In this study , we show that a subtle manipulation in the behavioural task can have profound consequences for which brain regions appear to support value comparison . We recorded whole-brain magnetoencephalography data whilst subjects performed a decision task . Value comparison-related 13–30 Hz oscillations were found in ‘goods space’ in ventromedial prefrontal cortex in one trial type , but in ‘action space’ in pre-motor and primary motor cortices in another trial type - despite information presented being identical across trial types . This suggests both decision mechanisms are available in the brain , and that the brain adopts the most appropriate mechanism depending upon the current context .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2013
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Trial-Type Dependent Frames of Reference for Value Comparison
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Few studies have investigated the relative influence of individual susceptibility versus household exposure factors versus regional clustering of infection on soil transmitted helminth ( STH ) transmission . The present study examined reinfection dynamics and spatial clustering of Ascaris lumbricoides , Trichuris trichiura and hookworm in an extremely impoverished indigenous setting in rural Panamá over a 16 month period that included two treatment and reinfection cycles in preschool children . Spatial cluster analyses were used to identify high prevalence clusters for each nematode . Multivariate models were then used ( 1 ) to identify factors that differentiated households within and outside the cluster , and ( 2 ) to examine the relative contribution of regional ( presence in a high prevalence cluster ) , household ( household density , asset-based household wealth , household crowding , maternal education ) and individual ( age , sex , pre-treatment eggs per gram ( epg ) feces , height-for-age , latrine use ) factors on preschool child reinfection epgs for each STH . High prevalence spatial clusters were detected for Trichuris and hookworm but not for Ascaris . These clusters were characterized by low household density and low household wealth indices ( HWI ) . Reinfection epg of both hookworm and Ascaris was positively associated with pre-treatment epg and was higher in stunted children . Additional individual ( latrine use ) as well as household variables ( HWI , maternal education ) entered the reinfection models for Ascaris but not for hookworm . Even within the context of extreme poverty in this remote rural setting , the distinct transmission patterns for hookworm , Trichuris and Ascaris highlight the need for multi-pronged intervention strategies . In addition to poverty reduction , improved sanitation and attention to chronic malnutrition will be key to reducing Ascaris and hookworm transmission .
The soil transmitted helminth ( STH ) infections , Ascaris lumbricoides , Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus are estimated to result in the loss of 39 million disability adjusted life years ( DALYs ) annually [1] and to have long-lasting implications for child physical and cognitive development [2]–[5] . To control the morbidity associated with these infections for high risk populations living in endemic communities , current public health programs focus on chemotherapy [6] through programs typically delivered to school children [7] . Although there is evidence from theoretical models [8] and community studies [9] that such efforts have spill-over effects that reduce transmission in the untreated portions of the population , ensuring control of infections in preschool children is particularly important given the impact of STH on early growth and development [5] , [10] . Many studies have shown that the individuals most heavily infected prior to anthelmintic treatment have a high reinfection burden , and thus that some individuals are predisposed to heavy infection [11] , [12] . Mechanisms used to account for this predisposition include genetic and nutritional components of susceptibility , behavioural patterns that directly promote contact with infective stages and factors that promote egg/larval survival in some domestic and peri-domestic environments . Susceptibility may be related to differences in the genetic regulation of B cell activation and immunoglobulin secretion [13] , and to poor nutritional status [14]–[16] through the impaired immune function that accompanies micro and macronutrient deficiencies [10] . Behaviours including geophagy [17] , [18] , poor hygiene [19] and not wearing shoes [20] reportedly increase an individual's risk of contact with eggs or larvae . Household level factors associated with poverty [21] , such as limited latrine access [16] , [21] , [22] , within household crowding [23] , [24] and low maternal education [21] , [25] , [26] increase environmental contamination with STH eggs and larvae and contribute to the patterns of household aggregation detected in community-level epidemiological studies [27] , [28] . It has also been shown that STH infections cluster at the regional and national scales [29] , [30] . Such clusters have been associated with biophysical and climatic features such as vegetation cover [31] , [32] , soil type [32] , [33] , rainfall , temperature and altitude [29] , [31] , [34] that influence egg and larval survival , and also with limited sanitation and hygiene infrastructure that promote transmission [35] . Given the challenge of disentangling the influence of individual , household and regional risk factors of STH transmission , spatial analysis is becoming more widely used in epidemiological studies . Three recent investigations included these methods and highlighted the relative importance of household-level socio-economic ( SES ) factors . In Uganda , a cross-sectional survey found that household variables influencing exposure play a greater role than host genetics in determining the distribution of hookworm infection intensity [36] . A longitudinal study assessing hookworm post treatment reinfection rates in Brazil found child sex and SES variables such as household construction were more influential than regional geographic variables such as rurality , altitude and soil moisture [37] . Another longitudinal study , in Bangladesh , found that household exposure risk factors accounted for more than half of the variability in household clustering of Ascaris infection and that after accounting for household clustering , individual predisposition to infection was minimal [38] . Thus it is well established that SES variables are influential in determining the spatial distribution and transmission of STH infections [36] , [38] . However , few studies have the detailed individual data to investigate the relative influence of individual susceptibility due to chronic undernutrition versus household exposure variables on STH transmission within a context of regional clustering of infection in preschool children . Our study was designed to examine the influence of spatial patterns of infection as well as household and individual factors on the transmission dynamics of Ascaris lumbricoides , hookworm , and Trichuris trichiura during two sequential reinfection cycles in preschool children in an impoverished , rural region of Panamá . Specifically , we aimed to: 1 ) compare reinfection dynamics among the three STH; 2 ) identify and characterize regional scale spatial clusters of high prevalence of infection; and 3 ) determine the relative contributions of individual factors ( age , sex , height-for-age , pre-treatment intensity , latrine use ) , household factors ( household density , asset-based household wealth index , household crowding , maternal education ) and a regional factor ( residence in a high prevalence cluster ) on reinfection intensity of STH infections in preschool children .
Ethical approval was obtained from the Instituto Conmemorativo de Gorgas in Panamá and McGill University in Canada . The study was conducted in accordance with the Guía para Realizar Estudios e Investigaciones en los Pueblos Indígenas de Panamá , including initial and result-sharing workshops with participants in each village . Written informed consent was obtained from primary caregivers during a household visit that included an explanation of study significance and participant requirements and rights , as well as an opportunity to ask questions in Spanish and Ngäbere . The comarca Ngäbe-Buglé is a semi-autonomous political region inhabited primarily by the Ngäbe and Buglé indigenous groups ( 2004 population estimate of 128 , 978 ) where over 90% of families live in extreme poverty ( < US$1 . 75/day ) [39] . In 2005 and 2007 , two forms of the Conditional Transfer ( CT ) program Red de Oportunidades began in the comarca , providing an additional US$50/mo in either cash ( 2007 ) or food vouchers ( 2005 ) in exchange for participation in health and education programs . The research reported here is part of a larger study that was conducted in the district of Besiko in two adjacent corregimientos ( Soloy and Emplanada de Chorcha ) , each accessible most of the year by one dirt road . As previously reported [40] , household density varied considerably . More densely populated regions ( >50 participant households/km2 ) were closer to a road and had better access to latrines , aqueducts and health facilities and had a higher average asset-based household wealth index ( HWI ) . Our study was designed to estimate STH infection and reinfection in 2 treatment and reinfection cycles during a 16 month sample period ( Cycle 1: 9 month reinfection period from July 2008 to April 2009; Cycle 2: 6 month reinfection period from April to October 2009 ) . The study recruitment protocol has been described previously [40] . In brief , households with children from 0–48 mo of age and living in extreme poverty ( defined as having participated in a CT program ) from 12 randomly selected villages split evenly between the 2 corregimientos were invited to participate in the study . All but 3 of the 265 eligible households that were approached agreed to participate . Spatial data were unavailable for 12 households ( missing or erroneous longitude and latitude coordinates ) . The household and demographic characteristics of the 12 households for which spatial data were unavailable did not differ from the other 250 participant households . Thus , the present analysis included a total of 250 households and 356 children ( 153 households with 1 eligible child , 88 with 2 , and 9 households with 3 eligible children ) . Stool samples were collected at 7 household visits , and additional data from questionnaires and anthropometry were provided during 3 additional household visits . Temporary migration for agricultural purposes was common in the participant population and therefore few children were available at all household visits . Labelled collection containers and detailed instructions were given to each caregiver during household visits on the day prior to fecal sample collection . Samples were collected from the home the following morning and transported on ice to the Parasitology Laboratory at the Hospital General del Oriente , Chiriqui , Panamá . The primary outcome in this study was intensity of infection , measured as epg . Hookworms were not identified to species level , however , Necator americanus is the predominant hookworm in Central America [41] . In Cycle 1 , data on epg were obtained only from duplicate Kato Katz preparations [42] whereas in Cycle 2 , both Kato Katz and the FLOTAC techniques [43] were used . For each nematode ( Ascaris , Trichuris and hookworm ) , a comparison of the diagnostic ability ( presence/absence ) between FLOTAC and Kato Katz was conducted using Cohen's Kappa statistic on samples that were assayed using both techniques . Sensitivity of each method ( expressed as a percentage ) was also analyzed by dividing the number of positives for a given method by the total number of positives identified by either method . Finally , the correlation of intensity estimates between the two methods was examined using Spearman Rank correlation coefficients . All statistical comparisons were conducted using STATA 11 . 1 ( College Station , TX ) . In all cases , the level of significance was set at p<0 . 05 . Binomial confidence limits ( 95% ) for prevalence data were determined using the Agresti-Coull calculation and comparisons were conducted using contingency tables and X2 tests . Continuous data were reported as the mean ± SEM , unless otherwise stated . Univariate comparisons between households within and outside high prevalence clusters , as well as between infected and uninfected individuals at both the 9 mo ( Cycle 1 ) and 6 mo ( Cycle 2 ) reinfection sample periods , were conducted using non-parametric Mann-Whitney , Kruskal-Wallis and Spearman correlation analyses due to the non-normal distribution of the data ( e . g . maternal education , HWI , age , epg ) . Univariate analyses related to reinfection included only those children who had provided a 9 mo/6 mo reinfection sample and who had received treatment ( Cycle 1 , n = 155; Cycle 2 , n = 200 ) . Egg Reduction Rates ( ERR ) were calculated as the mean percentage reduction in epg [50] , using epgs from the Kato Katz for Cycle 1 and from FLOTAC for Cycle 2 . Step-wise logistic regression models were used to examine which household risk factors were associated with presence in the high prevalence clusters detected by the SaTScan software ( hookworm and Trichuris at baseline of Cycle 2 ) . Step-wise negative binomial regression models were also performed to determine the impact of three sets of risk factors on reinfection epgs: 1 ) regional factor , namely residence in a high infection cluster; 2 ) household factors , namely HWI , household density , household crowding , maternal education ) ; and 3 ) individual factors , namely age , sex , HAZ , predisposition measured by pre-treatment epg , latrine use ) . These analyses were done for Ascaris reinfection in both Cycle 1 and Cycle 2 and for hookworm only in Cycle 2 when the more reliable FLOTAC epgs were available . No regression models were developed for Trichuris reinfection because of the low drug efficacy . Final models included variables with p<0 . 10 . The Huber estimator for robust standard error estimation was used to account for clustering at the household level . Multivariate analyses were limited to individuals who had received treatment and provided baseline and 9 mo or 6 mo reinfection samples and for whom a complete set of data on risk factors were available ( Cycle 1 , n = 100; Cycle 2 , n = 140 ) .
Although there was significant concordance between the methods for detection of all three parasites ( Ascaris: k = 0 . 91 , p<0 . 001; Trichuris: k = 0 . 66 , p<0 . 001; hookworm: k = 0 . 52 , p<0 . 001; n = 604 ) , FLOTAC was more sensitive than Kato Katz in the detection of hookworm ( 93% vs 45% of known positive samples , respectively ) and Trichuris ( 97% vs 56% of known positive samples , respectively ) . Spearman rank correlation analysis of intensity estimates among samples identified as positive by both methods ( n = 196 ) revealed a positive correlation between methods for Ascaris epg ( r = 0 . 79 , p<0 . 001 ) , hookworm ( r = 0 . 57 , p<0 . 001 ) and Trichuris ( r = 0 . 51 , p = 0 . 003 ) . Comparison of the mean intensity of infection revealed that estimates of Ascaris intensity measured by Kato Katz were higher than those measured using FLOTAC ( Kato Katz: 18393±1906; FLOTAC: 5364±1017; p<0 . 001 ) but mean intensity estimates for hookworm and Trichuris did not differ by method ( Hookworm: Kato Katz: 3601±826; FLOTAC: 385±72; p = 0 . 16; Trichuris: Kato Katz: 3357±1249; FLOTAC: 227±32; p = 0 . 39 ) . Due to the greater sensitivity of FLOTAC to hookworm and Trichuris infection , the similar diagnostic ability of both methods for Ascaris and the larger number of samples examined by FLOTAC in 2009 , analysis of infection prevalence and intensity used FLOTAC estimates whenever available . Average density of participating households was 35±2 houses/km2 and the household wealth index ( HWI ) was 0 . 21±0 . 04 . Latrines were available to 31% of households and piped water ( aqueducts ) to 34% of households . Households had an average of 5 . 4±0 . 2 people/room and mothers had 3 . 8±0 . 2 yrs of education . The average age of participating children at baseline of Cycle 1 was 31 . 4±0 . 9 months , 49% were female , and 72% were stunted . At the beginning of Cycle 2 the average child age was 36 . 9±0 . 9 months , 49% were female and 69% were stunted . Child demographic data presented here relates to the subset of children who received treatment in each Cycle , which differ slightly , but not significantly , from the total population reported elsewhere [40] . Although our first objective had been to compare infection and reinfection dynamics among the three STH infections over 2 consecutive reinfection cycles , the absence of FLOTAC data during Cycle 1 limited our analysis only to Ascaris . Ascaris was detected in 20% of children prior to ABZ treatment in Cycle 1 ( Figure 1 A ) with relatively low mean intensity ( Figure 1 B ) . Treatment with ABZ had an Ascaris ERR of 100% in the subsample for whom drug efficacy was assessed . Within 3 mo the prevalence of Ascaris in those who had received treatment had increased to 8% , half that recorded at baseline; by 9 mo , 34% of the children were infected and intensity of reinfection was three times higher than baseline values . At the baseline of Cycle 2 , 19% of all individuals who provided samples were infected . ABZ efficacy against Ascaris was 97% . Despite the low prevalence of Ascaris at the end of Cycle 2 ( 11% ) , the intensity had increased to pre-treatment levels ( Figure 1 B ) . Hookworm prevalence was 5% at the baseline of Cycle 1 ( Figure 1 C ) , with a low average intensity ( Figure 1 D ) . Drug treatment with ABZ eliminated infection in the 3 infected children . By 3 mo post treatment , hookworm prevalence ( Figure 1 C ) and intensity ( Figure 1 D ) were similar to baseline levels and by 9 mo post treatment both metrics had exceeded baseline levels . In Cycle 2 , ERR was 89% and both prevalence and intensity had returned to pre-treatment levels within 4 mo of treatment . Trichuris prevalence was only 1% at the baseline of Cycle 1 ( Figure 1 E ) and therefore it was not possible to calculate ERR . Three months after treatment , the prevalence remained very low , but by 9 mo post treatment , Trichuris prevalence ( Figure 1 E ) and intensity ( Figure 1 F ) reached the highest values detected during the study . A single dose of ABZ at the beginning of Cycle 2 led to only a 40% ERR for Trichuris . The 4 and 6 mo post-treatment prevalence and intensity for Trichuris did not differ from Cycle 2 baseline values .
Our study builds on past work in this rural indigenous area of Panamá and further characterizes the epidemiology of STH infections . As shown by previous empirical studies [51] , [52] , theoretical studies [8] and a recent meta-analysis [12] of STH infection dynamics , we found that prevalence and intensity of infection returned to baseline levels following treatment . Our use of spatial analysis uncovered distinctions among STH infections . Whereas spatial clusters of Trichuris and hookworm were detected , and overlapped in a region characterized by poor development ( as measured by low HWI and low household density ) no spatial clustering was detected for Ascaris , indicating its more homogenous dispersion throughout the region . Our multivariate regression models revealed differences between Ascaris and hookworm in the relative impact of household and individual factors in driving reinfection dynamics . Neither regional nor household variables emerged in the model for hookworm , and after controlling for baseline epg , the only individual factor that emerged was HAZ . Reinfection models for Ascaris also included individual factors ( baseline epg , HAZ , age , sex ) in one or both reinfection cycles , as well as two household factors , maternal education and HWI . Interestingly , in Cycle 1 , reinfection rates were higher in households with low HWI whereas the opposite pattern was seen in Cycle 2 . Our first objective was to compare reinfection dynamics among the three STHs . Due to differences in the sensitivity of FLOTAC and Kato Katz for hookworm and Trichuris infection intensity estimates , we have focused our comparison between reinfection cycles primarily on Ascaris , for which both diagnostic methods had similar sensitivity . For Ascaris , the reinfection profile was similar in both years , with epgs reaching pre-treatment levels within 6–9 mo . In contrast , our data indicate that reinfection with hookworm and Trichuris , measured as the change in epg , was more rapid during Cycle 1 between 3 and 9 mo post-treatment than during Cycle 2 . Four factors may have contributed to this difference . First , Cycle 1 included the wet season ( October – January ) whereas Cycle 2 overlapped only briefly with the wet season . Indeed , more wet days [53] and soil wetness [54] are considered conducive to Ascaris transmission and hookworm larval abundance . Second , the reinfection period was longer for Cycle 1 ( 9 mo ) than Cycle 2 ( 6 mo ) . Third , the Conditional Transfer programs in the region had incorporated anthelmintic delivery through school based programs for at least 1 year prior to this study . A slower rate of reinfection in subsequent treatment cycles is characteristic of an area with effective control [55] , [56] even in the segments of the population not targeted by treatment programs [6] , [9] . Fourth , at least in the case of Trichuris , the low efficacy of ABZ precludes us from assuming that the 6 mo epg at the end of Cycle 2 is driven by reinfection . Over time , epgs reach an equilibrium determined by the Basic Reproduction Ratio of the parasite and the host and environmental characteristics of the region thus Trichuris epgs after ABZ treatment may have been close to this equilibrium . Of further note is that the three STH species did not demonstrate the expected reinfection dynamics . Based on the longevity of the STH species , Ascaris and Trichuris are expected to reach baseline intensity more quickly than hookworm [51] however in our study , hookworm reinfection intensity reached baseline levels in 4 mo compared to the 6 mo that it took for Ascaris and Trichuris infections to reach pre-treatment intensity . We suggest that the lower treatment efficacy of a single dose of ABZ for hookworm ( ERR = 89% ) compared to Ascaris ( ERR = 97% ) and the higher baseline prevalence of hookworm infection ( 21% ) compared to Trichuris ( 10% ) led to increased transmission of hookworm by increasing the number of hookworm infectious stages in the environment relative to Ascaris and hookworm and thus increasing the efficiency of transmission [55] . The low efficacy of ABZ against Trichuris has been noted in other studies [57]–[60] and , in this Trichuris population , may be related to the genetic polymorphisms associated with benzimidazole resistance that have been detected in Trichuris eggs from our study area [61] . ABZ may not be the most appropriate drug to use for Trichuris in this region , and its continued use , even if directed towards control of Ascaris and hookworm could lead to an increase in the frequency of these polymorphisms and greater evolutionary pressure toward ABZ resistance in Trichuris . Our second objective was to identify and characterize spatial clusters of high STH prevalence . Two intriguing observations emerged . First , the location of hookworm and Trichuris high prevalence clusters significantly overlapped and the clusters shared the common characteristic of low household density . In our study area , we have previously reported that low household density was associated with lower HWI , as well as being farther from roads and health centres [40] , and thus we have considered low household density as an indicator of poor regional development or “remoteness” . The relationship between “remoteness” and clusters of high prevalence of infection could also be linked to characteristics of the physical environment [32] , [33] , to high risk activities that occur in these areas [21] , [62] , and to the lack of sanitation and hygiene infrastructure [40] . Surprisingly , residence in a high prevalence cluster did not emerge as a risk factor for hookworm reinfection intensity . This could be explained because the greater isolation of houses in the high prevalence cluster might reduce contact of children with hookworm larvae in neighbouring homes , compared with more densely spaced homes . Alternatively , by controlling for baseline epg , we may have reduced the influence of the regional scale clustering of infection . Second , no high prevalence clusters of Ascaris were detected . Ascaris was the most prevalent parasite in our study area . The long survival and the stickiness of Ascaris eggs [51] may facilitate their dispersion through the environment leading to more evenly distributed egg exposure , and may explain the generalized infection throughout the regions . Our final objective was to compare the relative contribution of individual , household and regional factors in the transmission dynamics of Ascaris and hookworm . Predisposition to heavy infection is characteristic of STH infection and has been noted throughout the developing world [44] , [45] , [63] , [64] . Our multiple regression analyses on epg of individual children further supported these findings as pre-treatment epg was a strong predictor of reinfection intensity for Ascaris and may have also influenced hookworm reinfection epg . Mechanisms to explain predisposition include the influence of individual traits on susceptibility such as genetic differences in immunity [65] or nutritional status [10] , as well as household factors that increase exposure to infection [38] . After controlling for baseline epg , HAZ emerged as an individual factor in models of reinfection intensity for hookworm ( Cycle 2 ) and Ascaris ( Cycle 1 ) . Most longitudinal studies examining STH infection and anthropometric outcomes have focused on the potential benefit of anthelmintic treatment on weight or height gain [66] , [67] rather than the impact of undernutrition on child susceptibility to infection or reinfection . The few studies that have specifically examined the latter relationship have either not controlled for potential confounding factors [14] or have found that the increased rates of reinfection in undernourished children are no longer significant when controlling for maternal literacy , income and latrine access [15] , [16] . We found , however , that after accounting for poverty related factors ( maternal education , HWI , latrine use ) that may influence child height-for-age , children who were shorter for their age ( a sign of chronic malnutrition ) became more heavily reinfected and thus may be more susceptible to hookworm and Ascaris infection . The fact that HAZ was associated with Ascaris reinfection in Cycle 1 but not Cycle 2 was an intriguing finding , as was the finding that younger ( not older children ) had higher epgs . It is possible that the relationship between stunting and reinfection intensity is easier to detect in younger children , given that intensity of infection increases rapidly with age [51] , [52] , [68] . Child latrine use was also associated with a lower reinfection burden , demonstrating the importance of sanitation for reducing infection and transmission , as has been shown previously [16] , [21] . In addition to individual level variables , household factors also contributed to Ascaris reinfection . Household risk factors commonly associated with poverty ( low maternal education , low HWI ) were related to increased reinfection intensity in either or both reinfection Cycles . Indeed , low maternal education [21] , [25] , [26] is commonly associated with greater infection burdens , likely due to poor home sanitation and hygiene practices as well as a reduced use of health services [69] . Interestingly , household poverty was associated with increased infection burden in Cycle 1 but a decreased infection intensity in Cycle 2 . Examining the spatial location of infected households at the end of Cycle 2 , we determined that the infections were primarily in an area of greater relative wealth . Thus it is possible that risk of infection was greater in that area and it wasn't HWI per se that influenced Ascaris reinfection dynamics in Cycle 2 . Of further note is that although lower household poverty was associated with greater reinfection burden , other poverty related variables that may be more directly linked to transmission ( latrine use , maternal education ) still demonstrated the expected relationship with infection burden . It is important to recognize that this study had a few limitations . First , we do not have data on the history of treatment prior to the baseline of Cycle 1 although we know ABZ was available in the area . Second , seasonal work-related migration reduced the number of preschool children available at the end of Cycle 1 . Fortunately many children were at home three weeks later . Hence the 3 wk mean epg in Cycle 2 incorporates data from children who had just received Albendazole and children who had not been treated . Also , we were able to provide ABZ treatment at 3 wks to those who were infected but logistics prevented us from confirming the ERR following this treatment . We assume that the available ERR data can be extrapolated to all the children , and therefore that hookworm epgs were reduced significantly after the additional children were treated at 3 wks . Third , a single ABZ treatment did not remove all Trichuris and hookworm parasites . For the reasons noted above , we were unable to determine whether the second dose of ABZ successfully cleared infection . Fourth , we likely underestimated Trichuris and hookworm prevalence and intensity in Cycle 1 because we did not use the FLOTAC technique . Although this limited our ability to compare reinfection dynamics between Cycle 1 and Cycle 2 , we were still able to comment on the period of most rapid transmission by verifying the trends observed with Kato Katz data for Cycle 2 ( data not shown ) . The observed greater sensitivity of FLOTAC than duplicate Kato Katz thick smears for low intensity infections characteristic of hookworm and Trichuris has been recorded previously in validation studies [70]–[72] and is believed to be due to the increased volume of sample used in the FLOTAC method ( 1 g ) compared to Kato Katz ( 41 . 7 mg ) . Use of both techniques in Cycle 2 alerted us that hookworm and Trichuris were more common in the region than previously recognized and furthermore , highlighted the low ERR for Trichuris after a single treatment with ABZ . In contrast to previous studies , however , Kato Katz and FLOTAC had a similar sensitivity for Ascaris infection . FLOTAC epg estimates were consistently lower than those calculated using Kato Katz for all 3 STH . This has also been noted previously , and could indicate that the Kato Katz technique overestimates epg due to the egg concentration that may occur while sieving the fecal sample [71] , [72] . A potential consequence is the over estimation of drug efficacy through ERR when using Kato Katz . Previous work that compared ERR values between FLOTAC and Kato Katz in a drug trial [73] also found that FLOTAC estimates resulted in lower ERR values than Kato Katz . An additional contributing factor could be the ability to process diarrhetic samples using FLOTAC but not Kato Katz . However , we did not detect any difference in mean FLOTAC epg between diarrhetic vs non-diarrhetic samples . When validated these findings have implications for the monitoring of preventive control programs , in particular for the perceived success of chemotherapy as well as early detection of drug resistance . Finally , it is likely that the absence of clusters in Cycle 1 was due to the low prevalence of infection in that cycle ( Hookworm: Cycle 1 = 5% , Cycle 2 = 22%; Trichuris: Cycle 1 = 1% , Cycle 2 = 10% ) which limited our ability to detect clusters . Our study of STH reinfection dynamics in the comarca Ngäbe Buglé of Western Panamá has emphasized that even within regions of extreme poverty , clusters of STH infections exist and that transmission is related to household level exposure variables as well as individual factors that may influence susceptibility . Our results have specific implications for public health interventions . First , the lower treatment efficacy of ABZ for Trichuris together with high infection levels during Cycle 2 calls attention to the importance of monitoring drug efficacy , especially against Trichuris , as well as the possibility of using multiple treatments or an alternative anthelmintic . Second , improving both the regional and household level sanitation and hygiene environment will be necessary to further reduce STH transmission . This will be especially important for the growth and development of stunted children who may be more susceptible to hookworm and Ascaris infection . Taken together , comprehensive control programs that combine short term morbidity control with the development of long term economic capacity , sanitation infrastructure and improved food security are necessary to make lasting improvements in child health in the comarca Ngäbe Buglé .
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Control of soil transmitted helminth ( STH ) infections is of central importance to improving preschool child health because these infections can have long lasting consequences on growth and development . Our study in indigenous Ngäbe preschool children in western Panama was conducted over a period of 16 months . We monitored reinfection dynamics of three STH infections ( Ascaris , Trichuris and hookworm ) over two reinfection cycles to gain an understanding of regional , household and individual factors that influenced transmission of these infections among preschool children . Despite the rural setting , where virtually all households live under conditions of extreme poverty , we identified spatial clusters of high prevalence of Trichuris and hookworm in the most remote and poorest area , whereas Ascaris was present throughout the study area . Preschool children who were chronically malnourished ( low height-for-age ) had a higher reinfection burden of Ascaris and hookworm . Household poverty ( low relative household wealth and maternal education ) and infrequent latrine use were also influential in Ascaris reinfection . This cross-disciplinary analysis of preschool child STH transmission in a poor rural setting provides pertinent information for STH control programs that aim to break the cycle of poverty and infection .
|
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2013
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Regional, Household and Individual Factors that Influence Soil Transmitted Helminth Reinfection Dynamics in Preschool Children from Rural Indigenous Panamá
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Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages . Toll-like receptor 4 ( TLR4 ) is expressed on the surface of macrophages and recognizes Gram-negative bacteria . Moreover , TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria , but the mechanisms remain unclear . Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter , TRAM , is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus . We find that TRAM forms a complex with Rab11 family interacting protein 2 ( FIP2 ) that is recruited to the phagocytic cups of E . coli . This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42 . Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation , two events that are both required for phagocytosis of Gram-negative bacteria .
Phagocytosis is a complex and versatile process that eliminates pathogens and is performed by specialized cells such as macrophages [1] . Phagocytosis requires cell surface receptors recognizing the pathogen [2] and Rho GTPases controlling local actin dynamics that drive engulfment [2–5] . Toll-like receptor 4 ( TLR4 ) recognizes lipopolysaccharide ( LPS ) present on Gram-negative bacteria [6] , and data from mouse macrophages show that TLR4 is required for the phagocytosis of E . coli [7 , 8] . Moreover , LPS-stimulated phagocytosis of E . coli occurs through actin polymerization controlled by Rho GTPases , Rac1 and Cdc42 , although the mechanisms are unclear [9] . In human macrophages , Rab11 is recruited to E . coli phagosomes and controls TLR4-mediated induction of interferon-β ( IFN-β ) [10] . Like all GTPases , Rab11 acts as a molecular switch alternating between active ( GTP-bound ) - and inactive ( GDP-bound ) forms [11] . In the active state Rab11 binds effector proteins such as the Rab11-family interacting proteins ( FIPs ) , allowing Rab11 to recruit cellular motor proteins [12] . FIP2 regulates intracellular transport within the recycling system and links Rab11 to actin motor proteins , like Myosin5B , to coordinate vesicle trafficking [13–16] . FIP2 also controls EGFR-mediated endocytosis [14] and EGFR-mediated internalization of Chlamydia pneumoniae [17] . Activation of TLR4 results in two different signalling pathways depending on cellular location and the recruited pair of Toll/interleukin-1 receptor ( TIR ) domain-adaptors [10 , 18 , 19] . At the plasma membrane , TLR4 binds MyD88-adaptor-like ( Mal ) and MyD88 to drive NF-κB activation and subsequent production of proinflammatory cytokines , such as TNF . From endosomes TLR4 binds TRIF-related adaptor molecule ( TRAM ) and TIR-domain-containing adapter-inducing interferon-β ( TRIF ) to initiate the production type I interferons , like IFN-β , through activation of the Interferon regulatory factor 3 ( IRF3 ) . A direct action of TRAM or TRIF in phagocytosis has not been established . Here we provide evidence that TRAM is a critical regulator of E . coli phagocytosis by a mechanism dependent on FIP2 . We find that TRAM interacts with FIP2 to drive actin filament formation at forming phagosomes through activation of Rac1 and Cdc42 . As a consequence , the TRAM-FIP2 complex is instrumental in controlling both phagocytosis and TLR4-mediated TRAM-TRIF signalling from E . coli phagosomes .
We have previously shown that E . coli-induced IFN-β mRNA expression is controlled by Rab11a and dependent on F-actin-polymerization [10] . Rab11 uses the FIPs as effector molecules to control endocytosis and endosomal sorting [20] . To identify if a single FIP could be involved in the regulation of E . coli-stimulated IFN-β mRNA expression , PMA differentiated THP-1 cells were silenced for FIP1 , FIP2 , FIP3 , FIP4 and FIP5 , and the effect on E . coli-stimulated IFN-β and TNF mRNA induction was analysed . Of all the FIPs investigated , FIP2 silening had a selective effect on the induction of IFN-β mRNA ( S1 Fig ) . FIP5 silencing reduced IFN-β mRNA expression to a similar extent as FIP2 , however , no selectivity was observed as TNF expression also was reduced under this condition ( S1 Fig ) . As shown , reducing FIP2 mRNA expression impaired IFN-β for both E . coli and LPS stimulations , however , the FIP5 mRNA expression was not affected under this condition of FIP2 silencing . Since FIP2 was involved in the control of E . coli-induced IFN-β mRNA induction , we next examined the role of FIP2 in F-actin and TRAM dynamics during E . coli phagocytosis in primary human macrophages . Surprisingly , TRAM and F-actin co-localized at the E . coli binding site on plasma membrane protrusions ( Fig 1A ) , and a similar phenotype was observed for FIP2 ( Fig 1B ) . These data suggest that TRAM and FIP2 are rapidly recruited to F-actin foci positive phagocytic cups containing E . coli . Indeed , TRAM and FIP2 co-localized in distinct spots on developing E . coli phagosomes 15 min after stimulation ( Fig 1C ) . After a 15 min chase ( 15+15 ) , where E . coli was removed by washing and the cells further incubated for 15 min , the amounts of FIP2 showed a marked decay while the amounts of TRAM showed a slight increase at the E . coli phagosomes ( Fig 1G and 1H ) . Pronounced accumulation of F-actin on the plasma membrane was largely observed at initial phases of uptake ( Fig 1D and 1E ) . In contrast , similarly stimulated Staphylococcus aureus macrophages did not show accumulation of TRAM or FIP2 on phagosomes , despite pronounced accumulation of F-actin ( Fig 1E and 1I ) . The observation that both TRAM and FIP2 were recruited to F-actin foci surrounding E . coli during phagocytosis , led us to investigate if FIP2 silencing could alter TRAM recruitment . FIP2-silenced macrophages were stimulated by E . coli as above , before 3-D imaging by confocal microscopy . The FIP2-silenced macrophages showed a marked reduction in both F-actin and TRAM recruitment to E . coli phagosomes ( Fig 1F and 1G ) . The lack of FIP2 recruitment to phagosomes in FIP2-silenced cells confirmed the specificity of the FIP2 antibody and efficient silencing ( Fig 1H ) . To investigate the distribution of TRAM and TLR4 on developing E . coli phagosomes in detail , 3-D stimulated emission depletion microscopy ( 3-D STED ) was used on primary macrophages stimulated by E . coli bioparticles for 15+15 min . At 70 nm resolution , TRAM showed a vesicular-tubular pattern towards developing phagosomes ( Fig 2A ) , while TLR4 appeared as an continous envelope ( Fig 2B ) . As E . coli was internalized , TRAM covered larger parts of the phagosome but still appeared vesicular-tubular , while TLR4 remained mainly as an envelope around the phagosome . As for TRAM , the recruitment of TLR4 to E . coli phagosomes was significantly reduced in the FIP2 silenced macrophages ( Fig 2C ) . Also , TRAM-silenced macrophages showed a reduction of TLR4 recruitment to E . coli phagosomes ( Fig 2D ) . Representative images of TLR4 are shown for cells that were treated with NS RNA and FIP2 siRNA or TRAM siRNA and stimulated for 15+15 min with E . coli ( S2A–S2C Fig ) . The FIP2- and TRAM-silenced macrophages contained significantly less phagosomes and these were frequently located on the plasma membrane . TRAM recruitment to E . coli during phagocytosis was also verified by live cell imaging of THP-1 cells expressing TRAM-mCherry . Vesicular-tubular TRAM structures were recruited to live E . coli during internalization , and accumulated as the bacteria entered into the cell ( S1 Movie and S2D Fig ) . To investigate if FIP2 recruitment to the E . coli phagosome was a TLR4 dependent process , we included mouse immortalized bone-derived-macrophages ( iBMDMs ) . FIP2 was frequently observed at E . coli phagosomes after 15 min of stimulation in both wild type and Tlr4-/- iBMDMs ( S3A and S3B Fig ) . Interestingly , the TLR4-/- iBMDMs showed significantly reduced FIP2 levels at the E . coli phagosomes at both 15 and 15+15 min of stimulation ( S2C Fig ) . Together these results demonstrate that TLR4 and TRAM are transported to E . coli phagosomes by a mechanism involving FIP2 . Because TRAM was found on FIP2 foci containing F-actin on E . coli phagosomes , we next investigated if TRAM could play a role in phagocytosis . For the study of comparison , we also included S . aureus . The number of internalized E . coli and S . aureus were quantified by 3-D imaging of primary human macrophages silenced for TRAM or MyD88 ( Fig 3A and 3B ) . TRAM silencing reduced the number of phagocytosed E . coli per macrophage with more than 60% at both investigated time points ( Fig 3A ) . TRAM silencing also affected S . aureus phagocytosis , particularly after 15+15 min where the reduction was approximately 50% ( Fig 3B ) . MyD88 silencing did not significantly reduce phagocytosis of either E . coli or S . aureus ( Fig 3A and 3B ) . Maturation of E . coli phagosomes was significant impaired by TRAM siRNA , whereas MyD88 silencing had no effect ( Fig 3C ) . Interestingly , both TRAM and MyD88 silenced macrophages showed slight , but significant , increase in S . aureus phagosome maturation . We next used THP-1 cells to verify our findings in primary human macrophages , as these cells show more efficient silencing of MyD88 than in primary macrophages ( S5A and S5B Fig ) . Immunoblots of THP-1 cells silenced for MyD88 did not show detectable MyD88 protein ( S5C Fig ) . Moreover , a functional MyD88 control in THP-1 cells confirmed that TNF and IL-6 mRNA expressions were strongly reduced in MyD88 silenced cells stimulated with Pam3CSK4 and LPS ( S5D Fig ) . The effect of TRAM siRNA on the uptake of E . coli in THP-1 cells was clear and significant and resembled the data obtained with primary macrophages ( S5E Fig ) . Also , flow cytometry analysis showed that TRAM silencing reduced phagocytosis of E . coli ( S5G Fig ) . In contrast to primary human macrophages , TRAM silencing in THP-1 cells did not significantly reduce the uptake of S . aureus bioparticles ( S5F Fig ) . Silencing of MyD88 in THP-1 cells did not result in significant reduction in uptake of either E . coli or S . aureus ( S5E–S5G Fig ) . To exclude the possibility that the observed effect of TRAM and MyD88 on phagocytosis , was due to the use of pHrodo-labelled killed bacteria , we also included live bacteria in this study . The phagocytosis of live E . coli and S . aureus was measured in a modified phagocytic killing assay [7] and monitored as colony-forming units ( cfu ) per cell . In line with the phagocytosis data of bioparticles in human macrophages and THP-1 cells , TRAM depleted THP-1 cells showed significant reduction in phagocytosis of live E . coli ( Fig 3D ) . Of interest , TRAM silencing also reduced uptake of live S . aureus ( Fig 3D ) . The effect of MyD88 silencing was not so clear with a weak reduction in E . coli uptake and in fact an increase in phagocytosis of S . aureus in this assay ( Fig 3E ) . We next used Tram-/- and Myd88-/- iBMDMs and flow cytometry to investigate if mouse macrophages showed a similar phenotype as human macrophages ( S5H Fig ) . While both TRAM and MyD88 deficient mouse macrophages showed impaired phagocytosis of E . coli , only the MyD88 deficient macrophages reduced S . aureus phagocytosis . The effect of TRAM- and MyD88 knock out on phagocytosis was also compared with knocking out TLR4 . Tlr4-/- mouse macrophages showed impaired uptake of E . coli , however , with less efficiency compared to TRAM- or Myd88-deficient macrophages ( S5I Fig ) . Phagocytosis of S . aureus was not reduced in the TLR4 deficient macrophages . Together these results show that TRAM has a strong and consistent phenotype in regulating phagocytosis of E . coli in human macrophages . TRAM silencing also reduced uptake of S . aureus bioparticles in primary human macrophages at early timepoints as well as impairing uptake of live bacteria in THP-1 cells . The involvement of MyD88 in phagocytosis of E . coli and S . aureus was less clear as differences between the human and mouse macrophages were observed . The observation that FIP2 and TRAM co-localized on forming E . coli phagosomes led us to investigate if TRAM and FIP2 could mutually interact . Indeed , co-immunoprecipitation analyses of THP-1 cells revealed that endogenous TRAM formed a complex with FIP2 ( Fig 4A ) . Interestingly , also Rab11 and TRIF were part of this complex which was markedly increased by E . coli stimulation . We next co-expressed TRAM and Rab11a in HEK293T cells , with and without FIP2 and found that TRAM and Rab11a formed a complex only when FIP2 was co-expressed ( Fig 4B ) . In line with this result Rab11a and TRAM did not form a complex when endogenous FIP2 was silenced ( Fig 4C ) . Moreover , FIP2 and TRAM could still interact in cells simultaneously silenced for Rab11a and Rab11b ( S4A Fig ) . In support of these results , the FIP2 I481E mutant [21] , containing a single amino acid mutation in the Rab11 binding domain of FIP2 , could not bind Rab11a , but was found to interact with TRAM ( S4B Fig ) . As expected , FIP2 bound strongly to Rab11a and the constitutive active GTP-bound Rab11aQ70L mutant but did not bind to the inactive GDP-bound Rab11aS25N mutant ( S4C Fig ) . Despite the lack of FIP2 binding to inactive Rab11aS25N , TRAM could still be found in complex with FIP2 . We next used the HEK293 cell model to investigate if FIP2 could be involved in the formation of enlarged LPS endosomes . HEK293 cells expressing human TLR4 , CD14 , MD2 , TRAM and Rab11 form enlarged Rab11 positive endosomes following LPS stimulation [22] . The data demonstrate that FIP2 , TRAM and constitutively active Rab11a are present on LPS endosomes ( S4D Fig ) . Cells co-transfected with the inactive form of Rab11a failed to form enlarged LPS endosomes and FIP2 appeared cytosolic ( S4E Fig ) . Taken together , these results suggest that FIP2 controls the localization of TRAM to enlarged LPS endosomes and that active Rab11a is needed for optimal FIP2 binding to TRAM . To identify the FIP2 binding site in TRAM we analysed a series of TRAM deletion mutants , which contained the N-terminal part of TRAM with 10–20 amino acid residues increments . While TRAM 1–68 and 1–79 did not bind FIP2 , a weak interaction was found with TRAM 1–90 that increased markedly with TRAM 1–100 , but not further with TRAM 1–120 ( Fig 4D ) . These data show that there is a FIP2 binding site located between the amino acid residues 80–100 in TRAM . This domain contains the acidic amino acid motifs E87/D88/D89 and E91/D92 , reported to be required for TLR4-mediated TRAM-TRIF signalling [23 , 24] . We next made a TRAM construct with the alanine substitutions E87A/D88A/D89A and D91A/E92A and investigated FIP2 binding . Both E87A/D88A/D89A and D91A/E92A mutants showed noticeably impaired FIP2 binding ( Fig 4E ) . Next , we made several FLAG-FIP2 variants containing the amino acids residues 1–512 ( wild type ) , and the deletion mutants 129–512 , 1–192 and 193–512 to locate the TRAM-binding site in FIP2 . While wild type FIP2 , FIP2 129–512 and FIP2 1–192 all showed TRAM binding , FIP2 193–512 failed to bind TRAM ( Fig 4F ) . To summarize , we found a sequence of 63 amino acids , located between positions 129–192 of FIP2 , to be responsible for TRAM binding . Of interest , FIP2 1–192 , which lacks Rab11 and Myosin5B tail binding [25 , 26] , showed an even stronger binding to TRAM . Taken together these results demonstrate that FIP2 binding to TRAM occurs via FIP2 , and not Rab11 , but Rab11 positively regulates TRAM-FIP2 complex formation . Since FIP2 interacted with TRAM , and FIP2 silencing specifically decreased E . coli induced expression of IFN-β ( S1A Fig ) , we next investigated if FIP2 was involved in phagocytosis . Indeed , primary human macrophages silenced for FIP2 showed more than 80% reduction of E . coli per cell at both 15 and 15+15 min , while S . aureus phagocytosis was only impaired in cells stimulated for 15+15 min ( Fig 5A and 5B ) . As observed in the TRAM-silenced human primary macrophages , the maturation of E . coli phagosomes was decreased while the phagosome maturation of S . aureus phagosomes was increased in FIP2 silenced cells ( Figs 3C and 5C ) . To validate the effect of FIP2 on phagocytosis in primary macrophages , we next used FIP2-silenced THP-1 cells which also showed a marked perturbation of E . coli and S . aureus phagocytosis ( Fig 5D and 5E ) . Analysis by flow cytometry showed that FIP2 silencing inhibited phagocytosis of both E . coli and S . aureus after 30 min of stimulation , while only E . coli phagocytosis was reduced after 60 min ( Fig 5F and 5G ) . Also , phagocytosis of live E . coli and S . aureus was significantly reduced upon FIP2 silencing ( Fig 5H ) . The effect of FIP2 silencing on the phagocytosis of E . coli and S . aureus was comparable to the F-actin inhibitor cytochalasin D ( S6C and S6D Fig ) . In THP-1 cells with lentiviral overexpression of FIP2 a marked increase in both E . coli and S . aureus phagocytosis was observed ( Fig 5I ) . As a control , FIP2 overexpression resulted in a strong increase in the amount FIP2 protein ( Fig 6C ) . Since FIP2 bridges TRAM and Rab11 , and complex formation was enhanced by E . coli stimulation ( Fig 4A ) , we also assessed the role of Rab11 in E . coli phagocytosis . We have previously shown that recruitment of both TRAM and TLR4 to the E . coli phagosomes in human macrophages are dependent on Rab11a , however , silencing of Rab11a alone did not affect phagocytosis [10] . To investigate if FIP2 controlled E . coli phagocytosis via Rab11 , we simultaneously silenced the Rab11 isoforms Rab11a and Rab11b in primary human macrophages . Indeed , this resulted in a significant and consistent inhibition of E . coli phagocytosis similar to the FIP2 and TRAM silenced macrophages ( S6E and S6F Fig ) . These data suggest that redundancy exists between Rab11a and Rab11b and that both isoforms must be targeted in order to affect phagocytosis . Together these results show that FIP2 is an important regulator of phagocytosis of E . coli . For S . aureus FIP2 seems to preferentially control phagocytosis at early timepoints , while the effect is lost at 60 min . Rho GTPases , like Rac1 and Cdc42 , are instrumental in regulating F-actin dynamics during phagocytosis [3] . Given the strong effect of FIP2 silencing on E . coli phagocytosis , we analysed Rac1- and Cdc42 activation in these cells . We made a construct encoding the Rac1/Cdc42 ( p21 ) binding domain ( PBD ) of the human p21 activated kinase 1 protein ( PAK ) fused to the GST protein . PBD binds specifically to the activated GTP-bound forms of the Rac1 and Cdc42 proteins [27] . We observed that FIP2 silencing had a marked inhibitory effect on E . coli-induced activation of both Rac1 and Cdc42 . Also , we noticed that FIP2 silencing reduced the amounts of Rac1 and Cdc42 proteins , whereas the mRNA levels were unaffected ( Fig 6A and S7A Fig ) . TRAM depletion did not reduce Rac1 protein , but lowered the amount of Cdc42 , ( Fig 6B ) , however , both Rac1 and Cdc42 mRNA expressions were significantly reduced by TRAM depletion ( S7B Fig ) . Furthermore , immunoblots of THP-1 cells overexpressing FIP2 showed increased amounts of both Rac1 and Cdc42 suggesting that FIP2 has a stabilising effect on both proteins . Indeed , we found that Rac1 is part of an immune-complex together with TRAM and FIP2 in primary macrophages ( Fig 6D ) . Altogether , these results demonstrate that FIP2 is a central effector molecule of phagocytosis through activation and stabilization of the Rho GTPases Rac1 and Cdc42 . As FIP2 was found to be a key regulator of E . coli phagocytosis , we would expect a decreased TRAM-TRIF signalling upon FIP2 depletion with siRNA . Thus , we examined how LPS- and E . coli-stimulated signalling was affected in FIP2 silenced THP-1 cells ( Fig 7A ) . Following stimulation , phosphorylation of the TANK-binding kinase-1 ( TBK-1 ) , IRF3 and IκBα were quantified and found to be markedly impaired in the FIP2 silenced cells , while the phosphorylation of p38 mitogen-activated protein kinase ( p38 MAPK ) was not markedly impaired ( Fig 7A and 7B , S8A Fig ) . Similar results were obtained using LPS for stimulation . Phosphorylation of TBK-1 at Ser172 , IRF3 at Ser386 and Ser396 , are all critical for IRF3 activation and induction of IFN-β [28 , 29] . In line with the phosphorylation patterns observed by Western blotting , the FIP2 silenced cells showed a markedly impaired induction of IFN-β with little effect on TNF ( Fig 7C ) . THP-1 cells with lentiviral-induced overexpression of FIP2 showed a 3-fold higher E . coli-stimulated IFN-β mRNA expression ( Fig 7D ) . In contrast , E . coli-stimulated TNF expression was relatively unchanged by FIP2 overexpression ( Fig 7D ) . Next , we investigated how the FIP2 silenced cells responded upon MDA5/RIG-I activation that also uses IRF3 to induce IFN-β mRNA expression . The cells were stimulated by poly I:C using lipofectamine transfection and LPS was included for comparison . When transfected , poly I:C triggers a TLR3-independent IRF3-mediated induction of IFN-β via cytosolic receptors MDA5 and RIG-I . MDA5/RIG-I stimulated IFN-β mRNA expression was reduced by only 3 . 5-fold , while TLR4 stimulated IFN-β mRNAs by LPS was reduced 22-fold after 4 h of stimulation ( Fig 7E ) . Taken together , these data demonstrate that FIP2 is a master regulator of LPS- and E . coli- mediated TLR4-TRAM-TRIF signalling , in addition to being a critical regulator of phagocytosis . Next , we addressed if inhibition of TLR4-mediated TRAM-TRIF signalling could alter macrophage E . coli phagocytosis . The TBK1 kinase operates downstream of TRIF and its activity is required for LPS-stimulated phosphorylation of IRF3 and production of IFN-β [28 , 29] . First , Western blot analysis was performed in THP-1 cells in order to compare the effect of two TBK1 inhibitors BX-795 and MRT67307 on E . coli-stimulated IRF3- and p38 MAPK-activation . Both inhibitors impaired IRF3 phosphorylation at Ser386 by more that 65% after 30 min of stimulation ( S8B and S8C Fig ) . In contrast , phosphorylation of p38MAPK was largely unchanged . Next , THP-1 cells and human primary macrophages were treated with the TBK1 inhibitors prior to addition of E . coli bioparticles . In cells with inhibited TBK1 activity , a marked reduction of phagocytosis of E . coli was observed after 15 min of stimulation ( S8D Fig ) . When comparing E . coli and S . aureus phagocytosis in MRT67307 treated THP-1 cells we found only E . coli phagocytosis to be significantly decreased ( S8E Fig ) . Also , when TBK1 was inhibited in primary human macrophages a significant and marked reduction of E . coli phagocytosis was observed ( S8F Fig ) . These results demonstrate that the early phagocytosis of E . coli , but not S . aureus , can be targeted by TBK1 kinase inhibition without affecting p38 MAPK activation . In order to examine the importance of FIP2 on bacterially induced gene regulation , we performed a targeted transcriptome profiling for immunologically relevant genes on RNA samples isolated from E . coli stimulated human macrophages from 7 donors . Hyper geometric Gene Ontology enrichment for biological processes was performed on genes differentially expressed during E . coli stimulation in FIP2 silenced cells . When compared to non-silenced macrophages , we found significant hits on downregulated genes involved in several cellular processes essential for innate immunity ( Fig 8A ) . FIP2 depletion had a modest effect on cytokine production , proliferation and activation of macrophages , and most prominent effects on genes regulating the LPS/bacterial stimulated responses , cell chemotaxis & migration , cyclic nucleotide mediated signalling , ion transport and intracellular trafficking ( Fig 8A ) . After 4 h of E . coli stimulation , the chemokines CXCL9 , CXCL10 and CXCL11 , together with IL12B ( IL12p40 ) were among the most downregulated genes in the FIP2 silenced macrophages ( Fig 8B and S3 Table ) . E . coli-stimulated IFN-β mRNA expression was at its highest after 2 h of stimulation and was among the 7 most downregulated genes after FIP2 silencing at this time point ( Fig 8B and S2 Table ) . In contrast , FIP2 silencing did not significantly alter E . coli-stimulated mRNA expression of TLR4 , CD14 , NF-κB1 , NF-κB2 and TNF ( Fig 8C and S1–S3 Tables ) . The results from the Nanostring experiment were verified by qPCR of selected cytokines . These data confirmed that IFN-β , CXCL9 , CXCL10 , CXCL11 and IL12B were significantly reduced in FIP2 silenced macrophages , while TNF , TLR4 and CD14 were not changed ( S9A and S9B Fig ) . The result from these experiments demonstrate that FIP2 silencing has a preference for reducing TLR4-stimulated induction of IRF3 target genes , which is likely to be a consequence of the impaired phagocytosis of E . coli .
In the present study , we show that TLR4 mediates phagocytosis of E . coli in macrophages via its adaptor TRAM . TRAM performs this function by interacting with FIP2 which subsequently activates the Rac1 and Cdc42 Rho GTPases for controlling actin-dynamics . A consequence of this is that FIP2 strongly regulates phagosomal signalling that involves IRF3 activation . Receptor recognition during phagocytosis launches signalling pathways that induce remodelling of the actin cytoskeleton and extension of membrane protrusions that surround the particle to form a phagocytic cup [30] . In early phases of E . coli phagosome formation , TLR4 is recruited to the phagocytic cup to provide a platform for subsequent TRAM-TRIF signalling [10] . Our findings demonstrate that TRAM recruitment to this platform requires FIP2 and that TLR4-TRAM-TRIF signalling is needed for phagocytosis . MyD88 is a universal signalling adaptor for TLRs , except TLR3 , and activates NF-kB , c-Jun kinase , and p38 MAPK [31] . In mouse macrophages , Blander and co- workers found that MyD88-mediated signalling is required for phagocytosis of E . coli and S . aureus and for phagosomal maturation [7] . In contrast to these findings , Yates and Russel showed that the phagosome maturation of beads coated with the TLR4 ligand LPS or the TLR2 ligand Pam3Cys occurs independently of MyD88-mediated signalling [32] . The controversy on the involvement of MyD88 in phagosomal maturation in murine macrophages may be due to variations in experimental models used . In previous studies on murine macrophages , the involvement of TRAM-TRIF signalling in phagocytosis and phagosomal maturation has not addressed . Our data suggest that in human macrophages TRAM , but not MyD88 , is involved in both uptake of E . coli as well as in phagosomal maturation . We found that murine macrophages deficient for TRAM or MyD88 showed a markedly reduced uptake of E . coli whereas only MyD88 affected phagocytosis of S . aureus . Apparently , mouse macrophages use both MyD88-dependent and MyD88-independent signalling for controlling phagocytosis of E . coli , whereas only MyD88 played a role for the uptake of S . aureus . The role of MyD88 in phagocytosis agrees with the data from murine macrophages published by Blander and co-workers [7] . The mechanism behind the species differences between human and mouse macrophages regarding the role of MyD88 in phagocytosis of Gram-positive bacteria are not clear . It has been shown that phagosomes in murine M1 macrophages become more acidic in mice compared to M1 macrophages in humans [33–35] . Thus , murine and human macrophages may behave differently in phagocytic processes . We observed that silencing of TRAM in fact reduced the uptake of both heat-killed S . aureus and E . coli in primary human macrophages , and both heat-killed and live bacteria in THP-1 cells . Several explanations may account for this effect of TRAM on phagocytosis . It is known that lipoproteins and lipoteichoic acids present in Gram-positive bacteria interact with TLR2 [36] . TLR2 can mediate signal transduction through TRAM-TRIF and IRF3 , in addition to MyD88 and IRF1 [37] . Also , TRAM has been reported to act as a bridging adapter with MyD88 to control TLR2-mediated induction of IFN-β via IRF7 [38] . These previous reported TLR2-dependent TRAM responses were observed after prolonged stimulation , whereas heat killed E . coli-stimulated IRF3 activation occurred rapidly within 30 min . The role of TRAM in phagocytosis of S . aureus may be uncoupled to TLR2 signalling . This is supported by our TBK-1 inhibitor data showing that phagocytosis of heat killed S . aureus is not reduced , whereas heat killed E . coli uptake was significantly inhibited . This may implicate that TRAM-TRIF signalling is linked to E . coli , but not S . aureus , phagocytosis . Moreover , TRAM was strongly recruited to heat killed E . coli , but not S . aureus , phagosomes . Furthermore , silencing of TRAM reduced E . coli phagosome maturation , but had no inhibitory effect on S . aureus phagosomes . Of interest , we found that TRAM silencing reduced mRNA expression of both Rac1 and Cdc42 as well as reducing the amount of Cdc42 protein . Given the fact that RhoGTPases are so instrumental in actin dynamics we suggest that TRAM may regulate phagocytosis of both E . coli and S . aureus by controlling the levels of Rac1 and Cdc42 in macrophages . FIP2 was found to be a master regulator of E . coli uptake . Phagocytosis of S . aureus was also reduced by FIP2 silencing , however , the effect seemed weaker and appeared to be lost after 60 min . Overexpression of FIP2 increased markedly the internalization of both E . coli and S . aureus . Silencing of FIP2 lead to decreased amounts of activated Rac1 and Cdc42 as well as reduced amounts of the proteins , without affecting their mRNA expression levels . Conversely , overexpression of FIP2 in THP-1 cells resulted in an increase in Rac1 and Cdc42 proteins . We suggest that FIP2 controls the RhoGTPases through ubiquitination and proteasomal degradation . Several studies have shown that Rho GTPases are regulated by post-translational modifications such as ubiquitination [39–41] . Our data also suggest that Rac1 may be stabilized through its interaction with FIP2 and TRAM . The profound effect of FIP2 on Rac1 and Cdc42 stability will have important functional consequences on actin dynamics and phagocytosis . This statement is supported by our data demonstrating that FIP2 silencing markedly reduces F-actin and TRAM on E . coli phagosomes . Moreover , data from Dong et al [42] have shown that FIP2 affects actin cytoskeleton dynamics in cancer cells , however , the mechanisms behind this effect was not addressed in their study . Since FIP2 is regulating both activation and stability of Rac1 and Cdc42 it is conceivable that it controls phagocytosis of heat-killed and live E . coli and S . aureus bacteria . In our experiments we have used siRNA technology to deplete TRAM and FIP2 . We made several THP-1 knock out cell lines using CRISP/Cas9 technology targeting TRAM and FIP2 . However , we experienced problems with these cell lines related to stability and compensatory mechanisms . Thus , we found it more reliable to reduce gene expression by siRNA , instead of using the CRISPR/Cas9 technology , which allowed comparison of the THP-1 cell system with primary human macrophages . TRAM can interact with proteins that do not contain a TIR domain [43] . In a recent study we reported that SLAMF1 binds to TRAM and regulates its transport to E . coli phagosomes and IFN-β release but does not affect phagocytosis [44] . In the current paper we show that TRAM also interacted with FIP2 . The binding of FIP2 to TRAM was not dependent on Rab11 , however , Rab11 was found to be a part of the FIP2-TRAM complex . Moreover , TLR4 activation increased the amount of endogenous FIP2 and TRAM complexes suggesting that TLR4 may augment FIP2-TRAM interaction by activation of Rab11 . The domain in TRAM involved in interaction with FIP2 was mapped to the amino acid residues 80–100 . Structural analysis shows that human TRAM and TRIF form a BB-loop–mediated homodimer at amino acid residues P116 and C117 , critical for TRAM and TRIF dimerization and subsequent signalling [23 , 45] . Moreover , Funami and co-workers reported that the E87/D88/D89 motif in TRAM is indispensable for TRAM-TRIF dimerization while the D91/E92 motif is not [24] . Our data demonstrate that both the E87/D88/D89 and D91/E92 motifs are critical for FIP2 interaction . The fact that the complex formation between TRAM , FIP2 , Rab11 and TRIF was increased by TLR4 stimulation suggest that FIP2 does not interfere with binding of TRIF to TRAM . Furthermore , we located the TRAM binding domain in FIP2 , between the amino acid residues 129–192 , that does not contain the motifs required for Myosin5B or Rab11 binding [25 , 26] . Our data suggest that the interaction between TRAM and FIP2 is required both for uptake of E . coli and for phagosomal maturation in primary human macrophages . In these cells Rac1 was found to be in complex with FIP2 and TRAM which may explain the close relationship with this complex and phagocytosis . In summary , we describe a novel function of TRAM in the regulation of phagocytosis of Gram-negative bacteria . Our model suggests that FIP2 exists in a preformed complex with TRAM-TRIF that is rapidly recruited to the E . coli binding site and enhanced by TLR4 activation . This allows FIP2 to activate Rac1 and Cdc42 resulting in F-actin formation at the phagocytic cup which together with TLR4-mediated TRAM-TRIF signalling is required for uptake of the bacteria .
The following ligands , bacteria and inhibitors were used: pHrodo Red E . coli K12 BioParticles ( P35361 ) , pHrodo Red S . aureus BioParticles ( A10010 ) from Invitrogen , E . coli K12 ( DH5α ) and S . aureus ( ATCC® 10832D-5™ ) . Ultrapure LPS from E . coli K12 ( tlrl-eklps ) and Poly I:C HMW ( tlrl-pic ) from InvivoGen . Live DH5α E . coli expressing pZE27GFP was a gift from James Collins ( Addgene plasmid 75452 ) . TBK1 inhibitors MRT67307 and Bx-795 from Sigma-Aldrich . THP-1 cells ( monocytic cell line derived from acute monocytic leukemia ATCC® TIB-202™ ) was maintained in RPMI-1640 ( ATCC® 30–2001™ ) complemented with 2-mercaptoethanol to 0 . 05 mM and 10% fetal calf serum ( FCS ) ( 10270106 GIBCO ) at 37 oC and 5% CO2 . THP-1 cells were differentiated in growth medium supplemented with 40–60 ng/mL phorbol 12-myristate 13-acetate ( P8139 Sigma-Aldrich ) . Human monocytes were isolated from buffy coats ( Department of Immunology and Transfusion Medicine , St Olavs Hospital ) and differentiated into macrophages in RPMI1640 supplemented with 50 ng/mL recombinant human M-CSF ( 216-MC-025 R&D systems ) , 10% pooled human A+ serum ( Department of Immunology and Transfusion Medicine , St Olavs Hospital ) , 700 μM L-glutamine ( Sigma-Aldrich ) and 20 μg/mL Gensumycin ( Sanofi-Aventis ) at 37 oC and 5% CO2 . Medium was changed on day 3 and 5 after seeding . HEK293T cells ( Human epithelial cells ATCCCRL-11268 ) and HEK293-TLR4mCherry cells ( were made by us as described in [10] ) and maintained at 37 oC and 8% CO2 in DMEM supplemented with 10% FCS , 1 μg/mL of Ciprofloxacin Hydrochloride ( CellGro® ) . 0 . 5 mg/mL G418 ( Geneticin , Life Technologies ) were used for TLR4mCherry selection . Transfection of plasmids was performed using GeneJuice transfection reagent ( Novagen ) . The iBMDMs ( Immortalized bone-derived-macrophages ) from wild type , Tlr4-/- , Tram-/- and Myd88-/- C57BL/6 mice were made in the lab of Dr . Douglas T . Golenbock [46] and maintained as the HEK293T cells above . pHrodo-conjugated E . coli or S . aureus heat killed bacterial bioparticles were given to the cells in doses ranging from 7 . 5 to 65 particles per cell dependent on the cellular assay . Prior stimulation both LPS and the bacterial particles were sonicated and opsonized in medium containing 10% human A+ serum for 5 min at 37 oC . The LPS dose was 100 ng/mL . 5 ug/ mL Poly I:C was transfected with Lipofectamine®RNAiMAX . Live E . coli and S . aureus were grown to a density of OD600 = 0 . 35 , washed with PBS and given at a dose of 10–50 bacteria per cell . THP-1 or HEK293T cells were seeded 24 h before siRNA transfection at a density of 400 000–500 000 cells /well in 6-well plates ( NUNC ) in their respective growth medium containing no antibiotics . siRNA was transfected at a concentration of 16 nM or 8 nM using Lipofectamine®RNAiMAX transfection reagent ( Invitrogen ) for 48–72 h . PBMC derived macrophages were transfected with 32 nM siRNA on day 6 and 8 after seeding using Lipofectamine®3000 Transfection Reagent ( Invitrogen ) . Medium was changed to fresh antibiotic-free medium 2 h before the second siRNA transfection and the cells stimulated on day 10 . The AllStars Negative Control siRNA ( SI03650318 QIAGEN ) was used as a non-silencing control and termed NS RNA . Hs_RAB11A_5 , Hs_RAB11B_6 , Hs_RAB11FIP1_12 , Hs_RAB11FIP2_5 , Hs_Rab11FIP3_9 , Hs_RAB11FIP4_5 , Hs_RAB11FIP5_5 , Hs_TICAM2_2 and Hs_MyD88_5 validated siRNA , all from QIAGEN , were used to target Rab11a , Rab11b , Rab11FIP1 , Rab11FIP2 , Rab11FIP3 , Rab11FIP4 , Rab11FIP5 , TRAM and MyD88 mRNA , respectively . THP-1 expressing lentiviral encoding FIP2 was made by cloning FIP2 into the bicistronic lentiviral expression vector pLVX-EF1α-IRES-ZsGreen1 ( Clontech ) and co-transfect with packaging plasmids psPAX2 and pMD2 . G , kindly provided by the TronoLab ( Addgene plasmids 12260 and 12259 , to produce pseudoviral particles in HEK293T cells . Supernatants were collected at 48 h and 72 h , combined and concentrated using Lenti-X™ Concentrator ( Clontech ) . Subsequently , the viral particles were used for transduction of THP-1 wild type cells along with virus particles without FIP2 coding sequence and ZsGreen positive cells selected by fluorescence-activated cell sorting ( FACS ) and tested for FIP2 protein expression by Western blotting . A THP-1 cell line expressing TRAMmCherry were generated using lentiviral transduction . TRAMmCherry was first subcloned into a Gateway ENTRY vector , before recombination into pCDH-EF1a-GW-IRES-Puro [47] from this vector the constructs was packaged into lentivirus particles using third-generation packaging system , a gift from the TronoLab ( Addgene plasmids 12251 , 12253 and 12259 ) and according to [48] . Transduced cells were selected using puromycin ( 1 μg/mL ) . Total RNA was isolated from THP-1 cells or PBMC derived macrophages using QIAzol ( Qiagen ) or Isol ( 5 prime ) and chloroform extraction followed by purification on RNeasy Mini columns , including DNAse digestion ( Qiagen ) . cDNA was made from total RNA with Maxima First Strand cDNA Synthesis Kit for RT-qPCR ( Thermo Scientific ) . Quantitative real-time PCR ( q-PCR ) was performed with the PerfeCTa qPCR FastMix ( Quanta Biosciences ) in 20 μL reaction volume in duplicate wells and cycled in a StepOnePlus™ Real-Time PCR cycler ( Applied Biosystems ) . The following TaqMan Gene Expression Assays ( Applied Biosystems ) were used: IFN-β ( Hs01077958_s1 ) , TNF ( Hs00174128_m1 ) , Rab11a ( Hs00900539_m1 ) , Rab11b ( Hs00188448_m1 ) , Rab11FIP1 ( Hs00951195_m1 ) , Rab11FIP2 ( Hs00208593_m1 ) , Rab11FIP3 ( Hs006085_m1 ) , Rab11FIP4 ( Hs00400200_m1 ) , Rab11FIP5 ( Hs00392033_m1 ) , TBP ( Hs00427620_m1 ) , Rac1 ( Hs00251654_m1 ) , Cdc42 ( Hs00741586_mH ) , CXCL9 ( Hs00171065_m1 ) , CXCL10 ( Hs01124251_g1 ) , CXCL11 ( Hs04187682_g1 ) , IL12B ( Hs01011518_m1 ) , TLR4 ( Hs00152939_m1 ) , CD14 ( Hs02621496_s1 ) , IL6 ( Hs00985639_m1 ) and GAPDH ( Hs99999905_m1 ) . The level of TBP or GAPDH mRNA was used for normalization and results presented as relative expression compared to the control-treated sample . Relative expression was calculated using the Pfaffl's mathematical model [49] . TNF in supernatants from THP-1 cells was detected using human TNF-alpha DuoSet ELISA ( DY210-05 R&D Systems ) , IFN-β by VeriKine-HSTM Human Interferon-Beta Serum ELISA Kit ( 41410 PBL Assay Science ) . Phusion High-Fidelity DNA Polymerase ( Thermo Fisher Scientific ) was used for amplification of desired gene sequences . PCR products , or restricted vectors , were purified by QIAquick PCR purification and gel extraction kits ( QIAGEN ) . Endofree plasmid Maxi kit ( QIAGEN ) was used for endotoxin-free plasmids preparations . Sequencing of plasmids was done at Eurofins Genomics . Primers used for cloning are listed in Table 1 . pEGFP-FIP2 ( KIAA0941 sequence in pEGFP-C1 ) and pEGFP-FIP2ΔC2 [50] , FIP2 I481E [21] were used as templates . FIP2 and deletion mutants were subcloned into pCMV- ( DYKDDDDK ) -N vector ( Clontech ) . pLVX-EF1α-IRES-ZsGreen-FIP2 was made by restriction digest of the vector with EcoRI and ligation with EcoRI fragment from pEGFP-FIP2 . pcDNA3-hTRAM-YFP was a gift from K . Fitzgerald ( University of Massachusetts Medical School , Worcester , MA , USA ) , used for transfections or as template for subcloning of TRAM and TRAM deletion mutants into pCMV- ( DYKDDDDK ) -C Vector ( Clontech ) . Flag-tagged proteins and EGFP- , EYFP- or ECFP-tagged proteins were overexpressed in HEK293T cells , with or without co-expression of human ( h ) TLR4 , hCD14 and hMD2 encoding plasmids . After 48 h of transfection cells were washed with PBS and harvested in lysis buffer ( 150 mM NaCl , 50 mM TrisHCl pH 8 . 0 , 1 mM EDTA , 1% NP-40 ) supplemented with cOmplete™ , Mini , EDTA-free Protease Inhibitor Cocktail , PhosSTOP , 50 mM NaF , 2 mM NaVO3 ( Sigma-Aldrich ) and 2 . 5 U/mL Benzonase Nuclease ( Novagen ) . Cell lysates were incubated on ice before centrifugation at 18000 x g , 4 oC for 15 min , and co-immunoprecipitations performed using 30 μL of anti-FLAG M2 affinity agarose ( A2220 , Sigma-Aldrich ) with rotation for 4 h at 4 oC . After washing the immunocomplexes were eluted at 95 oC for 3 min in 40 μL 2 x LDS buffer ( Invitrogen ) . Agarose beads were removed by centrifugation at 10000 x g for 30 seconds and DTT added to 25 mM . The samples were heated for 10 min at 80 oC before subjected to SDS-PAGE and immunoblotting . For endogenous co-immunoprecipitations , 5 μg rabbit anti-TICAM2/TRAM ( H-85 Santa Cruz Biotechnology ) antibody or normal rabbit IgG was coupled to 1 . 5 mg magnetic Dynabeads® M-270 Epoxy ( Life Sciences Technology ) and incubated with cleared lysates from human primary macrophages or THP-1 cells at 4 oC for 2 h with rotation , before extensive washing in lysis buffer followed by elution by heating in 2 x LDS buffer as described . The partial sequence of the p21-activated kinase 1 ( PAK1 ) ( 67–150 a . a ) containing p21-binding domain ( PBD ) from pDONR223-PAK1 ( a gift from William Hahn & David Root , Addgene plasmid 23543 ) , was subcloned to pGex-2TK vector ( GE Healthcare Life Sciences ) using the primers 5’-AATTGGATCCAAGAAAGAGAAAGAGCGGCCAG-3’ and 5’-TATAGAATTCTCAAGCTGACTTATCTGTAAAGCTCATG-3’ with Phusion High-Fidelity DNA Polymerase ( F530 , Thermo Scientific ) before digesting the PCR product with Fast Digest enzymes BamHI and EcoRI ( Fermentas ) . The PAK1-PBD in pGEX-2TK was transformed to BL21 ( DE3 ) Competent E . coli to produce GST-PAK1-PBD- recombinant protein following manufacturer’s instructions . Purified GST-PBD-PAK1 was used as a probe for pull downs of activated Cdc42 and Rac1 . THP-1 cells were treated with FIP2 siRNA , TRAM siRNA or NS RNA and stimulated with E . coli bioparticles . Following stimulation , the cells were placed on ice and washed with cold PBS and lysed in lysis buffer ( 25 mM HEPES pH 7 . 2 , 150 mM NaCl , 5 mM MgCl2 , 1% Nonidet P40 , 5% glycerol , 100 μM GDP ) . The cells were detached by scraping and the lysates cleared by centrifugation and immediately mixed with the glutathione-agarose beads conjugated with GST-PAK1-PBD and the solutions gently rotated for 45 min at 4°C . The beads were collected by centrifugation and washed two times in lysis buffer before bound proteins were eluted by heating in LDS-sample buffer ( Invitrogen ) . GTP-bound forms of Cdc42 and Rac1 were resolved on 12% NuPage gels ( Invitrogen ) and detected by Western blot analysis using anti-Cdc42 antibodies ( Santa Cruz Biotechnology ) and anti-Rac1/2/3 ( Cell Signaling Technology ) . The top parts of the gels were stained by Simple stain ( Thermo Fisher Scientific ) and GST-PBD-PAK1 imaged on Carestream GelLogic 212 PRO . The Molecular Imaging software ( Carestream Health Inc ) was used for quantification of GST-PBD-PAK1 . Total Rac1 , Cdc42 , Rab11FIP2 and β-tubulin were used for the control of protein input in lysates used for the pull downs . Protein samples were run on pre-cast NuPAGE™ Bis-Tris gels ( Invitrogen ) with 1 x MES or MOPS buffer ( Invitrogen ) and transferred on nitrocellulose membranes , using the iBlot®2 Gel Transfer Device ( Invitrogen ) . Membranes were washed in TBS-T ( Tris Buffered Saline with 0 . 1% Tween-X100 ) and blocked with TBS-T containing 5% dry milk or 5% bovine serum albumin ( BSA , Sigma-Aldrich ) . Membranes were incubated with primary antibodies in TBS-T containing 1% dry milk or 1% BSA at 4°C overnight or for 2–3 days . The following primary antibodies were used: anti–FLAG M2 ( Sigma-Aldrich ) , anti-GFP ( Living Colors® Full-Length GFP Polyclonal Antibody , Clontech ) , anti-TICAM2 ( GeneTex ) ; anti-Rab11FIP2 ( ab180504 ) , anti-β-tubulin ( ab15568 ) , anti-IRF3 ( ab68481 ) and anti-phospho IRF3 S386 ( ab192796 ) and from Abcam; anti-phospho IRF3 ( S396 ) ( 4D4G ) and ( S386 ) , anti-phospho TBK1 ( S172 ) , anti-phospho IκBα ( I4D4 ) , anti-phospho p38MAPK ( T180/Y182 ) , anti-MyD88 ( D80F5 ) , anti-Rab11 ( D4F5 XP ) , anti-TRIF and anti-Rac1/2/3 all from Cell Signaling Technology; anti-Rab11FIP2 ( S-17 ) , anti-Cdc42 ( P1 ) and anti-PCNA ( FL-261 ) from Santa Cruz Biotechnology . Membranes were washed in TBS-T and incubated with secondary antibodies ( HRP-conjugated , DAKO ) for 1 h at room temperature in TBS-T containing 1% milk or BSA , developed with SuperSignal West Femto Substrate ( Thermo Scientific ) and captured with LI-COR Odyssey system . Images were analyzed by Odyssey Image Analysis software . THP-1 cells or human PBMC derived macrophages were seeded in 24-well glass-bottom plates ( MatTek Corporation ) and fixed with a 1:1 solution of methanol: acetone for minimum 1h at -20°C or 2% paraformaldehyde ( PFA ) as previously described [10] . Immunostaining was performed after blocking in 20% human serum in PBS , using the following primary antibodies diluted to 2 μg/mL in 2% serum in PBS: rabbit anti-TICAM2/TRAM ( H-85 ) , rabbit-anti-TLR4 ( H-80 ) , goat anti-Rab11FIP2 ( Santa Cruz Biotechnology ) . Normal rabbit IgG and goat IgG ( Santa Cruz Biotechnology ) were used as controls for antibody specificity . Highly cross-absorbed secondary antibodies used for confocal microscopy ( Invitrogen ) : Goat anti-Rabbit IgG ( H+L ) Alexa Fluor 647 ( A-21244 ) , Goat anti-Rabbit IgG ( H+L ) Alexa Fluor 488 ( A-11034 ) , Donkey anti-Goat IgG ( H+L ) Alexa Fluor 647 ( A-21447 ) and Donkey anti-Goat IgG ( H+L ) Alexa Fluor 555 ( A-21432 ) were used at a concentration of 1 μg/mL in 2% serum in PBS . Phalloidin Alexa Fluor 488 ( A-12379 , Invitrogen ) and Rhodamine Phalloidin ( R415 , Invitrogen ) were used for F-actin staining . Confocal images were captured using a Leica TCS SP8 ( Leica Microsystems ) equipped with a HC plan-apochromat 63×/1 . 4 CS2 oil-immersion objective and the LAS X software , using 488 nm , 561 nm and 633 nm white laser lines and the 405 nm laser for detection . Three-dimensional data was obtained from 12-bit raw imaging data used to the build individual Z-stacks for the individual E . coli or S . aureus , F-actin- , TLR4- , TRAM- and FIP2-channels . The Bitplane-IMARIS 8 . 4 . 2 software and the inbuild spot detection mode were used to define individual phagosomes . The data were presented as median fluorescence voxel intensity of phagosomes or antibody staining on phagosomes . The software produced numerical values that were tested using the GraphPad-PRISM 6 . 0 and found not to show a Gaussian distribution . Therefore , statistical significance was calculated by One-way ANOVA Kruskal-Wallis multiple comparison test , reporting multiplicity adjusted p values ( adj . p values ) . For single comparisons the Mann-Whitney test was used . Stimulated emission depletion ( STED ) microscopy was used to investigate the localization of TRAM on phagosomal membranes of fixed cells using a Goat anti-rabbit IgG STAR RED secondary antibody ( 2-0012-011-9 , Abberior ) and embedded in ProLong® Diamond Antifade Mountant ( P36970 , Invitrogen ) . STED images were acquired on a Leica TCS SP8 STED-3X microscope with a 100×STED objective ( HC PL APO 100×1 . 4 oil ) and STED 775 nm depletion laser combined with the 488 and 561 nm white laser lines in the regular confocal mode for the companion markers . 3D-STED images , 16-bit raw data , were deconvolved using SVI Huygens before generating single micrographs or performing 3D-rendering using the Bitplane-IMARIS 8 . 4 . 2 software . A flow cytometry-based phagocytic assay was used to measure the phagocytic efficiency of pHrodo-conjugated E . coli and S . aureus BioParticles in THP-1 cells , Tlr4-/- , Tram-/- , Myd88-/- iBMDMs from C57BL/6 mice [46] . According to the manufacturer , the pHrodo® dye conjugates are low-fluorescent outside the cell but fluoresce brightly in phagosomes following uptake . Prior to being added to cells the bacterial bioparticles were opsonized in 10% human A+ or normal mouse serum ( sc-45051 , Santa Cruz Biotechnology ) . After stimulation , cells in 6-well plates , were put on ice and washed with cold PBS before being detached by treatment with 500 μl of Accutase solution for 10–15 min ( Sigma-Aldrich ) and transferred into FACS tubes . The cells were washed with PBS followed by PBS containing 2% FCS . The fluorescence intensity was measured with a BD LRSII flow cytometer using the FACS Diva software ( BD Biosciences ) . Data were exported and analysed with FlowJo software v10 . 0 . 5 ( Tree Star ) . Cells were seeded at a density of 200 000 cells /well in in 24-well plates and serum-free medium was added to cells before infection . Live E . coli ( DH5α ) and S . aureus ( protein A negative subsp . aureus strain Wood 46 ) were grown to optical density of 0 . 35 at 600 nm , washed with PBS and given at a dose of 10–50 bacteria per cell in 4 to 5 biological replicates . Bacteria were centrifuged onto macrophage monolayers at 750 x g for 7 min at 4°C . Plates were warmed to 37°C in a water bath for 15 min and quickly transferred to ice where each well was washed 3 times with ice-cold PBS to remove extracellular bacteria . Warm medium with 10% FCS and 100 μg/ml gentamycin were added and cells incubated for 30 min at 37°C . Subsequently , the plates were transferred to ice and again washed 3x with cold PBS . To free phagocytosed bacteria cells were lysed in 1 ml sterile water . Viable counts were determined by plating 10 μl of 1 ml lysate ( diluted 10 to 20-fold ) onto LB agar plates that were incubated at 37°C overnight . Colony forming units ( cfu ) were counted and the number of bacteria per cell calculated . To normalize the cell number per well , the total protein concentration in cell lysates was calculated using the Pierce™ BCA Protein Assay Kit , according to manufacturer’s instructions . The data were found to follow the Gaussian distribution and statistical significance was therefore calculated by the ordinary one-way ANOVA Holm-Sidak´s multiple comparisons test reporting adj . p values . Total RNA from human macrophages from 7 healthy donors treated with NS RNA or FIP2 siRNA and stimulated with E . coli particles , were hybridized with the Nanostring HS_Immunology_v2_C2328 probe set and analysed according to the manufacturer´s protocol . Count data was collected at maximum resolution and imported to R/Bioconductor 3 . 4 . 1/3 . 5 [51] using the NanoStringNorm 1 . 1 . 21 package without its internal normalization functions . Probes with counts in the range of the average of all non-targeting probes plus 2 standard deviations were excluded from further analysis . The count data was voom transformed , cyclic loess normalized and analysed for differential expression in limma 3 . 32 . 5 [52] . The individual donors were used as blocking factor in the linear model . Genes showing an absolute expression log2-fold change > 1 . 25 and FDR < 0 . 05 compared to samples treated with NS RNA for each time point were considered differential expressed . Hyper geometric enrichment analysis for Gene Ontologies of Biological Processes ( GO BP ) was performed using clusterProfiler 3 . 4 . 4 [53] . Networks were constructed based on edges between GO BP terms sharing similar genes in the analysed gene set , node size was assigned according to the ratio of differential expressed genes in each GO term and coloured according to the absolute maximum log-fold change of genes in each GO term . The resulting network was imported to Cytoscape 3 . 4 . 0 for visualization . Human monocytes were isolated from peripheral blood mononuclear cells ( PBMCs ) as previously described [10] . Approval no . 2009/224 was received from the Regional Committees for Medical and Health Research Ethics ( REC Central , Møre og Romsdal , Sør-Trøndelag and Nord-Trøndelag counties ) for the use of Buffy coats to isolate PMBCs . Buffy coats were isolated from anonymized blood donors at the department of Immunology and Transfusion Medicine , St Olavs Hospital .
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The Gram-negative bacteria E . coli is the most common cause of severe human pathological conditions like sepsis . Sepsis is a clinical syndrome defined by pathological changes due to systemic inflammation , resulting in paralysis of adaptive T-cell immunity with IFN-β as a critical factor . TLR4 is a key sensing receptor of lipopolysaccharide on Gram-negative bacteria . Inflammatory signalling by TLR4 is initiated by the use of alternative pair of TIR-adapters , MAL-MyD88 or TRAM-TRIF . MAL-MyD88 signaling occurs mainly from the plasma membrane giving pro-inflammatory cytokines like TNF , while TRAM-TRIF signaling occurs from vacuoles like endosomes and phagosomes to give type I interferons like IFN-β . It has previously been shown that TLR4 can control phagocytosis and phagosomal maturation through MAL-MyD88 in mice , however , these data have been disputed and published before the role of TRAM was defined in the induction of IFN-β . A role for TRAM or TRIF in phagocytosis has not previously been reported . Here we describe a novel mechanism where TRAM and its binding partner Rab11-FIP2 control phagocytosis of E . coli and regulate IRF3 dependent production of IFN-β . The significance of these results is that we define Rab11-FIP2 as a potential target for modulation of TLR4-dependent signalling in different pathological states .
|
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2019
|
The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2
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The HIV-1 envelope glycoprotein , gp120 , undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry , which are being increasingly defined at the atomic level using isolated proteins . In comparison , antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells . Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function . Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells . Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events . We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding . This array comprises both neutralizing and non-neutralizing epitopes , the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function . Under non-permissive conditions for viral entry , both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions . Under entry-permissive conditions , epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents . These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step . The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo .
The attachment and entry steps in the Human immunodeficiency virus 1 ( HIV-1 ) replication process involve sequential interactions between viral envelope glycoprotein trimers and cell surface receptors [1] . Each interaction causes conformational alterations in the envelope structure that in turn enables a subsequent phase in the process [2–6] . Attachment begins when the gp120 component of the envelope trimer binds to cell surface CD4 . This causes the trimer to assume a structure ( CD4-induced or CD4i ) that allows gp120 to bind a co-receptor , typically CCR5 in the context of natural virus transmission [7–12] . Co-receptor engagement causes additional conformational rearrangements that translate to the gp41 viral transmembrane glycoprotein , which enables HIV-1-driven membrane fusion and viral entry . HIV-1 envelope-receptor interactions can drive membrane fusion between infected and uninfected cells or virions and target cells . The latter is thought to occur either by direct fusion with target cell membranes; by fusion with membranes of endocytotic vesicles [13 , 14]; or by a combination of such processes [15] , depending on the microenvironment in which the virus-cell interaction occurs [13] . Numerous experiments with isolated HIV-1 envelope proteins or HIV-driven membrane fusion systems have suggested that the HIV-1 envelope experiences significant changes in epitope presentation as it progresses through the course of HIV-1 attachment and entry [16–21] . These patterns of epitope exposure define the key determinants for HIV-1 susceptibility to the antiviral effects of anti-envelope humoral immunity . A great deal of effort has been applied toward elucidating conserved neutralizing domains expressed on free virions prior to host cell attachment . In gp120 , the most broadly reactive domains include the CD4 binding site [22–29] , sequences encompassing a high mannose cluster ( 2G12 [30] ) and glycosylated regions of V1V2 loop structures [31–38] . Other highly conserved epitopes exist within gp120 but are poorly antigenic on free virions . These include the co-receptor binding site and other ( CD4i ) domains that are fully exposed only after reaction of gp120 with soluble CD4 [39–52] . Very little is known about the antigenic nature of HIV-1 virions residing on the surfaces of permissive cells although several lines of evidence suggest that they are linked with antibody-mediated antiviral activities . A number of studies including our own have shown that CD4+ , CCR5+ cells coated with gp120 or whole viral particles are susceptible to Fc-receptor dependent , antibody-mediated antiviral activities , such as antibody-dependent cellular cytotoxicity ( ADCC ) [53–55] or antibody-dependent cell-mediated viral inhibition ( ADCVI ) [56] , or trogocytosis [57–59] . It is particularly noteworthy that CD4i epitopes enable ADCC against cell-bound virions [55] even though they are hidden within the trimers on free virions as measured by numerous approaches [24 , 48 , 51 , 60–67] . These findings indicate that cell-bound virions exhibit unique and unexpected epitope profiles linked with the development of humoral anti-envelope responses , some of which have antiviral activity . As examples , multiple studies have linked ADCC against gp120 with protection from infection in non-human primate ( NHP ) models [68–77]; with reduced risk of infection in the RV144 vaccine trial [78]; with decreased risk of mother-to-child HIV-1 transmission [79]; and lower viral loads during HIV disease progression [54 , 80 , 81] . These observations that CD4i epitopes are involved in humoral effector functions conflict with evidence that CD4i epitopes on viral trimers are occluded from antibodies by steric constraints extant before and after virion attachment to target cells [43 , 82–84] . To reconcile this question , we developed microscopy-based methods to interrogate the timing , duration and extent of gp120 epitope exposure as a consequence of virus-cell surface interactions . We focused on virions located on the outer membrane surfaces of freshly targeted cells as they are the most likely candidates for substantive interactions with the anti-HIV-1 humoral response . We find that an array of conserved gp120 domains comprising ADCC targets and CD4i domains are rapidly exposed on cell-bound virions along with constitutively exposed neutralizing domains . Such epitope exposure profiles provide insights for understanding relationships between HIV-1 replication and previously reported humoral immune functions .
To define epitope exposure patterns specific to bound virions , it was first necessary to establish which epitopes are exposed on unliganded HIV-1JRFL virions . Previously we used FCS to do this with untagged ( i . e . , without SNAP-ICAM-1 or CLIP-Vpr ) virions in solution [51] . Briefly , this system is based on molecular diffusion rates , which are proportional to the cube root of molecular weight . As a consequence , virion-bound antibody diffuses much more slowly ( Db ~ 8μm2/sec ) than free antibody ( Dnb ~ 65μm2/sec ) . The proportion of a fluorescence-tagged antibody that ‘slows down’ in the presence of a much larger virus species reveals the extent cognate epitope exposure on HIV-1 surface trimers . These studies showed that CD4i epitopes ( e . g . 17b ) are poorly exposed except when virions are treated with soluble CD4 ( sCD4 ) . Other CD4i epitopes such as A32 remain unexposed in the presence and absence of sCD4 , in accordance with earlier studies [16 , 17] . Similar experiments were carried out to confirm that the SNAP-ICAM-1/CLIP-Vpr-tagged particles expressed the same epitope patterns seen with untagged virions . Alexa Fluor 647-conjugated antibodies were used as probes for this purpose . These included Mab A32 [85–88] against a CD4i epitope in the gp120 C1 domain , Mab C11 [85 , 89] against a CD4i epitope in the gp120 C5 domain , Mab 17b [88 , 90] against a CD4i epitope in the co-receptor binding site , Mab b12 [23 , 91–93] against a constitutively expressed epitope in the CD4 binding site and Mab 2G12 [30 , 94–97] against an N-linked glycan ( N332 ) in the outer domain of gp120 . An anti-respiratory syncytial virus ( RSV ) antibody , Synagis , was used as a negative control for nonspecific Mab binding . Only a minor fraction ( < 20% ) of Mabs C11 , A32 and 17b exhibited slower diffusion coefficients indicative of virion binding ( Fig . 1 ) , in accordance with our previous findings [51] . Mab 17b binding was markedly improved in the presence of 100μg/ml sCD4 ( ~50% ) , while binding signals for Mabs A32 and C11 were not substantially increased , as predicted by previous studies [16] . In comparison , a large fraction ( > 50% ) of Mabs b12 and 2G12 exhibited virion-bound diffusion coefficients; such binding for Mab b12 was competitively reduced by sCD4 . As expected , none of the Mabs exhibited binding to virions devoid of envelope ( HIV-1ΔEnv ) ; Synagis showed no binding to either type of particle . Matching observations were made with HIV-1JRFL virions attached to Poly-L-Lysine-coated cover glass probed with Alexa Fluor 488-labeled Mabs ( representative images shown in S1 Fig ) . The virions were fluorescently tagged via SNAP-ICAM-1 and CLIP-Vpr using , respectively , membrane-impermeable Surface Alexa Fluor 546 and membrane-permeable CLIP-Cell Alexa 360 as described in Methods . The possibility of interference from free gp120 was addressed by tests with conjugated polyclonal D7324 immunoglobulin , which marks the presence of free gp120 . Incubation of the substrate-bound virions with this reagent failed to produce binding signals ( representative image in S2A Fig ) indicating that free gp120 was unlikely to be a confounding factor . In comparison , Mab binding signals were clearly seen for the b12 and 2G12 epitopes , whereas no staining was seen with Mabs A32 and C11 ( S1 Fig ) . Weak Mab 17b staining was seen on a subset of substrate-captured virions . This variance with FCS is likely to be a peculiar aspect of the capture format , which can be less accurate for probing epitope exposure versus solution binding [51] . Fluorescently tagged HIV-1JRFL virions ( Alexa 546 labeled-SNAP-ICAM-1; Alexa 360-labeled CLIP-Vpr ) were incubated with either TZM-bl cells ( CD4+; CCR5+ ) or HeLa-CD4 cells ( CD4+; CCR5– ) for 0 to 240 minutes at 37°C or 4°C , then washed , fixed with 4% paraformaldehyde , stained with Alexa Fluor 488-conjugated test Mab , and examined by confocal microscopy ( see Methods ) . TZM-bl cells are derived from HeLa cells [98–101] but typically express 10-fold higher surface levels of CD4 versus HeLa-CD4 cells as determined by flow cytometry . Putative surface-bound virions were captured for analyses within regions of interest ( ROI ) that were defined as follows ( also see Methods for details ) . First , the ROIs were selected based on the presence of a SNAP-ICAM-1 signal . Second , the ROI borders were configured to define where fluorescent signals fell to apparent background . Third , target cells were stained with phalloidin ( which binds to cortical F-actin ) in order to distinguish between the intracellular space and the cell surface . Only surface-bound particles , designated as such according to their orientation determined by phalloidin signals examined in lateral ( XY ) and axial ( Z ) orientations were considered for analysis . An example tracing of cell peripheries versus the virus—cell interface is shown in S3 Fig . For each selected ROI , a randomly selected mock ROI ( i . e . one not containing an apparent fluorescent signal ) was selected on a region of the same cell to define background fluorescence signals . All putative virion ROI fluorescence values were corrected for such background measures ( see Methods ) . Fig . 2 shows representative images of ROI ( selected using the criteria described above ) in tests with either neutralizing Mab 2G12 ( Fig . 2A ) or Synagis negative control ( Fig . 2B ) conjugated to Alexa Fluor 488 . As with free virions ( Fig . 1 ) , Mab 2G12 reacted with cell surface-bound virions starting within five minutes of virus-cell attachment ( Fig . 2A ) . This was interpreted to indicate that envelope spikes on a subset of virions were oriented away from the cell surface and therefore free to engage antibody . No such reactivity was seen with Synagis ( Fig . 2B ) . A series of experiments were carried out to probe bound virions with Mabs b12 , 2G12 , A32 , C11 , 17b , and negative control Synagis . Labelled HIV-1JRFL virions were bound to either TZM-bl cells or HeLa-CD4 cells for various periods of time up to 120 minutes at 37°C . All experiments were conducted under identical incubation conditions using the same preparation of labelled virus . To characterize the dynamics of HIV-1JRFL bound to each type of target cell , we first examined SNAP-ICAM-1 and CLIP-Vpr fluorescence data obtained from at least 1800 ROIs per time point from across antibody experiments . The detection of Vpr by the membrane-permeable CLIP-Cell Alexa 360 dye was not attributable to the presence of degraded virions with exposed contents , as there was no staining of virions with anti-p24 antibody unless the particles were first treated to permeabilize viral membrane ( representative images shown in S2A Fig ) . The raw intensity data was normalized , log-transformed , and then multiplied by 1000 for analysis using the FlowJo flow cytometry software as described in Methods . As shown in Fig . 3A , the selected virions reflected a range of SNAP-ICAM-1 and CLIP-Vpr signals on TZM-bl ( Fig . 3A , blue ) or HeLa-CD4 ( Fig . 3A , red ) target cells at five minutes post-attachment . The distribution of SNAP-ICAM-1 signal intensities fell within a 1 . 5 log range but was roughly the same on both target cell types and did not appreciably broaden or constrict over time in either case . In comparison , the CLIP-Vpr signals dispersed over more extended periods of time on the TZM-bl cells; in large measure because of the steadily increasing appearance of virion populations with low or no Vpr signal ( Fig . 3A ) . To more precisely examine the observed changes in CLIP-Vpr signals , Boolean gating analysis ( using FlowJo ) was used to partition virions into three populations representing high , low or no Vpr signal based on data for the 5-minute time point: Vpr ( 0 ) , corresponding to data points with no Vpr signal; Vpr ( low ) corresponding to data in the bottom 5th percentile of signal intensity; and Vpr ( high ) corresponding to the upper 95th percentile of the data set . These gates were then transposed onto HIVJRFL data sets at subsequent time points . Overall , the temporal analyses confirmed that ROIs circumscribing particles with low or no Vpr signal , which were rare early after cell binding , increased in frequency over time on the surfaces of TZM-bl cells ( Fig . 3B ) . After 120 minutes of contact with TZM-bl cells , roughly 28% of virions had low or no Vpr signal . The fraction of surface-bound virions with high Vpr signal decreased reciprocally ( Fig . 3B , Left panel ) . Changes in the Vpr ( low ) population were statistically significant for all adjacent time points ( two-sample Kolmogorov-Smirnov test; p value corrected for multiple comparisons < 0 . 05 ) except for the 15 minute versus 30 minute times ( S1 Table ) . On HeLa-CD4 cells , bound virions with a low Vpr signal remained relatively sparse ( roughly 5 to 8% ) and did not increase over time . Only a negligible amount of virions with no Vpr signal ( <1% ) was apparent at any time ( Fig . 3B , right panel ) . Collectively , these data indicate that HIV-1 particles with low or no Vpr signal were very sparse in the initial virus preparation but evolved over time once bound to cell surfaces , in a cell type-specific manner . We next asked whether the time-dependent appearance of ROIs with low or no Vpr signal on TZM-bl cells arises from protein-protein interactions or from virion-cell membrane intermixing . At low temperatures ( i . e . less than 23°C ) , protein interactions can occur but membrane mixing cannot [42 , 102 , 103] . Accordingly , we examined the appearance and distribution of HIV-1JRFL pseudovirus populations with high , low or no Vpr signals in the context of extended periods ( up to 240 minutes ) of interaction with TZM-bl cells maintained at 37°C or at 4°C . These experiments were also conducted using the same preparation of labelled virus as in the previous experiments . As seen in Fig . 3C , at 37°C the fraction of ROIs with no Vpr signal increased over time on TZM-bl cells to reach roughly 40% of the total population examined . Notably , the fraction ( 11 to 15% ) of bound particles with low Vpr signals remained relatively constant from 120–240 minutes at 37°C . In comparison , at 4°C , particles with no Vpr signal were very rare on TZM-bl cell surfaces ( < 1% of the total population ) even after prolonged incubation times . Particles with low Vpr signals were observed ( 5 to 7% of the total population ) and did not change appreciably over time . Collectively , the data indicate that certain populations of cell-bound virions exhibit changes in content depending on time , temperature and target cell type . We next evaluated gp120 epitope exposure on the virions bound to either TZM-bl or HeLa-CD4 cells as revealed by reactivity with b12 , 2G12 , A32 , C11 , and 17b anti-gp120 Mabs . Synagis was used as a negative control . At least 300 ROIs were defined per test antibody on virions bound to either target cell . Surprisingly , anti-CD4i Mabs A32 ( Fig . 4A ) , 17b ( Fig . 4B ) , and C11 ( Fig . 4C ) bound to attached virions in a manner similar to Mabs b12 and 2G12 ( see Fig . 2 ) . Fig . 4 shows a comprehensive perspective of Mab binding signals ( expressed as arbitrary units of intensity; a . u . i ) over time derived from ROIs on TZM-bl cells or HeLa-CD4 cells ( panels D and E , respectively ) . The distribution of signals depended on target epitope , cell type and virus-cell interaction time . The broadest signal distributions were observed with Mabs 2G12 , A32 and 17b signals on virions bound to TZM-bl cells for 120 minutes ( Fig . 4D ) . Overall , the geometric mean signals from the negative control Mab Synagis were at least 1 . 5 logs lower than the values observed with any anti-gp120 Mab under all matched test conditions . These differences were significant ( Kruskal-Wallis test; p < 0 . 0001 ) . As with virions attached to coverslips , free gp120 did not appear as a confounding factor on HIV-1JRFL virions bound to target TZM-bl cells , as experiments with D7324 antibody were negative ( representative images shown in S2B Fig ) . Further , virion degradation appeared to be minimal as there was also no anti-p24 staining within bound virion ROIs , including the Vpr ( 0 ) subpopulation ( representative images shown in S2B Fig ) . As an additional control for nonspecific antibody binding patterns ( i . e . ones that might occur in the absence of viral receptors ) , the tagged HIV-1JRFL virions were co-cultured with parental HeLa cells ( CD4- and CCR5-negative ) for 30 minutes at 37°C , then fixed and stained with Alexa Fluor 488-conjugated test Mab as described above . As the washing step of the above procedure removed all virions from these cells , confocal analyses were performed on particles left to settle on cells at random . ROIs were defined as described above . Under these conditions , Mab signals were seen for the constitutively expressed epitope 2G12 , but not with Mabs A32 and C11 . There was marginal staining of some particles with CD4i Mab 17b ( S4 Fig ) . Next , we examined the exposure of gp120 epitopes after longer periods of virus interactions ( 120 , 180 , and 240 minutes ) with TZM-bl cells at either 37°C , or 4°C . The cells were fixed and the attached virions stained with antibodies as in the previous experiments . At least 200 ROIs ( defined as in the previous experiments ) were surveyed for each experimental condition ( S5 Fig ) . Under all conditions , the geometric mean fluorescence signal observed with Synagis did not change over time and was similar to what was observed during shorter time frames ( see Fig . 4 ) . The geometric mean fluorescence signals for all test Mabs was significantly lower on virions bound to TZM-bl cells at 37°C ( S5 Fig , black ) than at 4°C ( S5 Fig , blue ) ( Kruskal-Wallis test: p < 0 . 0001 ) . The difference in test Mab intensity at these two different temperatures increased with increasing co-culture time , from less than 1 log to approximately 2–3 logs . Most Mab staining levels were maintained at 4°C through the 240-minute incubation period , with the exception of Mab A32 where mean fluorescence signals dropped about 1 log between 180 and 240 minutes . Fig . 5 summarizes the geometric mean fluorescence signals obtained from 5 to 120 minutes ( shown in Fig . 4 ) of virus interaction with the two target cell types at 37°C , compared with signals measured on TZM-bl cells after longer time points ( comprehensive data arrays shown in S5 Fig ) at the two different temperatures . Overall , the compiled data show that temporal changes in antibody staining signals were dependent on assay conditions . Signals were relatively static on HeLa-CD4 cells at 37°C and on TZM-bl cells that were temperature arrested at 4°C , although in the latter case the Mab A32 and 17b signals declined by roughly one to one-half log between 120 and 240 minutes . The most extensive changes in Mab binding signal were seen on virions bound to TZM-bl cells ( CD4+; CCR5+ ) at 37°C . Excepting Mab C11 , fluorescence signals for the test Mabs began to decline after 60 minutes of virus attachment under these conditions . The most extensive changes were apparent with Mabs A32 and b12 . Between 5 and 240 minutes there was a roughly 2–3 log drop in geometric mean fluorescence binding signals , reaching the background signal delineated by Synagis at the later time point . Mab C11 signals began to decline after 120 minutes of virus attachment to TZM-bl cells at 37°C and decreased roughly two logs by 240 minutes . The temporal declines in Mab staining signals on TZM-bl cells under entry-permissive conditions of 37°C were explored in greater detail . In particular , we investigated whether this was associated with the gradual appearance of multiple cell-bound virion subpopulations that occurred most extensively under these conditions . To do this , Mab fluorescence signals were extracted from the Vpr ( high ) , Vpr ( low ) and Vpr ( 0 ) subpopulations ( see Fig . 3 ) using FlowJo software . For each Mab , mean fluorescence intensity signals observed within these subpopulations were then plotted over time ( Fig . 6 ) . The highest intensities for all test Mabs were recorded on the Vpr ( high ) subpopulation ( Fig . 6 ) . The lowest levels of Mab staining occurred on the Vpr ( 0 ) subpopulation that appeared after 60 minutes of virus- TZM-bl cell interactions ( see Fig . 3 ) . Mabs staining on the Vpr ( low ) populations on each cell type was intermediate and more dynamic . Mab b12 signals in this population decreased one-half log within 30 minutes of attachment then steadily declined to near background levels ( defined by Synagis negative control measures ) by 240 minutes . Mab 2G12 signals were more persistent , decreasing less than one-half log after 60 minutes and remained above background levels for 240 minutes . Signals from anti-CD4i epitope Mabs , A32 , C11 , and 17b dropped at least one to one-half log by 120 minutes; approaching background levels ( Synagis equivalent levels ) by 240 minutes of virus-cell attachment . Notably , the Vpr ( 0 ) population marginally reacted with Mab 2G12 at all the time points; even less reactivity was observed with Mabs b12 or C11 . Somewhat higher binding signals in the Vpr ( 0 ) population were observed with Mabs A32 and 17b at 60 minutes of virus-cell attachment , but were lost with increasing times . Thus , it is likely that the general decline in Mab binding signals observed for the entire population of ROIs on TZM-bl target cells under entry-permissive conditions ( see Fig . 5 ) could be explained in part by the temporal emergence of the low or no Vpr subpopulations . Superresolution microscopy techniques can be used to study structural and functional aspects of HIV replication [104–112] at levels of resolution compatible with the size of a retroviral particle . Accordingly , to provide a more detailed validation of the confocal microscopy we analyzed the TZM-bl/HIV-1JRFL system used here with three-color , three-dimensional direct stochastic optical reconstruction microscopy ( 3D dSTORM ) . This method provides a 10-fold increase in resolution ( 20nm lateral and ~50nm axial resolutions [113] ) over standard confocal microscopy . The lateral resolution was determined by the average full width at half maximum ( FWHM ) measurements of all three fluorophores used . Neutralizing Mab 2G12 and anti-CD4i Mabs A32 and 17b were selected as epitope probes . Fluorescently tagged HIV-1JRFL virions ( Alexa 546 labeled-SNAP-ICAM-1; Alexa 360-labeled CLIP-Vpr ) were incubated with TZM-bl cells stained with a non-HIV neutralizing anti-CD4 antibody ( Alexa 647-conjugated OKT4 ) for 30 minutes at 37°C . Co-cultures were then fixed with 4% paraformaldehyde , stained with Alexa Fluor 488-conjugated test Mab ( 2G12 , A32 , 17b or Synagis ) , and mounted in imaging buffer for examination using the Nikon N-STORM superresolution microscope ( see Methods ) . The 3D dSTORM images were acquired and single molecule fitting and Gaussian images were rendered using the N-STORM software NIS Elements , where ROIs containing SNAP-ICAM-1and Mab signals proximal to CD4 staining were selected ( representative images shown in Fig . 7 & S6 Fig; S1 , S2 , and S3 Movies ) . Consistent with the confocal microscopy results ( Fig . 2 , Fig . 4 & S4 Fig ) , neutralizing Mab 2G12 ( Fig . 7 , S1 Movie ) and CD4i Mabs A32 ( S6 Fig , S2 Movie ) and 17b ( S6 Fig , S3 Movie ) produced a signal in proximity to SNAP-ICAM-1 and cell surface CD4 . Importantly , scaling in the lateral plane versus the axial imaging range showed that SNAP-ICAM-1 and Mab signals fell within a 100–200 nm area , consistent with the size of a single retroviral particle ( Fig . 7A , Fig . 7B , S6A Fig & S6B Fig , bottom panels ) . Overall , these data agree with the confocal imaging of ROIs in the TZM-bl/HIV-1JRFL system , showing that both neutralizing and CD4i epitopes are found on bound particles . Notably , the superresolution ROIs indicated that Alexa 488-conjugated Mabs 2G12 , A32 and 17b generated comparable ranges of “blinking” fluorescence signals ( localized events; Fig . 7C ) when attached to cell-bound virions . In accordance , the Mabs shared consistent signaling characteristics when calibrated in the context of superresolution ROIs comprising a constrained gp120 antigen ( see Methods and S7 Fig ) . The geometric mean of the localized events measured for Mab 2G12 was roughly two fold lower than the mean for Mabs A32 and 17b ( S7 Fig , Panel A ) . There were no such differences between Mabs A32 and 17b . However , the distribution of localized events among ROIs was similar for all Mabs ( S7 Fig , Panel B ) . ROIs with two localized events were most frequently detected with all antibodies; followed by ROIs with four localized events . ROIs containing larger numbers of localized events were relatively scarce ( S7 Fig , Panel B ) .
Abundant evidence indicates that the susceptibility of HIV-1 to the antiviral effects of humoral immunity is dependent on how and when epitopes are expressed on the viral envelope glycoproteins . Intensive research has revealed a few sites of vulnerability on free virions , which comprise constitutively expressed epitopes with variable degrees of conservation among strains . On HIV-1 gp120 , these include the CD4 binding site [23 , 82–84 , 91 , 114] and glycan structures present on the variable loops [30 , 61 , 94 , 115 , 116] . More highly conserved regions including a variety of CD4i epitopes also exist on gp120 but appear not to be exposed on free virions [12 , 51 , 117–122] . Much less is known about the gp120 epitopes exposed on HIV-1 after attachment to target cell surfaces although the available evidence suggests that it differs substantially from that of free virions . Certain anti-envelope antibodies that bind poorly to free virions are capable of directly neutralizing infection , suggesting that exposure of neutralizing epitopes occurs after target cell attachment [24 , 51 , 63 , 123] . More recently , we [55] demonstrated that virions bound to target cells present targets for Fc receptor-dependent antiviral mechanisms such as ADCC . Some of the more potent gp120 targets for such activity are located within the C1 region [55 , 124 , 125] , which demands cell surface CD4 engagement for exposure [52 , 60 , 85 , 86 , 126 , 127] . Additional targets include CD4i epitopes within the co-receptor binding site that also require cell binding for full exposure [16 , 17 , 51 , 52 , 55 , 122 , 128–130] . Such findings contrast with previous reports and in silico molecular models suggesting that CD4i epitopes are always occluded from immunoglobulin [43 , 82–84] because of steric constraints at the cell surface . Importantly , this view of gp120 is derived largely from the crystal structures of soluble envelope glycoproteins and/or cryo EM images of engineered soluble trimers [131 , 132] or free virions [22 , 133–135] . However , such information may not fully reflect the antigenic profile of HIV-1 virions as they proceed through its attachment and entry steps . HIV-1 epitope exposure on target cell surfaces has been successfully studied using confocal microscopy in systems where various reactants are fluorescently labeled . Such studies concerned epitope exposure during HIV-1 envelope-driven cell-cell fusion [16 , 17 , 136]; few studies have examined the disposition of surface bound particles [110 , 137] . Standard confocal microscopy is incapable of the resolution needed to accurately position different fluorescence signals on single retroviral particles . Even under ideal conditions ( e . g . , a high numerical aperture and optimized laser alignment ) the resolution of standard confocal microscopy is limited to approximately half of the wavelength of the excitation laser , the shortest of which ( 200 – 250nm ) is larger than the size of an HIV-1 particle ( 0 . 145–0 . 181 μm ) as indicated by high-resolution methods including electron microscopy or optical trapping [138 , 139] . Nevertheless , standard confocal microscopy can be adapted to derive multiple fluorescence measures emanating from ROIs comprising bound virions . Furthermore , confocal microscopy is sufficiently robust to capture temporal information from populations of bound virions in order to reveal changes in the dispositions of HIV-1 components versus surface epitopes [16 , 17 , 137] . In the present study , we explored this question using virions expressing surface ( SNAP-ICAM-1 ) and internal ( CLIP-Vpr ) fluorescent tags that can also be stained with Mabs conjugated to harmonious fluorescent labels ( see Methods ) . Multi-parameter fluorescence data for populations of virions imaged over time was then normalized for Boolean gating in order to reveal concurrent changes in the dispositions of proteins expressing the various fluorescent signals . HIV-1 has been reported to engage in endosomal entry [14 , 15 , 140–145] and/or endosomal recycling [146] in addition to direct virus-cell membrane fusion at the cell surface . A recent study by Herold et al . [147] showed that productive HIV-1 entry occurs predominantly at the plasma membrane , and does not require endocytosis . We focused exclusively on surface-bound virions since they are the most plausible targets for various humoral anti-HIV-1 effector mechanisms and/or for other antiretroviral agents designed to block viral entry . Further , the antigenic profiles of internalized virions are likely to be clouded by overlapping processes of endosomal versus lysosomal uptake [143] potentially involving productive infection or virion degradation , respectively . Accordingly , ROIs were selected for collection of fluorescence information based on outer membrane surface orientation along with a calibrated size corresponding to the apparent size of a retroviral particle . Although particles engaged in endosome-related processes may have been present in the system used here , they would not have been captured in ROIs since cell permeabilization occurred after anti-gp120 Mab treatment , surface fixing , and washing . ROIs without Mab signals were considered only if their orientation was on the extracellular surface . Three dimensional superresolution imaging showed that ROIs on the cell surface were proximal to CD4 and contained fluorescent signals comprising the dimensions of a retroviral particle ( Fig . 7 and S6 Fig ) . However , we cannot completely eliminate the possibility that some of the ROIs defined by confocal imaging ( e . g . , those with more irregular contours ) occasionally contained two or three HIV-1 particles captured in close proximity by the target cells . The overarching immunological feature indicated by our analyses was that virions bound to target cells rapidly expressed an array of conserved gp120 epitopes including ones ( e . g . , A32 , 17b , and C11 ) that were not exposed on the same virions when probed in solution ( Fig . 1 ) . Further , the signals obtained with Mabs specific for these epitopes were generally on par with what was observed with Mabs against constitutively exposed epitopes such as b12 and 2G12 ( Fig . 2 , Fig . 4 , and Fig . 7 ) . The exposure of the 2G12 epitope in our experiments agrees with previously published superresolution microscopic studies [110] showing that this epitope is expressed on surface bound virions . In this context exposure of the b12 epitope , which forms part of the CD4 binding site on gp120 , is most likely attributable to the presence of epitopes on the virion face oriented away from the target cell . All gp120 epitopes examined here exhibited a time-dependent reduction in immunoreactivity with cognate antibody ( Fig . 4 , Fig . 5 , S5 Fig ) . However , such changes varied among the entire population of bound virions and were linked to conditions permissive for downstream membrane fusion and entry events . This was evident from findings that epitope exposure patterns were relatively static on HeLa-CD4 cells not expressing co-receptor or on TZM-bl cells held at low temperatures that prohibit membrane mixing ( Fig . 5 , Fig . 6 , and S5 Fig ) . Based on these data , we posit that the broad decrease in gp120 epitope immunoreactivity seen on entry-permissive cells is linked to the presence of CCR5 co-receptor and downstream replication steps including the entry process itself . Possible mechanisms include the occlusion of epitopes via the repositioning of envelope spikes on attached virions , as was indicated by cryo-electron microscopy [134 , 148] . An alternative albeit more speculative explanation is that the fully “opened” structure of CD4- and co-receptor-bound gp120 is uniquely susceptible to proteolytic degradation . Since our analyses were deliberately focused on extracellular processes , such degradation would have to occur at the outer cell surface to explain our findings . In this regard , it was intriguing that CLIP-Vpr signals declined and/or disappeared over time within subsets of particles attached to entry-permissive TZM-bl cells ( Fig . 3 ) . The expansion of these subsets occurred after 30 minutes of virus-cell incubation at 37°C , which is consistent with previous observations of a similar “lag time” before transition state envelope structures and evidence of membrane fusion are detected [16 , 17 , 137 , 149 , 150] . Importantly , such particles were very rare at early time points and therefore were not dominant artifacts in the virus preparations used for our experiments . Further , the population with no detectable CLIP-Vpr signal appeared only on the TZM-bl cells expressing both CD4 and CCR5 and not on the CCR5 negative HeLa-CD4 cells ( Fig . 3 ) . Conversely , this population did not appear on TZM-bl cells at 4°C , where membrane fusion was temperature-arrested . Only a fraction of the particles bound to TZM-bl cells at high temperature acquired the low or no Vpr signal profile over time ( Fig . 3C ) . Nevertheless , the occurrence of these particles is consistent with conventional models of virion fusion with the plasma membrane [151–160] . In this case , receptor-driven membrane mixing processes are predicted to release virion content into the target cell thus generating particles with low or no Vpr signal . However , loss of Vpr at the cell surface is at odds with models require endosomal uptake during or after CD4 and co-receptor engagement [13 , 15] for fusion , entry and infection . These models would predict that Vpr should be retained within all particles that rest on the target cell surface . Our data are consistent with another proposed scenario [13 , 150] in which some particles progress beyond hemifusion at the plasma membrane to create small pores which release a limited amount of virion content . Vpr is a plausible component of such release if a fraction of the protein is located beneath the viral envelope as has been reported [161 , 162] . The transducing properties of Vpr might further promote movement out of the virion and into the cell [163] . An alternative possibility is that content release occurs when virions contact adjacent surfaces of the cell; e . g . , as might occur between tightly spaced microvilli [164–166] . In this situation , lateral forces could enable a process of “fusion from without” [13 , 167] . Finally , our observations might reflect processes that lead to the specific loss of Vpr signal . Exploration of such questions will require future studies that simultaneously track multiple intra-virion components . Another noteworthy feature of the bound particles with low or no Vpr signals was that they poorly expressed the gp120 epitopes examined here ( Fig . 6 ) . Thus , the processes on TZM-bl cells that led to the temporal decline in CLIP-Vpr signals may have concurrently impacted the disposition of the viral envelope . The broad loss of epitope reactivity with antibody is consistent with either the extensive degradation or loss of gp120 as has been studied previously [168–173] . Alternately , in view of the potential mechanisms for CLIP-Vpr signal reductions discussed above , epitope loss may occur as part of an active or abortive entry processes . It must be noted that certain features of epitope exposure were less apparent in the entire population of bound particles ( Fig . 5 ) compared to what was seen in subpopulations ( Fig . 6 ) . For example , within the entire HIVJRFL virion population ( Fig . 5 ) epitope reactivity with anti-gp120 antibodies declined with increasing attachment time on TZM-bl cells . Examinations of subpopulations ( Fig . 6 ) revealed that Mab staining was retained on Vpr ( high ) but progressively lost on Vpr ( low ) or ( 0 ) particles ( Fig . 6 ) . Precise links between SNAP-ICAM-1 , CLIP-Vpr and Mab signal patterns are difficult to establish with population-based studies such as this one . However , live-cell imaging of fluorescent signals from single particles in real time may reconcile such questions . Data from the current study provide a foundation for such future efforts . Emerging techniques of superresolution microscopy have been increasingly applied toward studies of HIV replication [104–112] . The application of one such technique; three dimensional , three-color dSTORM , allowed us to obtain a more refined view of the TZM-bl cell-bound HIVJRFL virions examined in this study . This approach revealed that ROIs such as those selected for confocal microscopy comprised SNAP ICAM-1-and anti-gp120 Mab fluorescence signals that were co-localized within an area less than 200nm ( Fig . 7 & S6 Fig ) in size , consistent with measures of HIV virions by cryo-electron microscopy [138] . Further , these signals occurred within biologically relevant proximity to signals from anti-CD4 antibody bound to cell surface CD4 . Mabs 2G12 , A32 and 17b produced such co-localized signal patterns , in agreement with the fluorescence signals measured by confocal analyses ( Fig . 2 & Fig . 4 ) of cell-bound virions . An interesting question concerned how many gp120 epitopes on a cell-bound virion might react with cognate Mabs under these conditions . This was approached by first calibrating the number of localized events produced by each of the anti-gp120 test Mabs when bound to a delimited amount of known target antigen . The methods we used ( see Methods and S7 Fig ) were deliberately conformed to preserve intact antibody interactions with native gp120 structures . The latter feature was accomplished via D7324 antibody capture of a single chain gp120-CD4 complex ( FLSC ) that presents a stabilized CD4-induced structure [174] . A caveat was that such ROIs could variably contain one or two target antigens captured by a D7324 antibody . Thus , the total localized events measured could derive from the formation of either one or two immune complexes with the conjugated anti-gp120 Mabs . However , this possibility applied equally to all Mabs tested . In the calibration system , Mab 2G12 trended toward slightly fewer localized events compared to Mabs A32 and 17b although in all cases ROIs with two localization events were most frequently observed ( S7 Fig ) . In comparison , the differences between numbers of localized events generated by the Mabs on TZM-bl cell-bound virions was not significant ( Fig . 7C ) , in accordance with the similar signal intensity levels detected with the antibodies in confocal microscopy ( Fig . 4D ) . Accounting for these considerations , we could estimate that TZM-bl cell-bound virions reacted with roughly 5–10 Mab 2G12 molecules; 3–6 Mab A32 molecules; or 4–8 Mab 17b molecules . In agreement with these estimates , previous superresolution microscopy studies using Stimulated Emission Depletion ( STED ) [110] , indicated that on average Mab 2G12 bound to 7 trimeric spikes on a mature HIV virion . Other published studies using protein purification [175] and electron tomography [176] suggest that there are between 7 and 14 , or from 8 to 10 spikes per HIV-1 particle , respectively . Taken together , the patterns of gp120 epitope exposure revealed by imaging of cell-bound HIV-1JRFL virions are inconsistent with models in which CD4i epitopes are predicted to be fully occluded from antibodies at the cell surface [43 , 82 , 84] . There are two possible explanations for this disagreement . The simplest one is that the CD4i and other epitopes are not buried at interfaces where virions contact target cell membranes to the extent that interactions with immunoglobulins are prevented . The second , more speculative possibility is that HIV-1 binding to cell surface receptors propagates conformational changes across the virion that impact envelope spikes distal to the cell contact zone . Such plasticity has been observed with other enveloped viruses [177–183] . A related question concerns why antibodies to CD4i epitopes such as A32 are not directly neutralizing even though the cognate epitopes are exposed at the cell surface . The exposure of such epitopes without direct neutralizing consequences was previously established for HIV-driven cell-cell fusion [16 , 17] . Likely explanations pertinent to cell-bound virions include: epitope exposure occurs on trimers that fail to fully enable membrane fusion machinery; epitope exposure that occurs after the membrane fusion process has been committed; epitope exposure that occurs distal to the cell contact zone as suggested above . Further exploration of these possibilities in the context of HIV-1 attachment will require more advanced spatial analyses using superresolution microscopy and other molecular techniques . Importantly , the gp120 imaging patterns elucidated here for attached virions are entirely consistent with previous findings that certain anti-CD4i epitope Mabs mediate potent ADCC activity in vitro against virions bound to target cells [55] . This accordance supports the concept that there may be multiple opportunities for humoral responses to counter HIV-1 infection after attachment . Following the observations made here , it could be envisioned how Fc receptor-dependent modes of humoral immunity might locate and destroy cells recently targeted by HIV-1 for replication . In this case , the ensuing effector cell activity might have a protective effect even if it is directed toward replication defective particles if other virions on the same cell manage to initiate productive replication . However , the efficacy of such responses in vivo is obviously dependent on the number of virions that attach to any given target cell , the durability of epitope exposure during initial stages of HIV-1 replication , and the proximity of effector cells to the epicenter of HIV-1 replication . Our data suggest that gp120 epitope exposure on attached virions is transient but sustained for periods of time that might allow immune mechanisms to impact infection under certain conditions . Information from clinical trials of candidate HIV-1 vaccines along with systematic testing of anti-gp120 Mabs in various animal models of HIV-1 infection could help to reconcile this question .
HeLa cells , which express CXCR4 but not CD4 or CCR5 ( CD4- , CCR5- ) [7 , 8 , 184] , HeLa-CD4 clones ( CD4+ , CCR5- ) stably transfected to express CD4 , and TZM-bl cells expressing CD4 and CCR5 ( CD4+ , CCR5+ ) were used . TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program , Division of AIDS , NIAID , NIH: TZM-bl from Dr . John C . Kappes , Dr . Xiaoyun Wu and Tranzyme Inc . [98–101] . The HeLa-CD4-LTR-β-gal cell line with high CD4 expression was also obtained through the AIDS Research and Reference Reagent Program , Division of AIDS , NIAID [185] . Both cell lines were maintained in Dulbecco modified Eagle medium ( DMEM; Gibco-BRL ) supplemented with 10% heat-inactivated fetal bovine serum ( FBS ) , 2 mM L-glutamine , and antibiotics , with 0 . 1 mg of G418 ( Gibco-BRL ) /ml , and 0 . 05 mg of hygromycin B/ml supplements for HeLa-CD4 cells in a 37°C incubator with 5% CO2 . CD4 and CCR5 content of these cells was quantified using the QuantiBRITE PE fluorescence quantitation kit ( BD Biosciences ) . TZM-bl cells had 1 . 7 x 105 CD4 and 5 . 4 x 104 CCR5 molecules/cell , while HeLa-CD4 cells had 1 . 8 x 104 CD4 molecules/cell and no CCR5 . To make pSNAP-ICAM-1 , human ICAM-1 was extracted from the pCDM8-ICAM-1 vector ( Addgene , Cambridge , MA ) using PCR , and cloned into the SNAP-tag expressing pSEMXT-26m plasmid ( New England Biolabs , Ipswich , MA ) at the 3’ end of the SNAP-tag coding region using the SbfI and BamHI restriction sites . To make pCLIP-Vpr , the Vpr coding region was extracted from pEGFP-Vpr vector obtained through the NIH AIDS Research and Reference Reagent Program , Division of AIDS , NIAID [186] by PCR , and cloned into the pCLIPm vector ( New England Biolabs ) at the 3’ end of the CLIP-tag coding region using the SbfI and BamHI restriction sites . pSNAP-ICAM-1 and pCLIP-Vpr were sequenced by the Biopolymer Laboratory of the University of Maryland School of Medicine and assigned GenBank accession numbers Banklt1758508 Seq1 KM555100 and Banklt1758535 Seq1 KM555101 , respectively . CCR5-tropic HIV-1JRFL pseudoviruses were generated by co-transfecting HEK 293T cells with ( i ) pSG-3ΔEnv virus backbone with an Env deletion obtained through the AIDS Research and Reference Reagent Program , Division of AIDS , NIAID [187 , 188]] , ( ii ) pCAGGS-JRFL , a plasmid containing JRFL Env , obtained through the AIDS Research and Reference Reagent Program , Division of AIDS , NIAID [189] ( iii ) pSEMXT-ICAM-1 ( expresses SNAP-tagged ICAM-1 captured by virions from the cell membrane during viral budding [190 , 191] , and ( iv ) pCLIP-Vpr ( expresses CLIP-tagged Vpr marking virion content ) [157–160] . Transfections were accomplished using FuGENE 6 ( Roche , Indianapolis , IN ) transfection reagent at a 3:1 reagent-DNA ratio . Pseudovirus-containing supernatant was harvested after 3 days , and concentrated about 10-fold by incubating with PEG-it virus precipitation solution ( System Biosciences , Mountain View , CA ) for 18 hours at 4°C as recommended by vendor . The antigen content of pseudoviruses was quantified using p24 and gp120 ELISAs , and their TCID50 was obtained as previously described by Li et al . [192] . HIV-1JRFL pseudoviruses with gp120 to p24 ratio of 1:10–1:50 , and 200 , 000–500 , 000 TCID50/mL ( FCS ) or 1 x 106–3 x 106 TCID50/mL ( Confocal and Superresolution microscopy ) were used . We probed anti-Env epitope exposure on free HIV-1JRFL as well as on cell-attached virions by examining the binding properties of fluorescently-labeled cognate human monoclonal antibodies ( Mabs ) . 2G12 mAb was purchased from Polymun Scientific ( Vienna , Austria ) ; b12 , A32 , C11 and 17b , were expressed from plasmid clones using an IgG1 backbone for heavy-chain variable regions and either a κ- or λ-chain expression vector for light-chain variable regions by transfecting HEK 293T cells . Mabs were purified from culture supernatants by protein-A chromatography . A32 and 17b were initially provided by James Robinson , Tulane University , New Orleans , La . The humanized monoclonal anti-respiratory syncytial virus ( RSV ) antibody , SYNAGIS® ( MedImmune LLC , Gaithersburg , MD ) , and human plasma IgG ( Calbiochem , La Jolla , CA ) were used as non-specific negative controls . D7324 ( Aalto BioReagents , Dublin , Ireland ) , an antibody that binds the C terminus of monomeric gp120 , and an HIV-1 p24 antibody ( Abcam Ab9071 , Cambridge , MA ) , were used to assess gp120 dissociation from HIV-1JRFL trimers and exposure of capsid , respectively . OKT4 , an antibody against CD4 that does not neutralize HIV-1 , was purchased from BioLegend ( San Diego , CA ) . These Mabs were fluorescently labeled with Alexa Fluor 647 ( for FCS experiments ) and Alexa Fluor 488 ( for confocal and superresolution microscopy experiments ) using monoclonal antibody labeling kits from Molecular Probes ( Eugene , OR ) following manufacturer instructions . Briefly , 100 μg of Mabs were labeled with Alexa Fluor reactive dyes which have a succinimidyl ester moiety that reacts efficiently with their primary amines and form stable dye-protein conjugates . The labeled Mabs were separated from unreacted dye by centrifugation through a spin column at 1100 x g . The dye:protein ratio of the recovered Mabs was measured using a UV-vis spectrometer ( Nanodrop 2000 , Thermo-Scientific ) . Conjugated Mabs used in our experiments had an optimal ratio range of 4–6 ( Alexa Fluor 488 ) or 3–5 ( Alexa Fluor 647 ) moles of dye per mole of Ab . Fluorescence Correlation Spectroscopy ( FCS ) is a methodology that allows real-time detection of protein-protein interactions in solution , by measuring diffusion and reaction kinetics of fluorescently-labeled biomolecules [193] . The binding of Alexa Fluor® 647-labeled Mabs b12 , 2G12 , A32 , C11 , 17b , and anti-RSV antibody , Synagis ( negative control ) to HIV-1JRFL was monitored by tracking diffusion of their fluorescent label across the observation area , where unbound antibodies will diffuse much faster than those that bound viral particles as described in [51] . Briefly , HIV-1JRFL pseudovirions were diluted to 10μg/mL p24 equivalent in a 100-μL reaction volume ( gp120:p24 ratio of 1:50 ) , and were first incubated with 100μg/mL non-specific IgG1 ( 1 . 5μL of a 7mg/mL stock ) for 90 minutes at 37°C to block non-specific binding . Then 1μL of the test Alexa Fluor® 647-conjugated Mab ( 4 . 5–6 . 6μg/mL ) was introduced and allowed to interact with pseudovirions for 90 minute at 37°C . In experiments where CD4-induced conformational changes were studied , HIV-1JRFL pseudovirions were pre-incubated with 100μg/mL sCD4 ( Biogen ) ( 1 . 5μL of a 7mg/mL stock ) along with the non-specific IgG1 for 90minutes at 37°C before the addition of test Alexa Fluor 647 Mab . For spectroscopic measurements , 11μL of the reaction mixture was loaded onto an FCS slide reservoir , sealed , then placed on the Picoquant MicroTime 200 , a time-resolved confocal microscope ( inverted ) , with a high numerical aperture ( NA = 1 . 3 ) oil objective ( 100× magnification ) . The samples were excited with λex = 635 nm laser , and fluorescence signals from the Alexa Fluor 647 Mabs were collected over 60 seconds in a constant detection volume that is continuously replenished . PicoQuant Symphotime software was used to generate the autocorrelation function of the fluorescent fluctuations of the Alexa Fluor® 647 Mab signal . The autocorrelation function of fluorescence intensities is given by the product of the Mab intensity at time t , I ( t ) with the intensity after a delay time τ , I ( t+τ ) , typically in the range from 10-2 to 102 ms , averaged over the 60 seconds of measurement . For experiments where only the Alexa Fluor 647- Mabs were in the sample , the autocorrelation function was fitted with the pure diffusion model equation for single species , where a diffusion coefficient of 65μm2/sec was extracted for the 150kD IgG molecules . In the presence of HIV-1JRFL pseudovirions , the autocorrelation was fit to a two-species diffusion model , where one species had the unbound Mab diffusion coefficient of 65μm2/sec ( Dnb ) , and the second species with a diffusion coefficient of 8μm2/sec ( Db ) , represented Mabs bound to the 100nm HIV-1JRFL . These equations were also used to obtain the amount of Mab exhibiting the slower diffusion rate as a measure of the percentage of virus-bound Mabs in each reaction mixture . The derivation of all the equations is described in [51] . 4 . 0 x 105 cells ( HeLa , TZM-bl , or HeLa-CD4 ) were attached to 22-mm2 glass coverslips ( Fisher Scientific ) and incubated overnight at 37°C in 5% CO2 . The next day , HIV-1JRFL pseudovirions expressing SNAP-ICAM-1 and CLIP-Vpr were fluorescently labeled with their respective substrates , membrane-impermeable SNAP-Surface Alexa 546 ( Red , Exmax: 558nm , Emmax: 574nm ) , and membrane-permeable CLIP-Cell Alexa Fluor 360 ( Blue , Exmax: 357nm , Emmax: 437nm ) ( New England Biolabs ) , by incubation for 20 minutes at 37°C . TZM-bl and HeLa-CD4 cells grown on coverslips were co-cultured with 0 . 5mL of 1 x 106–3 x 106 TCID50/mL ( equivalent to 7 . 0 x 105–2 . 1 x 106 PFU; ~ 1–3 MOI ) of these fluorescently-labeled HIV-1JRFL pseudovirions for the indicated times ( 5 , 15 , 30 , 60 , 120 , 180 , or 240 minutes ) at either 37°C or 4°C , washed , then immediately fixed with 4% Paraformaldehyde ( Electron Microscopy Sciences , Hatfield , PA ) for 15 minutes . Experiments with receptor-negative parental HeLa cells eliminated the wash step , as this operation removed all viral particles ( due to the absence of cell surface receptors ) . Thus , for microscopy the particles were left settled on the cells . Non-specific interactions were blocked by incubating coverslips with 10% normal goat serum ( Thermo Scientific , Rockford , IL ) and 100μg/mL non-specific IgG1 solution for 30 minutes at 4°C , then the specific epitopes were recognized by incubating cells with 5μg/mL Alexa Fluor 488 ( Green , Exmax: 495nm , Emmax: 519nm ) -conjugated Mabs ( b12 , 2G12 , A32 , C11 , 17b , or Synagis ) for 1 hour at 4°C . For actin staining , after the addition of Mabs , coverslips were post-fixed with 4% Paraformaldehyde for 10 minutes , permeabilized in 0 . 2% Triton X-100 ( Sigma ) then incubated with Alexa Fluor 647-conjugated Phalloidin ( Invitrogen ) for 30 minutes at room temperature . Coverslips were mounted in Fluoromount ( Sigma ) and observed using the Zeiss Laser Scanning Microscope ( LSM ) 5 DUO . To verify that our results were independent of the fluorophore choices , we alternatively labeled HIV-1JRFL with SNAP-Surface Alexa 546 ( Red , Exmax: 558nm , Emmax: 574nm ) and CLIP-Cell BG505 ( Green , Exmax: 505nm , Emmax: 532nm ) , and recognized anti-Env epitopes with cognate Mabs conjugated with Alexa Fluor 350 ( Blue , Exmax: 346nm , Emmax: 442nm ) . To view unbound virions by microscopy , 22-mm2 glass coverslips were coated with 0 . 1% Poly-L-Lysine w/v in water ( Sigma ) overnight at room temperature . The Poly-L-Lysine was removed and coverslips were washed with 1X PBS . Fluorescent-tagged HIV-1JRFL pseudovirions ( SNAP-Surface Alexa 546 and CLIP-Cell Alexa Fluor 360 ) were added and incubated for 2 hours at 4°C . Unbound virions were then washed off , and virions were fixed in 4% paraformaldehyde , non-specific interactions blocked with 10% normal goat serum , and test Alexa Fluor 488-conjugated Mabs added as described above . Virus—cell interactions were imaged using the Zeiss Laser Scanning Microscope ( LSM ) 5 DUO using a 63X oil-immersion lens at room temperature , where signals from the dually labeled virus and anti-HIV-1 antibody signals were tracked in 3D by taking 512 by 512 XY scans , 0 . 279018 μm/pixel size , in 0 . 2-μm increments in the Z-direction . Images were taken at 5 or more randomly selected regions that span the coverslips . To minimize cross-talk between the different fluorescence labels , the multi-tracking setting of the Zeiss Zen software was used , which sequentially illuminated and detected one fluorophore at a time [194] . The image settings were saved and re-used for subsequent experiments in order to compare differences in virus and antibody intensity levels . Images were processed using MetaMorph ( Molecular Devices ) program . Maximum projection images were used to include fluorescence signals from all the Z planes . Viral particles on the surface of target cells were identified using SNAP-ICAM-1 signals . Orientation on the outer cell membrane surface was determined by Phalloidin staining signals ( see S3 Fig . ) . Regions of interests ( ROIs ) were traced around ICAM-1 SNAP Surface Alexa 546 signals . The ROIs perimeters were configured to define where fluorescent signals fell to apparent background . Such boundaries were more expansive than the full width at half maximum measures of point spread function ( 200–250 nm for our instrument ) , but were desirable as a means to include all the intensity signals for unbiased and comprehensive comparisons . This approach was rehearsed using 0 . 1μm carboxylate-modified red microspheres ( FluoSpheres; Invitrogen ) , which have similar excitation and emission spectra as the SNAP-ICAM-1 label ( Exmax: 540nm , Emmax: 560nm ) and a diameter matching the lower end of the size range [138 , 139] reported for HIV particles ( 0 . 145–0 . 181 μm ) . As such , the microspheres provided a serviceable gauge , although they are designed to be intensely fluorescent with little or no photobleaching and are likely to exhibit greater image resolution compared to the randomly incorporated SNAP-ICAM-1 label on virus surfaces . Configured as stated above , ROIs determined for microspheres settled on to target cells exhibited a median diameter of 0 . 72 ± 0 . 09 μm . The ROIs for putative cell-bound virions exhibited a similar median diameter of 1 . 32 ± 0 . 17 μm; ROIs for virions bound to coverslips exhibited a median diameter of 1 . 25 ± 0 . 15 um . These concordant sizes indicated that in general single entities versus large “clumps” of virions were interrogated on target cell surfaces . However , the possibility that we occasionally surveyed multiple particles located in close proximity cannot be completely eliminated . To correct for background fluorescence , each ROI meeting the above criteria was compared to a mock ROI . These were randomly selected regions of equivalent diameter on condition-matched cells that did not have apparent SNAP-ICAM-1 signals . Background intensity levels in all 3 color channels were recorded and subtracted from corresponding ROI measures . For the displayed images , each fluorescent channel was deconvolved using the nearest neighbor algorithm in MetaMorph to decrease the noise in the system . SNAP-ICAM-1 , CLIP-Vpr , and antibody relative intensity values were exported from MetaMorph as excel files and graphed using GraphPad Prism ( version 5 . 04 for Windows ) or SigmaPlot ( version 12 . 0 for Windows ) . For all data points to appear in the log scale , intensity values less than 1 were amended to be equal to 1 . Since the virus and antibody intensity signals did not have a normal distribution , the non-parametric Kruskal-Wallis was performed for statistical analysis , using GraphPad Prism . To quantify the cell-bound particle and antibody signals , fluorescence intensity data of HIV-1JRFL ( SNAP-ICAM-1 & CLIP-Vpr ) , and test Mabs were imported into the flow cytometry software , FlowJo X . This allowed standard Boolean gating of the data in order to examine differences in the various treatment groups . To achieve this , a same day experimental set was normalized , and then log transformed to offset the treatment of the input data as log values . Finally , the numbers were multiplied by 1000 to spread the data set to the capacity of the software [the number of pseudocolor palettes of a 12-bit image ( 212 = 4096 ) ] . Conditions similar to the confocal experiments were used for direct stochastic optical reconstruction microscopy ( dSTORM ) experiments . 4 . 0 x 105 TZM-bl cells were seeded on 20-mm2 glass-bottom MatTek dishes ( MatTek Corp . , Ashland , MA ) and incubated overnight at 37°C in 5% CO2 . The next day , HIV-1JRFL pseudoviruses expressing SNAP-ICAM-1 and CLIP-Vpr were fluorescently labeled with SNAP-Surface Alexa 546 and CLIP-Cell Alexa Fluor 360 , respectively as described above . The TZM-bl cells were co-cultured with 0 . 5mL of 3 x 106 TCID50/mL of these fluorescently-labeled HIV-1JRFL , and with 5μg/mL Alexa 647-conjugated OKT4 ( a non HIV neutralizing anti-CD4 antibody ) for 30 minutes at 37°C , then immediately fixed with 4% Paraformaldehyde for 15 minutes at room temperature . Non-specific interactions were blocked by incubating coverslips with 10% normal goat serum and 100μg/mL non-specific IgG1 solution for 30 minutes at room temperature . Epitopes of interest were probed with 5μg/mL Alexa Fluor 488-conjugated antibodies for 30 minutes at room temperature . Dishes were then post-fixed in 4% Paraformaldehyde for 15 minutes at room temperature and stored in 1X PBS at 4°C until imaging . Three-color , 3D dSTORM imaging was carried out using the Nikon N-STORM microscope ( Nikon Instruments Inc . , Melville , NY ) . Using the 100X CFI Apo TIRF oil-immersion objective ( 1 . 49 NA ) , 256 by 256 XY scans , 0 . 165 μm/pixel size , were acquired , and 3D images were obtained using the astigmatism method of 3D localization [113] . The 647nm , 561nm , and 488nm laser lines were used to excite Alexa 647-conjugated OKT4 , SNAP-ICAM-1 labeled with SNAP Surface Alexa 546 , and Alexa 488-conjugated Mabs , respectively . The 405 laser was used to obtain TIRF images of the CLIP-Vpr Alexa 360 signal . The oxygen-scavenging imaging buffer was 14mg glucose oxidase and 50μL of 17mg/mL catalase ( Sigma ) in 200μL Component A ( 10mM Tris , 50mM NaCl ) ; Component B ( 50mM Tris-HCl , 10mM NaCl , 10% glucose ) ; and 1M cysteamine ( MEA ) . Single molecule fitting and Gaussian images were rendered using the N-STORM software NIS Elements ( version 4 . 30 . 01 ) . The localization precision for all three fluorophores was determined to be 20 nm using full width at half maximum ( FWHM ) , with a 50 nm axial resolution . After high resolution images were obtained , ROIs were defined around single virions with ICAM-1 and Mab signals within 200nm . Virus-bound Mab signals and the number of localized signals were recorded . The calibration method was designed to equate localized events ( Alexa Fluor 488 fluorophore signal “blinks” ) with a defined number of dye-conjugated antibody molecules within a superresolution ROI . To do this , MatTek dishes were coated with 5μg/mL D7324 in 1X PBS overnight at room temperature and were used to capture 0 . 5μg/mL full length single chain ( FLSC ) , a monomeric gp120-sCD4 complex stably expressing CD4i epitopes [174] . Thus , the substrate-bound bivalent antibody in an ROI could theoretically capture one or two FLSC antigens; which in turn could capture one or two conjugated Mabs . Importantly , the capture format was selected because it preserves the native structure of the target antigen by avoiding chemical modifications or direct adsorbance to substrate , either of which can perturb epitope presentation . Alexa Fluor 488-labeled Mabs 2G12 , A32 , or 17b ( 5μg/mL ) were incubated with the captured FLSC for 30 minutes at room temperature , fixed and imaged by dSTORM . ROIs were circumscribed around distinct Gaussian signals , and the corresponding number of localized events was extracted . The number of localized events measured within an ROI was then taken to reflect the presence of one or two conjugated Mabs . As this qualification applied to all target epitopes , cross-comparisons of Mabs tested under identical conditions were feasible .
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A major strategy for blocking HIV-1 infection is to target antiviral antibodies or drugs to sites of vulnerability on the surface proteins of the virus . It is a relatively straightforward matter to explore these sites on the surfaces of free HIV-1 particles or on isolated viral envelope antigens . However , one difficulty presented by HIV-1 is that its surface proteins are flexible and change shape once the virus has attached to its host cell . To date , it has been difficult to predict how cell-bound HIV-1 exposes its sites of vulnerability . Yet the antiviral activities of certain antibodies indirectly suggest that there must be unique sites on cell-bound HIV-1 that are not found on free virus . Here , we use new techniques and tools to determine how HIV-1 exposes unique sites of vulnerability after attaching to host cells . We find that the virus exposes a remarkable array of these sites , including ones previously believed hidden . These exposure patterns explain the antiviral activities of various anti-HIV-1 antibodies and provide a new view of how HIV-1 might interact with the immune system . Our study also provides insights for how to target HIV-1 with antiviral antibodies , vaccines , or antiviral agents .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2015
|
Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells
|
In our daily lives timing of our actions plays an essential role when we navigate the complex everyday environment . It is an open question though how the representations of the temporal structure of the world influence our behavior . Here we propose a probabilistic model with an explicit representation of state durations which may provide novel insights in how the brain predicts upcoming changes . We illustrate several properties of the behavioral model using a standard reversal learning design and compare its task performance to standard reinforcement learning models . Furthermore , using experimental data , we demonstrate how the model can be applied to identify participants’ beliefs about the latent temporal task structure . We found that roughly one quarter of participants seem to have learned the latent temporal structure and used it to anticipate changes , whereas the remaining participants’ behavior did not show signs of anticipatory responses , suggesting a lack of precise temporal expectations . We expect that the introduced behavioral model will allow , in future studies , for a systematic investigation of how participants learn the underlying temporal structure of task environments and how these representations shape behavior .
Our ability to represent time and to generate complex actions and plans based on this representation are central to all aspects of our behavior . Knowing when to act , precisely , is clearly crucial for our survival [1 , 2] . Thus , it is not surprising that the question of how we perceive and represent time gains an increasing interest in neurosciences [3–5] . However , in comparison to our understanding of spatial cognition , the neural basis of time perception is still poorly understood [6] . Here , we address the question of how the brain , in principle , can use knowledge about a hidden temporal structure of a task when making decisions . Traditionally , the question how we learn the structure of the world and use these representations for decision making , has been investigated from the perspective of reinforcement learning [7 , 8] . The focus of these investigations has been on how learning is driven by prediction errors [9] , defined as a mismatch between expected and observed outcomes of one’s actions . More recent studies on human behavior in dynamic environments [10–13] have demonstrated that the relative precision of one’s prior beliefs and current sensory information weights prediction error signals [14–21] . These findings indicate that humans update their beliefs about the structure of the world akin to a rational ( Bayesian ) agent [22 , 23] . This suggests that one can approximately describe human behavior using the methodology of probabilistic inference . The key advantage of the probabilistic inference framework over the standard reinforcement learning modelling approach is that one can embed the knowledge about the structure of the world and the uncertainty about that knowledge within a generative model that describes the known rules that shape the dynamics of the world . A potential limitation of previous approaches—both probabilistic and reinforcement learning based—is that they do not take into account the underlying temporal structure of the task . Recently a series of studies have demonstrated the relevance of learned temporal associations on attention , perception , and sensory integration [24–27] . Experimental paradigms employed in this studies utilized observable temporal structure within trial . Similarly , in [28 , 29] authors demonstrate that learning of the underlying temporal structure of the task is used by human participants ( or animal subjects ) to anticipate the moment in time at which rules are most likely to change . In contrast to the experimental design which use within trial temporal structure , here participants were exposed to a hidden temporal structure across trials . Motivated by these findings , we propose a way to extend current probabilistic models of behavior , aimed at describing decision making in dynamic environments , to incorporate an explicit representation of the underlying temporal structure in the form of prior beliefs about state durations . In particular we will focus on the case when the state durations are not directly observable , but inferred across multiple trials using observable changes in action outcomes . The two essential components of the proposed behavioral model are ( i ) the update of beliefs about states and duration derived using approximate inference under hidden semi-Markov models [30 , 31] and ( ii ) the update of beliefs about actions , that is the planning process , cast as an inference problem [32–34] . As a test bed for illustrating the key properties of the model and for demonstrating its applicability to experimental studies we adopted a probabilistic reversal learning task [35] . This task and its variants have been frequently used in human and animal studies to investigate key properties of flexible behavioral adaptation in dynamic environments [29 , 36–38] . In a typical setup participants first learn to associate a particular choice with a reward , followed by a sequence of reversals of reward contingencies . The interesting question is typically how fast participants adapt their choices to these new contingencies . In the work here , we address the question , whether participants actually use the underlying temporal task structure to predict the moment of the reversal so that behavior is adapted faster as compared to the case when the reversal is unexpected . Using the reversal learning task , we first demonstrate using simulations that we can link sub-optimal behavior in changing environments to a misrepresentation of the underlying temporal structure of the changes . Subsequently , when fitting the behavioral model to experimental data , we relate the measured behavior of each participant to model parameters that define the beliefs about the temporal structure of reversals . Strikingly , our results suggest a heterogeneous distribution of the task representation across participants where some participants correctly inferred the latent temporal structure of the task , whereas others did not . We discuss potential reasons for this finding and how the presented approach enables systematic investigations of how participants learn the underlying temporal structure of task environments and how these representations shape behavior .
All participants provided written informed consent and were paid on an hourly basis . The Medical Faculty of Leipzig University approved the study . A probabilistic reversal learning task is typically structured as follows . An agent is presented with two choices A and B where each choice is associated with a probability of receiving a reward or punishment . For example , initially choice A has high probability pH and choice B low probability pL of getting a reward . Importantly , after several trials the reward contingencies switch , such that choice B now corresponds to the high reward probability choice . Participants are not informed about the switch and they have to infer that a change occurred and adapt their behavior . Here we used a multiple reversals design in which reversals occur several times during the experiment . Furthermore , the reversals occur at predefined trial numbers and are independent of participant’s performance . This reversal schedule was successfully used in past studies in healthy as well as patient samples and is well suited to detect inter-individual differences in behavioral adaptation [39 , 40] . For the model-based analysis , we used a subset of behavioral data—consisting of 22 healthy participants—from a previously published fMRI study [39] . In the experimental task participants were deciding between two cards shown on a screen , each showing a different stimulus ( a geometric shape , e . g . rectangle , triangle , etc . ) as shown in Fig 1A . The reward probabilities associated with the two choice options were anti-correlated on all trials: whenever reward probability of choice A was high ( pH = 0 . 8 ) the reward probability of choice B was low ( pL = 0 . 2 ) , and vice versa . Note that pH = 1 − pL on all trials . Reward contingencies change as follows: they were stable for the first 55 trials , afterwards they changed four times after 15 or 20 trials , and remained stable for the last 35 trials , see Fig 1B . In total , the experimental block consisted of 160 trials . The location of each stimulus on the screen ( right or left side ) was randomized over trials . After each choice the stimulus was highlighted and depicted for 1 . 5 s minus the reaction time . The feedback in form of reward ( won 10 Eurocents ) or punishment ( lost 10 Eurocents ) was shown for 0 . 5 s . If no response occurred during the decision window , the message “too slow” was presented , and no outcome was delivered . During the inter-trial interval , a fixation cross was presented for a variable duration ( jittered and exponentially distributed; range 1–12 . 5 s ) . All 22 participants underwent a training session during which they had the opportunity to learn the statistics of the rewards associated with high and low reward probability choices . The set of stimuli used in the training phase differed from the one used during the testing phase . The participants were instructed that they could either win or lose 10 cents on each trial , and that they will be paid the total amount of money they gained during the testing phase at the end of the experiment . Each participant performed 20 training trials without any reversal of reward contingencies . Before the start of the testing phase participants were told that reward probabilities might change over the course of the experiment . No other information about reversals or the correlation of choices and outcomes was provided . Thus , the participants had no explicitly instructed knowledge about the anti-correlated task structure before the experiment . To derive the probabilistic model of behavior we start by defining a generative model of the task that formalizes an agent’s beliefs about the structure and the dynamics of the task environment . The update rules are then obtained using ( approximate ) Bayesian inference . The assumption here is that participants learn to represent the latent task structure . This structure consists of hidden states which define two possible configurations of the environment: in one configuration stimulus A corresponds to a high reward choice , in the second , stimulus B corresponds to a high reward choice . Note that the notion of state used here differs from what is typically used in reinforcement learning models , in which states are cues that are associated with values over time . Here , the states are hidden and not directly observable . Furthermore , they capture a context , which defines how two stimuli ( option cues ) are related to actions and outcomes of those actions . Importantly , the task environment transits from one state to another in a probabilistic manner . Here we will consider two assumptions about the dynamics of state transition probabilities: ( i ) the state transition probability is constant and independent of the moment of the previous change , as is the case under hidden Markov models ( HMM ) [44]; ( ii ) the state transition probability is time dependent and linked to the moment of the last change , which we will represent using hidden semi-Markov models ( HSMM ) [31 , 45] . As the HMM corresponds to a special case of HSMM , in which state transition probabilities are constant , it is sufficient to define the behavioral model based on the HSMM assumption . In what follows we will define the components of the generative model ( observation likelihood and state dynamics ) and derive the corresponding update rules . To formalize the presence of sequential reversals , that is , transitions from one task configuration to another , we define the state transition probability as p ( s t + 1 | s t ) = { 1 - δ , ifs t = s t + 1 δ , ifs t ≠ s t + 1 ( 4 ) where δ denotes probability of transiting between distinct hidden states ( e . g . from st−1 = ¬R to st = R . This representation of the state transition process corresponds to a standard HMM previously used in reversal learning tasks [29 , 38 , 46–48] . Note that under this formulation of the state transition matrix the agent implicitly assumes that the interval ( the elapsed number of trials ) between two reversals follows a geometric distribution . Hence , the probability that any between reversal interval is of length d is given as p ( d ) = ( 1 - δ ) d - 1 δ , whered ∈ { 1 , 2 , 3 , … } ( 5 ) with an expected dwelling time in each task configuration ( mean interval length ) set to μ = 1 δ . To derive agents with arbitrary beliefs about between reversal intervals we will use a special type of hidden semi-Markov models , the so-called explicit duration hidden Markov models ( ED-HMM ) [49 , 50] . This will allow us to identify individual differences in the beliefs about the temporal task structure and to investigate what impact these beliefs might have on an agent’s performance . The ED-HMM embeds the generative model with the representation of state durations , that is , the state dwelling time dt ( the time spent in each state , ¬R or R ) . In other words , an agent will be able to form expectations about the number of trials between consecutive reversals . Under the ED-HMM we define the state transition matrix as follows p ( s t | s t - 1 , d t - 1 ) = { I 2 , if d t - 1 > 1 , J 2 - I 2 , if d t - 1 = 1 , ( 6 ) where I2 denotes the 2 × 2 identity matrix and J2 denotes the 2 × 2 all-ones matrix . The above relations describe a simple deterministic process for which the current state st remains unchanged as long as dt−1 > 1 and switches to alternative state ( e . g . if st−1 = A then st = B ) with probability one when dt−1 = 1 . The transitions between state durations follow a deterministic countdown ( dt = dt−1 − 1 ) as long as dt−1 > 1; once dt−1 = 1 the subsequent value dt is sampled from the prior over state durations p0 ( dt ) , that is , from the beliefs over between reversal interval . We can write this formally in the form of the transition probability as p ( d t | d t - 1 ) = { δ d t , d t - 1 - 1 , if d t - 1 > 1 , p 0 ( d t ) , if d t - 1 = 1 . ( 7 ) In Fig 2 we illustrate conditional dependencies between states , durations , reward probabilities , and outcomes in the form of a a graphical model . Although , the semi-Markov formalisms allows for defining state dependent prior beliefs p0 ( dt|st ) , here , to reduce model complexity , we have assumed that the priors are independent of the current state , thus we set p0 ( dt|st ) = p0 ( dt ) for any st ∈ {¬R , R} . In practice , prior beliefs about state durations p0 ( dt ) can have an arbitrary form , however for the purpose of inferring the participants’ representation of between reversal intervals we will use a parametric distribution , specifically the negative binomial NB distribution defined as p 0 ( d t ) = N B ( d t ; δ , r ) = ( d t + r - 2 d t - 1 ) ( 1 - δ ) d t - 1 δ r , ( 8 ) where δ ∈ [0 , 1] and r > 0 . The NB distribution has several convenient properties . First , in the case r = 1 we obtain the geometric distribution ( see Eq ( 5 ) ) , hence we recover the HMM model described above . Second , for r > 1 the NB distribution exhibits a nonzero mode which shifts towards the expected value μ = r + δ ( 1 - r ) δ as a function of a decreasing variance σ = ( 1 - δ ) r δ 2 , which is illustrated in Fig 3 . Hence , the parameters of the negative binomial distribution will allow us to quantify an agent’s specific belief in the regularity of reversals . The lower the variance σ , the more an agent believes that reversals occur at regular intervals . Importantly , the variance of prior beliefs p0 ( d ) has direct effects on the temporal expectation profile ( that a reversal will occur at some future trial τ ) . In Fig 4 we show the dependence of the expected reversal probability δτ on the variance σ , and under fixed mean μ = 20 of prior beliefs about the between-reversals interval . The expected reversal probability δτ is defined as δ τ = p ( s τ = R | s τ - 1 = ¬ R ) = ∑ d , s p ( s τ = R | s τ - 1 = ¬ R , s 1 = s , d 1 = d ) p 0 ( d ) p ( s ) , ( 9 ) where τ > 1 , and p ( s = ¬R ) = 1 . Hence , δτ corresponds to the transition probability ( from non-reversal state ¬R into reversal state R ) at some future trial τ starting from the non-reversal state s1 = ¬R . Note that for prior beliefs p0 ( d ) with large variance ( σ = μ ( μ − 1 ) ) , we get a constant transition probability , which corresponds to the HMM formulation of the state transition probability . In contrast , for prior beliefs with low variance ( σ = μ ) one obtains a trial dependent transition probability with values alternating between the low and high probabilities in a periodic manner . This temporal dependence of the transition probability will affect the inference process . The agent will become insensitive to subsequent reversals occurring few trials after the initial reversal , and highly sensitive to reversals occurring twenty to thirty trials after the initial reversal . For model inversion we use a variational inference scheme [51–53] which allows us to derive the update rules for the inference process akin to the ones presented in Eq ( 2 ) . After making a choice ct and observing outcome ot at trial t , the agent updates its beliefs over reward probabilities ρ → , states st , and durations dt following Bayes’ rule p ¯ ( ρ → , s t , d t ) ∝p ( o t | ρ → , s t , c t ) p ˜ ( ρ → ) p ˜ ( d t | s t ) p ˜ ( s t ) ( 10 ) where we used the following notation p ˜ ( x ) = p ( x | o t - 1 : 1 , c t - 1 : 1 ) , and p ¯ ( x ) = p ( x | o t : 1 , c t : 1 ) —for x ∈ { ρ → , s t , d t }—to denote prior and posterior beliefs , respectively , at time step t . If we express prior beliefs as p ˜ ( s t = ¬ R ) = θ ˜ t , p ˜ ( d t = d | s t ) = p d s t , p ˜ ( ρ → ) = ∏ c ∈ {A , B} B ( ρ c ; a t - 1 c , b t - 1 c ) , ( 11 ) where B ( x; a , b ) denotes a beta distribution , we can define the approximate posterior in similar form p ¯ ( ρ → , s t , d t ) ≈ q ( ρ → ) q ( d t | s t ) q ( s t ) , ( 12 ) where we use the following parametrization of the factors of the approximate posterior q ( s t = ¬ R ) = θ t , q ( d t = d | s t ) = p ¯ d s t , q ( ρ → ) = ∏ c ∈ {A , B} B ( ρ c ; a t c , b t c ) . ( 13 ) The prior expectation at the next trial t + 1 depends on the current posterior q ( st , dt ) via the sum product rule p ˜ ( s t + 1 , d t + 1 ) = ∑ d t , s t p ( s t + 1 , d t + 1 | s t , d t ) q ( s t , d t ) . ( 14 ) The update of the agent’s beliefs about reward contingencies for each choice ρ , current state st , and the number of trials until the next reversal dt is completely defined by the update rules of the sufficient statistics of the posterior , namely parameters θt , a t c t , and b t c t . We will obtain the update rules for these parameters using variational inference . The parameters of the approximate posterior can be found as minimizers of the variational free energy defined as F [ q ] = D K L ( q | | p ¯ ) -lnp ¯ ( o t ) = ∑ s t , d t ∫ dρ → q ( ρ → ) q ( s t , d t ) lnq ( ρ → ) q ( s t , d t ) p ( o t | ρ → , s t , c t ) p ˜ ( ρ → , s t , d t ) . ( 15 ) The posterior beliefs q that minimize the free energy F can be obtained using the following relations q ( ρ→ ) ∝p˜ ( ρ ) e〈 lnp ( ot|ρ→ , st , ct ) 〉q ( st ) , q ( st , dt ) ∝p˜ ( st , dt ) e〈 lnp ( ot|ρ→t , st , ct ) 〉q ( ρ→t ) . ( 16 ) To obtain update rules similar to the delta learning rules of the RW model we simplified the above iterative procedure needed to estimate the posterior parameters . To break the cyclic update we will assume that one can first update beliefs about q ( st , dt ) by setting 〈 lnp ( ot|ρ→ , st , ct ) 〉q ( ρ→ ) ≈lnp˜ ( ot|st , ct ) and then use the obtained posterior beliefs about states and durations to estimate the reward probabilities q ( ρ → ) . Using this simplification we obtain the following update rules p¯dst=pdst , θt=θ˜tθ˜t+elnp ( ot|st=R , ct ) p ( ot|st=¬R , ct ) ( 1−θ˜t ) , ( 17 ) where θ ˜ t = ( 1 - θ t - 1 ) p d = 0 R + θ t - 1 ( 1 - p d = 0 ¬ R ) . Note that the conditional posterior q ( dt|st ) corresponds to the conditional prior p ˜ ( d t | s t ) , as p ¯ d s t = p d s t , hence it remains constant during the update of beliefs . However , the prior expectations ( at the next time step ) about state duration will be linked to the inference process via the state-duration transition matrix ( see Eqs ( 6 ) and ( 7 ) ) . Subsequently , we update the beliefs about the choice reward contingencies as a t c t = a t - 1 c t + θ t o ¯ t , b t c t = b t - 1 c t + θ t ( 1 - o ¯ t ) , a t P ^ · c t = a t - 1 P · c t + ( 1 - θ t ) o ¯ t , b t P ^ · c t = b t - 1 P · c t + ( 1 - θ t ) ( 1 - o ¯ t ) , ( 18 ) where o ¯ t = o t + 1 2 ( o ¯ t ∈ {0 , 1} ) . To demonstrate the relation of the above update rules to the ones of the DU-RW model , we transform the shape parameters ( a t c t , and b t c t ) of the posterior beta distribution into the mean μ t c t and the samples size ν t c t as ν t x = a t x + b t x , μ t x = a t x ν t x , ( 19 ) where x ∈ { c t , P ^ · c t } . Expressing the expected reward probability μt as a function of the expected value Vt , that is , as μ t x = V t x + 1 2 , we get the following set of update equations V t c t = V t - 1 c t + α t c t ( o t - V t - 1c t ) V t P ^ · c t = V t - 1 P ^ · c t + κ t α t P ^ · c t ( o t - V t P ^ · c t ) ( 20 ) where α t x = θ t ν t x , and κ t = 1 - θ t θ t . Although in form similar to the DU-RW learning rules , a notable difference is that the fictive prediction error is of the same sign as the actual prediction error . The reason for this is that under the generative model the fictive learning signals are a product of the agent’s uncertainty about the current state of the world , that is , the current configuration of reward contingencies and not the uncertainty about the anti-correlation of reward contingencies on different choices . This alternative representation could be introduced into the generative model by introducing beliefs about a dependence between high and low reward probabilities . However , we will leave this extension for future work as it increases model complexity and does not contribute to our analysis of how temporal representations influence behavior . Here we will describe the response model , which defines the mapping from the agent’s beliefs to responses , based on the active inference framework [54] . We will demonstrate how a response likelihood that is often used in reinforcement learning models ( in the form of a softmax transform ) can be derived within this framework . We do this for didactic purposes to show how one can formally relate active inference to a well-known reinforcement learning account . The core concept of active inference is that agents generate behavior that is most likely to minimize the expected free energy , that is , that tends to maximise , at the same time , the extrinsic and the epistemic value of agents’ choices [55] . The expected free energy can be defined as G t c = E q ( o t | c ) [- U ( o t ) - D K L ( q ( x | o t , c ) | | p ˜ ( x ) ) ] , ( 21 ) where x = ( ρ → , s t , d t ) , U ( ot ) denotes the utility of future outcomes ( reward or punishment ) , and DKL denotes the Kullback-Leibler divergence between the posterior ( conditioned on possible future outcome and action ) and prior expectations at the time step t . Note that we have assumed that the behavior is characterized by planning only a single time step into the future . Importantly , the response model maintains the causal structure of the task , where at trial t an agent first makes a choice ct and only afterward observes an outcome ot . The first term on the right hand side of Eq ( 21 ) is typically denoted as the extrinsic value of an action ( policy ) whereas the second term is denoted as epistemic value or information gain . If we express the utility U as U ( o t ) = λ o t , where ot ∈ {−1 , 1} , and λ > 0 , the extrinsic value becomes 〈 U 〉q=λV˜tc , V˜tc=θ˜tVt−1c+ ( 1−θ˜t ) Vt−1P^·c . In practice , for sufficiently large λ the behavior will be fully driven by the extrinsic value , as each independent observation ot carries little information about the underlying hidden states ( a sequence of observations is required to identify the precise past moment of a reversal ) . In other words , each individual choice leads to a small information gain . Thus , we will assume that the expected free energy can be approximated as G t c ≈ - λ V ˜ t c . The optimal action ( choice ) corresponds to the one that minimizes the expected free energy , thus c t = argmin c G t c = argmax c V ˜ t c . Still , for describing participants’ behavior we have to assume that the action selection process is corrupted by external sources of noise; e . g . mental processes irrelevant for the task at hand . Therefore , we will soften the requirement of minimizing the expected free energy using the softmax transform , which is typically used to define the response likelihood [56 , 57] . The choice probability then becomes p ( c t = c | o t - 1 : 1 ) = e - β V ˜ t c + ln p 0 ( c ) ∑ u e - β V ˜ t u +lnp 0 ( u ) , ( 22 ) where β denotes the response precision and p0 ( c ) the response bias . These two parameters are the free parameters of the response model and allow us to capture participants’ deviation from optimal behavior when fitting models to the data . Finally , in the case of the DU-RW model , we will use the same form of the response likelihood as above , with the difference that the choice value V ˜ t c = V t c . When fitting models to behavior we have focused our analysis on either the DU-RW or the ED-HMM model , where we have used in both cases a hierarchical prior over free model parameters ( see below for details ) . We will not consider the SU-RW and HMM models in model comparison as they represent special cases of the DU-RW and the ED-HMM model , respectively , which are obtained in the limit of κ → 0 , in the case of the DU-RW model , and r → 1 in the case of the ED-HMM model . Hence , we can easily recover these special cases from the estimates of the posterior distributions of the free model parameters , which are summarized in Table 1 . Note that the parameters ν 0 A and ν 0 B of the ED-HMM model ( see Eq ( 19 ) ) , which define the initial number of observations , have been fixed to values which reflect that participants underwent a 20 trials long training session . Hence , setting ν 0 A = ν 0 B = 10 reflects our assumption that participants selected both options in equal amounts during the training session . To estimate the posterior distribution over free model parameters we have used the above defined response model as observation likelihood of the behavioral model . Hence , the likelihood of behavior ( the sequence of a participant’s responses ) is defined as p ( c T : 1 n | o T : 1 n , Λ , m ) = ∏ t = 1 T p ( c t n | o t - 1 : 1 n , Λ , m ) , ( 23 ) where c T : 1 n denotes the sequence of responses of the nth participant ( n ∈ {1 , … , 22} ) , o T : 1 n denotes the sequence of observations ( wins or losses ) that the nth participant made , Λ denotes the set of free model parameters , and m ∈ {1 , 2} denotes the corresponding behavioral model . To define the prior distribution over model parameters we have used the so-called horseshoe prior as a weakly informative hierarchical prior . If we denote the ith model parameter ( where i ∈ {1 , … , 6} ) of the nth participant as λ i n , the horseshoe prior is defined as p ( λ i n , τ i ) = C + ( λ i n ; 0 , τ i ) C + ( τ i ; 0 , 1 ) ( 24 ) where C+ ( x; 0 , γ ) denotes a half-Cauchy distribution with scale parameter γ . Hence , the full hierarchical prior for the model m can be expressed as p ( λ , τ | m ) = ∏ n = 1 22 ∏ i = 1 6 C + ( λ i n , 0 , τ i ) C + ( τ i ; 0 , 1 ) . ( 25 ) Note that τi acts as a hyper-prior , which plays the role of regulating the prior scale , for the corresponding free parameter , over the whole group . As λ i n is a positive definite variable , we have used specific transforms to relate each λ i n parameter to the corresponding model parameter ( see Table 1 ) . The posterior probability over model parameters can be obtain from the Bayes rule as follows p ( λ , τ | C , O , m ) ∝ ∏ n = 1 22 p ( c T : 1 n | o T : 1 n , λ , m ) p ( λ , τ | m ) , ( 26 ) where C = { c T : 1 1 , … , c T : 1 22 } , and O = { o T : 1 1 , … , o T : 1 22 } . However , the exact posterior distribution of the model parameters and their hyper-priors is analytically intractable , hence we have applied the variational mean-field approximation , in which one assumes that the posterior is fully factorized p ( λ , τ | C , O , m ) ≈ ∏ i ∏ n q ( λ i n | m ) q ( τ i | m ) . ( 27 ) The full hierarchical model was implemented in the probabilistic programing library PyMC3 [58] . The PyMC3 library provides an interface to multiple state-of-the-art inference schemes . In our specific case , for estimating the approximate posterior distribution over model parameters , we have used the PyMC3 implementation of the automatic differentiation variational inference ( ADVI ) [59] . ADVI is a stochastic black-box variational inference scheme which returns an approximate estimate of the posterior distribution in a fully factorized form . To attribute participants’ behavior to a specific behavioral model we have assumed that models can be treated as random effects ( variables ) , that is , the attribution of a model to participants’ behavior can differ across participants [60] . Furthremore , we have based Bayesian model comparison on the posterior predictive model evidence [61] instead of the full model evidence , that is marginal likelihood . The motivation for basing model comparison on the posterior predictive model evidence comes from the fact that the posterior predictive model evidence is less sensitive to a prior specification of free model parameters: the approximate estimate of the predictive evidence is based on the samples from the posterior distribution which is already constrained by the subset of the behavioral data . This procedure provides for a more robust model comparison and testing results as it resolves some issues arising when using weakly-informative or misspecified prior probabilities [61] . The posterior predictive model evidence is defined as a marginal expectation of the subset of behavioral responses CT:k condition on the behavioral responses up to the kth trial ( Ck:1 ) and the full set of outcomes presented to participants ( O ) . Formally , we can define the posterior predictive model evidence as p ( C T : k | C k : 1 , O , m ) = ∏ n p ( c T : k n | C k : 1 , O , m ) p ( c T : k n | C k : 1 , O , m ) = ∫ dλ ∏ t = k T p ( c t n | o t - 1 : 1 n , λ n , m ) q ( λ n | m ) ≈ 1 N ∑ l = 1 N ∏ t = k T p ( c t n | o t - 1 : 1 n , λ l n , m ) ( 28 ) where q ( λ n | m ) = ∏ i q ( λ i n | m ) , and N = 104 . Note that to estimate the posterior predictive model evidence we have first estimated the approximate posterior over free model parameters on a reduced data set consisting of the pre-reversal and reversal phases ( k = 125 ) . Then , we generated N samples from the posterior distribution to estimate the posterior predictive model evidence on the remaining part of behavioral data CT:k which covers only the post-reversal phase ( see Fig 1 ) . We then used the posterior predictive model evidence as the likelihood of the random effect model proposed in [60] . The goal here is to identify the posterior probability that each of the two behavioral models can generate the same sequence of behavioral response as participants . Here we have defined the random effect model as the following mixture model p ( C T : k , π , γ | C k : 1 , O ^ ) = p ( C T : k | C k : 1 , O ^ , π ) p ( π | γ ) p ( γ ) , ( 29 ) where p ( C T : k | C k : 1 , O ^ , π ) = ∏ n = 1 22 ∑ m n = 1 2 p ( c T : k n | C k : 1 , O , m n ) p ( m n | π ) p ( m n | π ) = ∏ l = 1 2 π l δ m n , l , p ( π | γ ) = 1 B ( 1 γ , 1 γ ) ∏ l = 1 2 π l 1 γ - 1 p ( γ ) = C + ( γ ; 0 , 1 ) ( 30 ) Similar to the role of the hyperpriors on the parameters of the behavioral model , γ plays here the role of a regularization parameter that allows for data driven constraints of the random effects assumption . If the marginal posterior probability over γ shrinks towards zero , this implies that data supports a null hypothesis which states that all models are present in the population with the same frequency and any differences we observe are purely chance driven [60] . As above , the random effects model selection was implemented in PyMC3 , where the estimation of the posterior was performed using the PyMC3 implementation of the ADVI procedure . A fully factorized approximate posterior p ( π , γ | C , O ) ≈ q ( γ ) q ( π ) ( 31 ) provides us with an estimate of the posterior model probability q ( π ) . The posterior model probability can then be used to identify group level posterior estimate over possible model frequencies in the group of participants and the participant specific posterior model probability .
To illustrate how the agent’s performance depends on the agent’s prior beliefs over the between reversal interval p0 ( d ) we will start by comparing two special cases of the ED-HMM based behavioral model using synthetic data . In the first case , we will fix the variance σ of the prior beliefs p0 ( d ) to σ = μ ( 1 − μ ) , which we named irregular reversal interval ( IRI ) agent . In the second case , we will fix the variance to σ = μ , which we named regular reversal interval ( RRI ) agent . In addition , we will compare the performance of the two probabilistic behavioral models to the single ( SU ) and dual update ( DU ) Rescorla-Wagner ( RW ) models introduced in Eqs ( 1 ) and ( 2 ) , respectively . To illustrate the behavioral difference of different computational models we have modified the experimental setup and introduced two conditions in which reversals occur either at regular or irregular intervals , as shown in Fig 5 . These two conditions allow us to make clear distinction between different behavioral models with respect to their behavior in the presence or absence of regularities . In all simulated experiments we fixed the number of trials to T = 160 and the number of experimental blocks to n = 1000 . Hence , each simulated agent repeated the experiment n times , where at the beginning of each experiment the free model parameters ( besides mean and variance ) of the ED-HMM based agents ( IRI and RRI ) were set to the following values a ˜ 0 A = 8 ; b ˜ 0 A = 2 , a ˜ 0 B = 2 ; b ˜ 0 B = 8 , V 0 A = 0 ; V 0 B = 0 , p ˜ ( d 1 ) = p 0 ( d 1 ) . ( 32 ) In other words , simulated agents have a loose initial knowledge of the underlying reward probabilities , but do not know the initial configuration of the task ( whether the environment is initially in the reversal or the no-reversal state ) . Fig 6 shows the dependence of the agent’s performance on its prior beliefs about the between reversal interval in two different environments . We have defined the performance as the fraction of correct choices ( the choice associated with the higher reward probability ) . The irregular environment corresponds to the interval duration drawn from the geometric distribution with mean μ = 20 and variance σ = μ ( μ − 1 ) , and the semi-regular environment corresponds to interval durations drawn from the negative binomial distribution with mean μ = 20 and variance σ = μ , as illustrated previously in Fig 5 . The IRI agent exhibits stable performance levels independent on the environment or a belief misspecification ( incorrect mean interval duration , or incorrect variability around the mean ) . In contrast , the performance of the RRI model strongly depends on the correctness of the prior beliefs . These results make the rather intuitive point that if one is unfamiliar with the temporal structure of the environment , assigning high uncertainty to the expected state duration ( as is the case for the IRI agent ) ensures reasonably high levels of performance . However , if the environment changes at regular intervals , it is worthwhile to build an accurate representation of the temporal structure , as the performance levels can drastically increase ( in this example from 81% to 87% of median performance levels ) . Fig 7 shows the comparison of the performance distribution between four models; the probabilistic agents IRI and RRI , and the two reinforcement learning based agents with single update ( SU-RW ) and dual update ( DU-RW ) learning rules . The free model parameters ( α and κ , see Table 1 ) of the SU-RW and DU-RW agents were fixed to those values that maximize the average performance levels in each environment . Importantly , when comparing the performance distribution of the DU-RW agent to the performance distribution of the IRI agent we find similar average performance per trial which is stable across environments . This finding suggests that in spite of subtle differences in learning rules ( see Eqs ( 2 ) and ( 20 ) ) the two agents generate very similar behavior . To investigate the accuracy of the procedure for model comparison which we described in Model fitting and model comparison , we have simulated behavior of the ED-HMM based and the reinforcement learning based agents on the experimental task , and applied the same model inversion procedure which we used for the analysis of behavioral data below . In Fig 8A we show the average performance per trial for each agent type estimated over n = 1000 repetitions of the experimental task . Fig 8B shows the probability of assigning behavior of each type of agents to the correct model type . The confusion matrix was estimated over n = 100 simulated experimental blocks , where in half of the experimental blocks ED-HMM based agents ( IRI and RRI ) were used to generate behavioral responses and in the other half of the experimental blocks the reinforcement learning agents ( SU-RW and DU-RW ) were used to generate behavioral responses . The rather high mixing probability of the confusion matrix suggests that the model comparison will have difficulties distinguishing properly between the models . Nevertheless , adjusting the experimental design to contain larger number of trials , that is , more data points for estimating posterior and model evidence , is likely to make model comparison more accurate . Here we will present model comparisons and model fitting based on the behavioral data of 22 participants . As the SU-RW model was shown in previous studies to provide a worse account for behavior than the DU-RW model [40] , we do not expect the SU-RW model to explain the behavioral data better than the other models . Moreover , as our simulations indicate that the HMM and DU-RW models provide for comparable response patterns ( see Fig 7 ) , we will use only the DU-RW model as a reference model , as this model was used in a previous study based on the same data [39] . To circumvent a potential sensitivity of the model evidence to the specification of the prior distribution for the free model parameters , we have based our analysis on the posterior predictive model evidence ( see Model fitting and model comparison for details ) . As the posterior predictive model evidence is estimated from the posterior distribution , rather than the prior ( see Eq ( 28 ) ) , it is more robust to mis-specification of the prior , given a sufficient amount of data in the predictive sample . Hence , to estimate the posterior predictive evidence we have split the behavioral data for each participant into two sets . The first set , containing the initial 125 trials , that is the pre-reversal phase and the reversal phase of the experiment ( see Fig 1 ) , we used for estimating posterior distributions of free model parameters ( see Table 1 ) . Note that as this set contains all but the last reversal , it should provide sufficient information to constrain the posterior model parameters . The reversal phase of the experiment is the only period which participants can use to shape their beliefs about the time structure of reversals . The second set , containing the last 35 trials , that is , the post-reversal phase ( see Fig 1 ) , we used for Bayesian model comparison and model validation . Critically , as no reversals are present in the post-reversal phase , this phase is especially suited for model selection: If participants believe that a reversal will occur at specific moments during the post-reversal phase this belief should be reflected in their behavior; e . g . , they might change their choices in anticipation of a reversal . In Fig 9 we show the results at both the group level and the individual level , using Bayesian model comparison based on the posterior predictive model evidence . Although the group level results suggest substantial evidence in favor of the DU-RW model ( see Fig 9A ) , we can still identify six participants for which we find higher model attribution ( a participant specific posterior model probability ) of the ED-HMM based model ( see Fig 9B ) . The exact values of the predictive log model evidence ( used for model comparison ) and per subject model attribution are shown in S1 Table . We next asked the question whether there is some systematic behavioral difference between these six and the remaining eighteen participants . Thus , we compared the averaged responses of participants belonging to the ED-HMM group and the participants belonging to the DU-RW group . In Fig 10 we show the choice probability averaged for each of the two groups . Tellingly , the ED-HMM group ( blue line ) exhibits a sudden switch toward the alternative choice approximately 20 trials after the start of post-reversal phase . Note that no reversals were induced in this phase . This result suggests that participants belonging to the group for which the ED-HMM model has higher posterior evidence behaved as if they were expecting another reversal 20 trials after the last one . This indicates that they have used the reversal phase to infer the reversal frequency and regularity . If the ED-HMM model indeed captures this aspect of behavior better than the DU-RW model , we would expect to see a similar trend in the response probabilities estimated from the two behavioral models . For this we estimated the response probability for each participant under each of the two models and averaged responses according to group . In Fig 11 we show the between model comparison of the estimated response probabilities for the two groups of participants . Although the mean response of the DU-RW model also shows a trough towards the alternative choice ( when conditioned on the participants in the ED-HMM group ) , we see a much wider excursion in the mean response probability obtained using the ED-HMM , explaining its higher predictive model evidence for this group of participants . Still , it is important to note that the presence of the trough in the mean response probability of both models suggest that the change of response probability towards the alternative choice was driven by the specific sequence of outcomes that participants observed during this time window of the post-reversal phase . In other words , it seems that the participants that were assigned to the ED-HMM group were sensitive to a short sequence of negative outcomes , as if they were expecting another reversal during the post-reversal phase . In Fig 12 we show the posterior estimates of the expected reversal probability δτ for all participants of the ED-HMM group . The expected reversal probability was estimated from State durations and corresponds to the measure illustrated in Fig 4 . Note that for four out of the six participants we find the peak of the reversal probability to fall before τ = 20 , that is , before the 20th trial within the post-reversal phase . For the other two we see a rather flat trajectory which suggests that these subjects behave close to the IRI agents . Although these results seem to be in contrast to our previous findings ( that these six participants anticipated the change on the 20th trial of the post reversal phase ) , the expected reversal probability does not correspond to the expected response probability for a specific participant . The relation between these two quantities is non-linear and is also shaped by the sequence of outcomes .
The key component of the proposed behavioral model is its conceptualization as an explicit duration hidden Markov models ED-HMM [30] , which involves an explicit representation of the between reversal intervals as the hidden structural variable . This representation results in an anticipation of specific moments of reversals . Such anticipation would be clearly advantageous for an agent , as it enables faster behavioral adaptation in cases when reversals actually do occur in a ( semi ) regular manner . The ED-HMM belong to more general group of hidden semi-Markov models which are often applied to the analysis of non-stationary time series [65–68] . In the context of decision making the semi-Markov formalism allows for temporal structuring of behavioral policies [69] . Importantly , semi-Markov dynamics was also applied to temporal difference learning to account for dopamine activity in cases when the timing between action and reward is varied between trials [70] . The proposed model builds upon recent approaches to model behavior in changing environments [11 , 71 , 72] and can be seen as a direct extension of hidden Markov models ( HMM ) which were applied to reversal learning tasks before [29 , 38 , 46–48] . In previous works , the HMM were used to identify the moment of reversal , changes in the beliefs about reversal probability , and the most likely moment in which agents reversed their behavior . This was crucial for understanding the effects of dopamine modulation on the underlying inference and consequently behavior . Although it is out of the scope of the present paper , it is possible to perform backward inference with hidden semi-Markov models ( HSMM ) , hence identifying the most likely moments of reversal in the past . Furthermore , we will explore in the future possible learning rules for parameters of the prior beliefs p0 ( d ) , similar to the work of [73] . Such extension would make the models also suitable for addressing questions related to changes in prior beliefs of state durations . In recent years , reinforcement learning models have found multiple applications in studies relying on a reversal learning task . For example , the classical Rescorla-Wagner model [74] , the dual update extension of the Rescorla-Wagner model ( as described in the present paper ) [39 , 40] , or models separating the prediction error signals on positive and negative prediction errors [75] . As we have shown here a reinforcement learning model generates behavior very similar to a probabilistic counterpart in relatively simple settings of the reversal learning task . Hence , we would expect that additional extensions of the considered dual update RW model could make the behavior of the reinforcement learning even more similar to the probabilistic model introduced here . Still , we can point out several advantages of probabilistic models of behavior over reinforcement learning models , in the context of decision making under uncertainty and in dynamic environments . The probabilistic modeling approach allows for a principle way of mapping complex knowledge about spatio-temporal task structure into a relatively simple set of learning rules ( as demonstrated here ) . In turn this provides clear functional interpretation of various prediction error signals , and corresponding adaptive learning rates , which are typically difficult to derive or motivate within the context of classical reinforcement learning . Specifically , we would say that prescribing to a probabilistic modeling approach is crucial for understanding interaction between representation of temporal structure and decision making . The sense of time and time representation at a neuronal level has been the focus of numerous studies in the past [76–82] . Studies investigated how neuronal circuitry might implement a robust internal clock [83] , and how such an internal clock can be utilized to estimate time elapsed between consequent events ( e . g . , between two tones ) . Although not described here , behavior in such and similar tasks can be modelled using the formalism of the hidden semi-Markov models . Estimating elapsed time between events ( here , reversals ) can be obtained using a backward inference process which provides an estimate on the most likely moment of the previous reversal . Nevertheless , it is an open question if the neural circuitry that explains behavior related to interval timing , which involves sub-second time scales , can also be useful to understand behavior which requires a temporal reasoning over much longer time scale . This is of specific interest for understanding how the brain makes accurate predictions about the expected moment of reward , and how this information is used to construct timed prediction error signals [84 , 85] . The reversal learning task has been shown to be especially useful for behavioral phenotyping , e . g . in areas of executive control [86–90]; for quantifying individual proneness towards impulsive and compulsiveness [91]; and defining individual vulnerability towards addiction [92] and other psychiatric disorders [35 , 93 , 94] . The experimental paradigms associated with a reversal learning task can differ substantially from the design presented here ( see Fig 1 ) . For example , the probability of reward and punishment for different choices could be correlated to some degree [47 , 95] , or the reversal schedule can be made dependent on the number of correct choices [96–98] . It is relatively straightforward to adapt the proposed model to any of these variants . For example , when the reversal schedule depends on the number of previously correct choices , one can make the duration transitions dependent on the belief that the previous choice was correct . This would relate state durations to the beliefs about the number of correct choices since the last reversal . Thus , we believe that in a combination with the proposed model , the reversal learning task opens a wide range of opportunities for linking anomalous beliefs in the time domain to different cognitive disorders . Distortions in the temporal organization of cognition and behavior have been implicated , for a long time , in a wide range of psychiatric conditions , perhaps most prominently in attention-deficit hyperactivity disorder , autism and schizophrenia [99] . Hence , the proposed model might help to integrate findings of patients’ aberrant behavior in simple time estimation tasks ( e . g . , a deficit to reproduce durations [100–102] with their known deficits in the decision-making domain [103] , such as , in variants of the reversal learning task . Over the recent years , the question of how people apply their knowledge about the underlying structure of the environment in their decision-making has been widely investigated and discussed . Studies have come to the rather unspecific notion that a reduction in model-based control based on using environmental structure is ubiquitous across different psychiatric symptoms and diagnoses [104 , 105] . We believe that the model at hand could be a starting point for developing a set of tasks particularly appropriate for refining this notion . For example , Bayesian theoretical accounts have conceptualized addictive behavior as decision-making based on an aberrant model of the world [106] . Both animal and human studies suggest that addicted patients might have a specific deficit to infer or simulate statistical regularities in the environment [40 , 107 , 108] . Such deficits suggest limitations in their ability to infer regularities , which consequently is observed as suboptimal decision-making . Reversal learning deficits and impairments in model-based control have also been shown in patients suffering from obsessive compulsive disorder ( OCD ) [109 , 110] . A subgroup of OCD patients suffers from an obsession for symmetry and organization . Similarly , obsessive compulsive personality disorder ( OCPD ) patients show an exaggerated focus on order and symmetry as well as rule-bound traits . Given this clinical picture , it is interesting to speculate that beliefs about regularities in the environment might differ between OCD/OCPD patients and controls . A reduced ability to infer and represent irregular changes in the environment might lead to suboptimal decision-making and distress in OCD/OCPD patients in the presence of irregular changes . Finally , recent studies have demonstrated that schizophrenia patients may be characterized by an “over-dominance” of an internal model [111] ( e . g . , in the framework presented here , overrating regularities in an irregular environment ) . When such an internal model is detached from the external input from the world ( i . e . , the true regularity structure ) , this mismatch might be involved in the development of psychotic states ( in paranoia , delusions , hallucinations ) . In summary , we have presented a novel probabilistic model for understanding human decision making behavior in changing environments . The proposed model is based on the explicit duration hidden Markov model , which forms a special case of more general hidden semi-Markov models . The presented results , obtained from both simulated behavior and the model-based analysis of behavioral data , suggest concrete applications of the proposed model in understanding human behavior in changing environments . Most notably , it might be possible to link the quality of the underlying representation of temporal task structure to behavior , thereby opening new directions for cognitive phenotyping .
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Although time perception and timed behavior are essential for our everyday experience , it is still unclear how the human brain represents the underlying temporal regularities of our dynamic environment . These regularities and their representations in the brain are important to generate well-timed behavior . When deciding on the sequence of actions to complete most of our everyday tasks like cooking , driving , or even brushing our teeth , it is essential to represent and keep track of the durations of different parts of the tasks . Here we introduce a behavioral model of decision making in environments in which a change is at least partially predictable by the time it took since the last change . We show that human participants are using such predictions in the so-called reversal learning task , which simulates abrupt but not immediately obvious changes of the environment . We find that some but not all participants harness previously experienced regularities in these changes to anticipate when the next change is going to happen . We expect that a wide range of similar questions of how humans and other animals use temporal expectations to make their decisions in a dynamic environment can be addressed using the new modelling approach .
|
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"Abstract",
"Introduction",
"Materials",
"and",
"methods",
"Results",
"Discussion"
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2019
|
Predicting change: Approximate inference under explicit representation of temporal structure in changing environments
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The bicoid stability factor ( BSF ) of Drosophila melanogaster has been reported to be present in the cytoplasm , where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes . BSF may also have other roles , as it is ubiquitously expressed and essential for survival of adult flies . We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein . We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage . Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs , accompanied by an enrichment of unprocessed polycistronic RNA intermediates . Furthermore , we observed a significant reduction in mRNA steady state levels , despite increased de novo transcription . Surprisingly , mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies , suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability . We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role , which is essential for maintaining mtDNA gene expression and oxidative phosphorylation .
The maternal to zygotic transition , during which control of development shifts from maternally contributed mRNAs to genes expressed in the zygote , is of considerable interest . The maternally contributed bicoid mRNA , encoding a protein important for formation of anterior body patterning , is dependent on regulatory mechanisms controlling the cytoplasmic stability and localization of the mRNA in the zygote . The bicoid stability factor ( BSF ) is thought to be involved in this process as it binds the 3′UTR of the bicoid mRNA [1] . Mutation of the BSF binding site leads to reduced abundance of bicoid mRNA , whereas a P element insertion mutation that leads to a drastic reduction in BSF protein levels does not affect the abundance or distribution of endogenous bicoid mRNA [1] . In another study , BSF was reported to have a role in regulation of early zygotic genes by binding a short consensus sequence in the 5′UTR of genes expressed in the early zygote [2] . It was also reported that BSF is essential , as a P element insertion in the bsf open reading frame is lethal in the homozygous form [2] . However , homozygous bicoid mutant flies are viable and BSF therefore is likely to have an additional role besides regulating bicoid expression [2] . Characterization of global gene expression patterns in flies have shown that BSF is ubiquitously expressed in adults , further indicating that the role of BSF may not be limited to embryogenesis [3] . Studies of the subcellular localization of BSF have shown that it is present in cytoplasmic particles in oocytes and surrounding nurse cells [1] and in the cytoplasm and nucleus in early embryos [2] . Bioinformatics analyses suggest that BSF has some homology to the mammalian LRPPRC protein [1] , [4] , which belongs to the pentatricopeptide repeat class of proteins [5] . The LRPPRC protein has been reported to have roles in cytoplasmic RNA transport [6]–[8] and nuclear transcription [9] , [10] , but its main localization is in the mitochondrial matrix [4] , [10] , [11] , where it has been suggested to stabilize mitochondrial transcripts [12] . The fly mtDNA is a small compact , circular molecule , encoding 13 essential polypeptides , which are components of the mitochondrial oxidative phosphorylation system , and RNA components of the mitochondrial translational system , which include 22 transfer RNAs and 2 ribosomal RNAs [13] . Mitochondrial transcription generates large polycistronic transcripts , which are processed by endonucleolytic cleavage to generate individual mRNAs [14] , [15] These mRNAs are subsequently polyadenylated [16] , in a process believed to take place in two steps . First , oligoadenylated transcripts are generated by addition of a short adenine tail to the 3‘ends of most mitochondrial mRNAs . The enzyme necessary for this oligoadenylation has not yet been identified . Second , the poly A polymerase enzyme will add up to <50 adenines to the oligoadenylated mRNAs to create the long polyadenylated tail [17] . The vast majority of the ∼103 mitochondrial proteins are encoded by nuclear genes , including the majority of the respiratory chain subunits , all proteins involved in replication and transcription of mtDNA and all proteins of the mitochondrial ribosome . The regulation of oxidative phosphorylation capacity is thus dependent on a crosstalk between two genomes and nuclear genes play a key role in this process as they regulate mtDNA expression at many different levels . The mitochondrial genomes of flies and mammals have the same gene content although there are substantial differences in gene order . The high level of conservation of mtDNA and important nuclear-encoded regulators of mtDNA expression suggests that key regulatory processes are similar in insects and mammals . We therefore hypothesized that BSF might have a role in fly mitochondria , in addition to its suggested regulatory roles in developmental processes . We report here that BSF is a bona fide mitochondrial protein involved in regulating mtDNA gene expression . Contrary to previous reports , our data demonstrate that BSF is involved in the maturation and polyadenylation of mitochondrial mRNAs and coordinates mitochondrial translation . We have thus identified an essential role for BSF in maintaining mtDNA gene expression and oxidative phosphorylation in flies .
We previously identified two possible fly homologues for the mammalian LRPPRC protein [4] . Here we present additional phylogenetic analyses showing that BSF is the most closely related homologue to LRPPRC in flies ( Figure 1A ) . Previous experiments have demonstrated a punctuated cytoplasmic localization of BSF in oocytes and a cytoplasmic and nuclear localization in early embryos [1] , [2] . We further addressed the subcellular localization of BSF by transfecting Schneider ( S2R+ ) and HeLa cells with a BSF-FLAG-GFP fusion construct . There was a perfect overlay between Mitotracker Deep Red and BSF-FLAG-GFP fluorescence with a co-localization rate of 91±1% in Schneider cells ( N = 5 ) and 94±3% in HeLa cells ( N = 7 ) ( Figure 1B ) , thus indicating that BSF is localized to mitochondria . We also performed subcellular fractionation experiments of tissues from adult flies and found that BSF was present in the mitochondrial fraction ( Figure 1C ) . Our results thus show that BSF is mainly localized to mitochondria , which is in good agreement with the known main localization of its mammalian homolog LRPPRC [4] . In order to analyze the in vivo function of BSF , we induced gene silencing by using the UAS-GAL4 system and two independent bsf RNAi-knock down fly lines , that target different , non-overlapping regions of the bsf transcript . In each case , the performed fly crosses generated the bsf knockdown lines wDahT;+;UAS-bsf-RNAi#1/daGAL4 or wDahT;UAS-bsf-RNAi#2/+;daGAL4/+ ( w;;UAS-bsfRNAi#1/daGAL4 or w;UAS-bsfRNAi#2/+;daGAL4/+ ) and two control lines wDahT;+;daGAL4/+ ( w;;daGAL4/+ ) , and wDahT;+;UAS-bsfRNAi#1/+;+ or wDahT;UAS-bsfRNAi#2/+;+ ( w;;UAS-bsfRNAi#1/+; or w;UAS-bsfRNAi#2/+ ) , which all were analyzed in parallel in the experiments . Ubiquitous knockdown ( KD ) , using the daughterless-GAL4 ( daGAL4 ) driver line , resulted in an up to 80% down-regulation of bsf transcript levels both in third-instar larvae and in adult flies ( Figure 2A ) . Western blot analyses revealed that the BSF protein was undetectable in third-instar KD larvae ( Figure 2B ) and KD flies ( Figure S1A ) , demonstrating a highly efficient KD of BSF protein expression in both bsf-RNAi lines . Ubiquitous BSF KD caused a delay in pupal development , with the majority of pupae from line 1 hatching , and only 15% from line 2 managing to complete their eclosure ( Figure 2C ) . KD flies had a significantly reduced climbing ability , suggesting muscle weakness ( Figure 2D ) . Female flies from both KD lines weighed less ( Figure 2E ) , whereas there was no consistent weight change in the males ( Figure 2F ) . Continuous bsf KD male/female crossings resulted in no offspring , and KD flies laid significantly fewer eggs in comparison with controls ( Figure 2G ) . Dissecting ovaries from adult bsf KD flies revealed a significant reduction in size in comparison with controls ( 45±21% v . 100±10% ) ( Figure 2H and Figure S1B ) , possibly contributing to the lower body weight of the female KD flies . Flies of both bsf KD lines had a drastically reduced life span compared with controls ( Figure 2I ) , with most flies surviving less than 30 days . Together , these results identify BSF as an essential protein for fly survival . To investigate the biochemical consequence of reduced BSF expression levels we measured respiratory chain enzyme activities in isolated mitochondria from larval tissue . We found that all respiratory chain complexes containing mitochondrially-encoded subunits had reduced enzyme activities . Complex I was the most affected , but complex I+III , complex II+III and complex IV activities were also profoundly reduced in bsf KD flies ( Figure 3A ) . Complex II , containing exclusively nuclear-encoded subunits , exhibited normal enzyme activity ( Figure 3A ) , strongly supporting the notion that loss of BSF has specific effects on mtDNA gene expression . We also analyzed mitochondrial respiratory capacity by monitoring oxygen consumption in permeabilized tissues extracted from third-instar larvae or thoraces of flies . Maximal oxygen consumption levels were significantly reduced in mitochondria from third-instar bsf KD larvae and flies in the presence of substrates entering the respiratory chain at the level of complex I ( CPI ) , but not with substrates ( SUCC-G3P ) that deliver electrons to complex II or glycerol-3-phosphate dehydrogenase , which both are upstream of complex III ( Figure 3B ) . There was significant reduction of oxygen consumption with combined substrates ( CPI-SUCC-G3P ) in adult fly mitochondria ( Figure 3B ) . We also observed a progression of the decrease in the uncoupled oxygen consumption with complex I substrates , when comparing third-instar larvae and flies ( Figure 3B ) . In summary , the results from measurements of enzyme activities ( Figure 3A ) and oxygen consumption ( Figure 3B ) show that complex I is the most affected of the oxidative phosphorylation complexes , perhaps due to its high number of mtDNA-encoded subunits . As a result of the respiratory chain deficiency the [ATP]/[ADP] ratio in adult bsf KD flies was reduced to 44% of ratio in controls ( Figure 3C ) . Furthermore , in adult bsf KD flies the [lactate]/[pyruvate] ratio was considerably increased ( Figure 3D ) , most likely as a consequence of the compromised respiratory function , which leads to increased lactic acid fermentation in order to maintain the reduction-oxidation homeostasis . We performed a detailed study on steady-state levels of mitochondrial transcripts in bsf KD lines by using both QRT-PCR and northern blot analyses . The levels of all analyzed mitochondrial mRNAs were reduced in third-instar bsf KD larvae ( Figure 4A , 4C , 4D and Figure S1C ) and flies ( Figure 4B ) , with the exception of ND6 , which was significantly reduced only in adult flies . QRT-PCR demonstrated slightly increased mtDNA levels in third-instar bsf KD larvae ( Figure 4E ) , showing that the reduced mitochondrial mRNA levels cannot be explained by mtDNA depletion . In contrast , the mitochondrially encoded small ribosomal subunit rRNA ( 12S rRNA ) was significantly increased , while the large ribosomal subunit rRNA ( 16S rRNA ) was slightly decreased ( Figure 4C and 4D ) . Additionally , there was a substantial increase in levels of all analyzed mitochondrial tRNAs in third-instar bsf KD larvae ( Figure 4F and 4G ) . There was no clear correlation between the level of a particular tRNA and the location of its gene in fly mtDNA . The presence of increased steady-state levels of tRNAs makes it unlikely that the reduction in levels of mRNAs are explained by reduced transcription as both types of mature transcripts are produced by processing of polycistronic precursor transcripts . We nevertheless assessed mitochondrial de novo transcription by performing in organello labeling experiments . Both third-instar bsf KD larvae ( Figure 5A and Figure S2B ) and bsf KD flies ( Figure S2A ) had a dramatic increase in de novo transcription , showing that the reduced mRNA levels must be explained by increased degradation . The increased mitochondrial de novo transcription and increased mtDNA copy number are likely parts of a compensatory mitochondrial biogenesis response induced by the respiratory chain deficiency , which , in turn , is caused by defective post-transcriptional regulation of mitochondrial mRNA stability in the absence of BSF . We further investigated whether the decrease in mRNA steady-state levels resulted in decreased mitochondrial translation by assessing de novo translation in isolated mitochondria . Surprisingly , third-instar bsf KD larvae demonstrated a selective increase in the synthesis of subunits of complex I ( ND1-6 and ND4L ) and complex IV ( COXI-III ) , whereas the synthesis of subunits of complex III ( Cyt b ) and complex V ( ATP6 ) remained unchanged ( Figure 5B ) . We observed an exceptionally large increase in levels of the COXII subunit of complex IV ( Figure 5B , asterisk ) . We also found an unidentified translation product migrating above the ND1 subunit of complex I ( Figure 5B , arrow ) . Interestingly , both of these aberrant translation products were almost invisible after a 3-hour chase with cold methionine ( Figure 5B , left panel ) , suggesting that they are subjected to an increased degradation shortly after synthesis . Decreased mitochondrial mRNA steady-state levels , in combination with increased de novo transcription and translation of specific mitochondrial polypeptides ( ND1-6 and COX1-3 ) led us to investigate the nature of the mature mitochondrial mRNAs . We analyzed the 5′-and 3′-ends of mitochondrial transcripts , using RNA circularization , followed by reverse transcription and direct sequencing or cloning and subsequent sequencing ( see Materials and Methods ) . The 5′ and 3′ ends of the 12S and 16S rRNA were identical in bsf KD and control larvae . However , we observed severely reduced poly A tail lengths of all mitochondrial mRNAs except Cytb and ATP6/8 ( Figure 5C and 5D ) . Interestingly , those mRNAs with reduced poly A tail length encoded the polypeptides that showed increased levels of de novo synthesis . We further analyzed the 5′ and 3′ends of two polycistronic transcripts each containing an mRNA with a retained tRNA at its 3′ end ( ND3 plus tRNA-Ala and COXII plus tRNA-Lys ) . In control samples these polycistronic transcripts were polyadenylated at their 3′-ends , showing that the polyadenylation process is not specific for fully processed mRNAs , but rather can occur at any free 3′end . In the bsf KD larvae , these polycistronic RNAs had severely reduced lengths of their polyA tail , thus suggesting that the lack of polyadenylation is a direct consequence of the loss of BSF and not a secondary effect of impaired translation . In the bsf KD larvae , we consistently failed to recover the mature COX III mRNA and only observed COXIII as part of a large polycistronic RNA . The 5′part of this RNA consisted of ATP6/8 and the 3‘part of COXIII . This RNA had undergone a correct processing at the 3′ end of COXIII but lacked polyadenylation . The lack of mature polyadenylated COXIII transcripts in the bsf KD larvae is thus likely explained by a combination of defective RNA processing and defective polyadenylation . Cloning and subsequent sequencing of the ND4/ND4L PCR product revealed several different RNA species in the bsf KD samples , indicating both polyadenylation and processing differences in comparison with control samples ( Figure 5D ) . In summary , sequencing of RNA 5′ and 3′-ends in third-instar bsf KD larvae revealed a severe mRNA maturation defect with reduced poly A tail lengths and an enrichment of unprocessed polycistronic RNA intermediates containing COXIII and ND4/ND4L sequences . Despite the observed compensatory increase of de novo transcription and translation , third-instar bsf KD larvae show a severe respiratory chain dysfunction presumably causing delayed hatching and reduced lifespan . Sequencing of the mitochondrial mRNAs showed reduced length of poly A tails and processing defects , which could affect translational initiation or lead to the production of abnormal polypeptides . Subunits of complex I and IV were translated at increased rates , but a subset of the newly synthesized subunits were nevertheless preferentially degraded ( Figure 5B ) , suggesting that they fail to assemble into mature complexes . We therefore assessed the levels of assembled respiratory chain enzyme complexes by using Blue-Native polyacrylamide gel electrophoresis ( BN-PAGE ) . We observed reduced levels of assembled complex I , complex III , complex IV and supercomplexes in third-instar bsf KD larvae ( Figure 6A and 6B ) and flies ( Figure S2C and S2D ) . The levels of assembled complex V ( ATP synthase ) were unaffected . This reduction in steady-state levels of assembled complexes was accompanied by reduced in-gel activity of complex I ( Figure 6A , Figure S2C ) and complex IV ( Figure 6B , Figure S2D ) . The BN-PAGE and complex I in-gel activity analyses showed reduced steady-state levels of supercomplexes and the presence of a smaller , partially assembled , form of complex I in third-instar bsf KD larvae ( Figure 6A , asterisk ) . Western blot analyses showed reduced levels of a nuclear encoded subunit of complex I ( NDUFS3 subunit ) , indicating and supporting the notion that there is a severe reduction in steady-state levels of assembled complex I in third-instar bsf KD larvae ( Figure 6C , Figure S2E ) and flies ( Figure 6D ) . There was no major reduction in steady-state levels of the nuclear-encoded complex V subunits ( α-subunit of ATP synthase ) in third-instar bsf KD larvae ( Figure 6C , Figure S1B ) and flies ( Figure 6D ) , suggesting normal assembly . Together , these results show that third-instar bsf KD larvae and flies fail to assemble sufficient levels of complex I , III and IV as well as supercomplexes consisting of these complexes , which explains the observed profound reduction in oxidative phosphorylation capacity .
BSF has previously been suggested to stabilize cytoplasmic mRNAs in oocytes and early zygotic cells during the first few hours of fly embryogenesis [1] , [2] . However , the ubiquitous expression of the BSF RNA [3] and the punctuate cytoplasmic localization in flies has prompted us to re-investigate the function of BSF in the fly . Surprisingly , we were able to demonstrate that the BSF protein is mainly localized to mitochondria , where it controls polyadenylation of specific mitochondrial mRNAs . Further , BSF plays a key role in the regulation of mtDNA gene expression , by coordinating mitochondrial translation in flies . GFP-tagged BSF localized to mitochondria in transfected tissue culture cells and cell fractionation experiments showed that BSF is mainly present in mitochondria of adult flies . Interestingly , BSF was not detectable in the cytoplasmic and nuclear fractions of adult flies . It is important to note that the mammalian homolog of BSF , LRPPRC , has also been reported to have roles in regulation of cytoplasmic mRNA transport [6]-[8] and in regulation of nuclear transcription [9] , [10] . However , convincing evidence proposes that the main proportion of LRPPRC is localized to mitochondria [4] , [10] , [11] . In one study of tissue culture cells , endogenously expressed LRPPRC was not detectable in highly purified nuclei lacking mitochondrial contamination [4] . These findings do not exclude that there is a small fraction of BSF and LRPPRC localized in other subcellular compartments besides mitochondria . However , the proposed extramitochondrial functions of BSF and LRPPRC should be revisited in a new set of experiments focusing on avoiding mitochondrial contamination of isolated nuclei and cytoplasmic extracts . Ubiquitous RNAi-induced reduction of endogenous bsf levels in flies causes mitochondrial dysfunction with decreased respiratory chain enzyme activities and reduced levels of assembled complex I , III and IV . This mitochondrial dysfunction leads to severe phenotypes , including delayed and incomplete eclosure , reduced fecundity , sterility and shortened life span . Mammalian cell lines with reduced LRPPRC levels have reduced mitochondrial mRNA steady-state levels and reduced mitochondrial translation [12] , [18] . Interestingly , downregulation of BSF protein levels in flies also leads to decreased steady-state levels of mitochondrial mRNAs but , in contrast with the results in mammals , translation is increased and aberrant translation products are generated . Some of the mitochondrial translation products that are synthesized at increased rates in flies are subject to increased degradation , suggesting that BSF might also act as a translational coordinator . Regulation of mitochondrial gene expression in response to different metabolic demands in animal cells is largely unknown , and most likely this regulation occurs at several different levels . The basic mtDNA transcription machinery has been defined [19] , but it is unclear how it is regulated . Processing of mRNAs is required for correct translation [20] , but the coupling between transcript processing and translation is poorly understood . Furthermore , mitochondria are in essence a prokaryotic system with no compartmentalization between transcription and translation , which makes it likely that both of these processes directly communicate . Here we demonstrate that the loss of BSF results in incorrect processing of the polycistronic precursor transcripts and a failure to polyadenylate a subset of both processed and polycistronic transcripts . This failure to mature mitochondrial mRNAs , in turn , leads to the destabilization of mitochondrial mRNAs and reduced steady-state levels , despite a simultaneously increased de novo transcription . In animals all mitochondrial transcripts are polyadenylated as a part of becoming a mature mRNA ready for translation , except for ND6 of vertebrates , which in humans and fish has a long 3′UTR without any poly A tail [16] , [21] . In the mammalian system it is believed that nearly all mitochondrial RNAs are oligoadenylated during transcription by a yet unknown enzyme , followed by the polyadenylation to approximately 50 adenines by the mitochondrial poly A polymerase ( mtPAP ) [22] . A number of mitochondrial transcripts require polyadenylation to generate functional stop codons , but otherwise the exact function of mRNA polyadenylation in mitochondria is unknown . While in bacteria and chloroplasts the poly A tail seems to promote transcript degradation , eukaryotes seem to polyadenylate nearly every fully processed cytosolic mRNA at the 3′ end , resulting in increased mRNA stability , increased translational efficiency , and promotion of transport of the processed mRNA from the nucleus to the cytoplasm [23] . A primary role of BSF in the maturation of mitochondrial mRNAs is supported by the recent observation that PPR proteins in trypanosomes affect polyadenylation of mitochondrial mRNAs . In this unicellular protozoon , the PPR proteins KPAF1 and KPAF2 associate with the mtPAP , stimulating mRNA polyadenylation and thereby coordinate stability and translation of mRNA [24] . Our results suggest that BSF is involved in the actual mRNA maturation process by controlling polyadenylation of specific transcripts . The mammalian BSF homolog LRPPRC has been shown to be stabilized by a second RNA-binding protein called SLIRP , in a direct interaction [12] . A SLIRP homolog has also been suggested to exist in flies , raising the possibility that such an interaction with a second RNA binding protein is also required in fly mitochondria . Interestingly , bioinformatics analyses identified an additional LRPPRC homolog , besides BSF , in Drosphila melanogaster [4] , suggesting that both homologs work together to control the polyadenylation and translation of different sets of mitochondrial mRNAs . This is supported by our observation that several mitochondrial transcripts , such as cytb and ATP6/8 , are still polyadenylated in the bsf KD lines . Loss of BSF also results in increased and aberrant translation , and reduced levels of assembled RC complex I , III and IV , suggesting that BSF coordinates mitochondrial translation and RC complex assembly . The coupling of transcription to translation is supported by studies of the yeast homologue of BSF and LRPPRC , PET309 [25] . This factor is implicated in activation of translation by binding to the 5′UTR of yeast mitochondrial mRNAs , thereby tethering mitochondrial polysomes to the inner mitochondrial membrane , which , in turn , is thought to ensure co-translational insertion of newly synthesized polypeptides into the assembling respiratory chain complexes [26] , [27] . Mitochondrial mRNAs without these 5′-UTRs are translated at normal levels followed by rapid degradation , suggesting that targeting to the inner mitochondrial membrane is necessary for stability of the newly translated peptides [28] . It is important to point out that most mRNAs encoded by metazoan mtDNA lack 5′UTRs and targeting of translation to the inner membrane of animal mitochondria , if this occurs , must therefore involve , at least partly , different regulatory mechanisms . Cytoplasmic mRNAs have several different types of destabilizing elements and it seems that translation is coupled to degradation of some classes of mRNAs [29] . If a similar mechanism exists in mitochondria , it would suggest that the increased de novo translation in BSF KD larvae results in increased degradation of the mitochondrial mRNAs . Some support for this proposed mechanism comes from characterization of a patient with a microdeletion between the genes for ATP6/8 and COXIII , which has been reported to cause incorrect processing of the corresponding mitochondrial mRNAs and severely reduced steady-state levels of the ATP6/8 transcript due to translationally induced deadenylation [30] . Additional support comes from the observation that human cells treated with the mitochondrial translation inhibitor thiamphenicol have increased steady-state levels of mitochondrial mRNAs [31] . However , decreased translation does not always increase mRNA steady-state levels as exemplified by the conditional mouse knockout for TFB1M , which abolishes mitochondrial translation , but does not affect the levels of most mitochondrial mRNAs despite activation of de novo transcription [32] . In conclusion , we present here the unexpected result that the BSF protein mainly is localized to mitochondria , where it controls the polyadenylation of specific mitochondrial mRNAs . In addition , our results suggests that BSF has a novel role in coordinating mitochondrial translation as loss of BSF leads to increased and uncoordinated translation with increase synthesis of unstable translation products . BSF thus has an essential role in regulating mitochondrial function in the fly .
For in vivo KD studies two independent non-overlapping UAS-bsf RNAi lines were used . w;;UAS-bsfRNAi#1 ( #10302R-I ) was obtained from the National Institute of Genetics ( Japan ) and w;UAS-bsfRNAi#2 ( #22839 ) was obtained from the Vienna Drosophila RNAi Center ( VDRC ) . Ubiquitous bsf knock down was achieved by crossing UAS-bsf RNAi lines to a daughterless-GAL4 ( da-GAL4 ) driver line . UAS-RNAi lines and daGAL4 driver lines were backcrossed for at least 6 generations into the white Dahomey background ( wDahT ) . All fly stocks were free from the endosymbiontic bacterium Wolbachia . Flies were propagated and experiments were conducted at 25°C on a 12 h∶12 h light∶dark cycle at constant humidity on a standard sugar-agar-yeast medium . Homologs of LRPPRC were collected using PSI-BLAST [33] against the RefSeq protein database at NCBI and aligned using ClustalX [34] with the BLOSUM matrix . Multiple sequence alignments were trimmed using the GBLOCKS server [35] with relaxed settings . The trimmed alignment was submitted to PhyML [36] using standard parameters and non-parametric bootstrap analysis with 500 replicates . The resulting tree was displayed using Dendroscope [37] and prepared for publication in Illustrator . Full-length bsf cDNA was obtained from the Drosophila Genomics Resource Center ( SD10676 , AY058795 ) . Two amino acid changing substitutions were identified in the bsf cDNA in comparison with the reference sequence ( FBtr0081087 ) . The corresponding mutations at nucleotide positions 415 and 710 were changed by site-directed mutagenesis of the cDNA , using the QuickChange II XL Site-Directed Mutagenesis Kit ( Agilent Technologies ) . A cDNA encoding FLAG-tagged BSF was cloned into the plasmid pAcGFP1-N2 ( Clontech , Mountain View , USA ) to generate the vector pbsfFLAG-AcGFP1 , which encodes a fusion protein consisting of BSF-FLAG with an in-frame addition of green fluorescent protein ( GFP ) to its carboxy-terminus ( BSF-FLAG-GFP ) . Schneider 2R+ and HeLa cells were transfected in microscopy dishes ( µ-Dish , ibidi , Martinsried , Germany ) with pbsfFLAG-AcGFP1 using the FuGENE HD Transfection Reagent ( Roche Diagnostics , Mannheim , Germany ) . The mitochondrial counterstaining was achieved by 100 nm MitoTracker Deep Red FM ( Invitrogen , Darmstadt , Germany ) . Live cell image acquisition was performed with a Leica TCS SP5-X confocal microscope ( Leica Microsystems , Wetzlar , Germany ) . The colocalization rate was determined by the software LAS AF ( Leica Microsystems , Wetzlar , Germany ) under the following conditions and calculations: threshold 30% , background 20% , colocalization rate [%] = colocalization area/area foreground , and area foreground = area image − area background . All fractions were isolated from adult wDahT flies . Mitochondrial , cytoplasmic and nuclear fractions were isolated by differential centrifugation as previously described [38] . The used primary antibodies were: HISTONE H3 ( Santa Cruz Biotechnology , dilution 1∶200 ) , Complex I-subunit NDUFS3 ( Mitoscience MS112 , dilution 1∶1000 ) , tubulin ( Sigma , dilution 1∶1000 ) and polyclonal rat antisera raised against BSF ( kindly provided by Professor MacDonald PM , Stanford University , dilution 1∶1000 ) . Protein bands were visualized with ECL western blotting reagents ( Bio-Rad ) . Western blot analyses were performed using whole fly or mitochondrial protein extracts according to the Cell Signaling Technology protocol ( CellSignaling ) . Additional primary antibodies used were: complex V ( Mitoscience MS504 , dilution 1∶5000 ) , VDAC ( Mitoscience MSAO3 , dilution 1∶2000 ) , actin ( Sigma , dilution 1∶1000 ) . For adult hatching rate measurements eggs were collected during a 3 hours time window and transferred to vials ( 80 eggs/vial ) to ensure standard larval density . Hatching of adult flies was monitored in regular intervals . After hatching , virgin females and males were collected and mated for 2 days . For lifespan analyses , 50–100 females per genotype were used at a density of 10 flies per vial . Flies were transferred to new vials every two to three days and dead flies were counted . For fecundity assays , 100 females were equally distributed in ten vials , transferred to new vials every day and the number of eggs was counted . Climbing assays were conducted and performance index was calculated as described [39] . 100 three-day-old males per genotype were tested for their climbing ability . DNA of third-instar larvae was extracted using the DNAeasy Kit ( Qiagen ) . Mitochondrial DNA levels were determined by quantitative real-time PCR ( QRT-PCR ) on a 7900HT Real Time PCR system ( Applied Biosystems ) , using SYBR green master mix ( Invitrogen ) . Reactions were carried out in triplicates per sample in a final volume of 20 µl with 5 ng of DNA and 10 pmol of specific primers ( primers are listed in Table S1 ) . Total RNA from third-instar larvae or adult flies was extracted using the Totally RNA KIT ( Ambion ) . Reverse transcription and QRT-PCR was performed using the High capacity RNA-to-cDNA kit ( Applied Biosystems ) and the Taqman 2x Universal PCR mastermix , No Amperase UNG ( Applied Biosystems ) , respectively . Custom-made TaqMan probes against Drosophila mitochondrial transcripts were obtained from Applied Biosystems and are listed in Table S1 . For Northern blot analyses , RNA was fractionated on 1 . 2% agarose gels and blotted to Hybond-N+ membranes ( Amersham Biosciences ) . Membranes were hybridized with 32P-labeled probes and afterwards exposed to PhosphoImager Screens and/or X-ray films . Labeling of mitochondrial double-stranded DNA probes and oligonucleotides was performed as described [40] . Primers and oligonucleotides used for Northern blot are listed in Table S1 . Isolation of mitochondria from third-instar larvae was performed as described [41] with modifications in buffer composition . Briefly , third-instar larvae were washed and gently homogenized in ice-cold MSB buffer ( 210 mM mannitol , 70 mM sucrose , 10 mM EDTA , 50 mM Tris , pH 7 . 5 ) using 15 ml Dounce homogenizers . Protein concentration was determined using Bradford assay and aliquots corresponding to 10 µg mitochondrial proteins were pelleted and resuspended in resuspension buffer ( 250 mM sucrose , 15 mM K2HPO4 , 2 mM MgAc2 , 0 . 5 mM EDTA and 0 . 5 g/L HSA , pH 7 . 2 ) . Biochemical activities of respiratory chain complexes were determined as described [42] . Third-instar larvae ( n = 10 ) or thoraces from adult flies ( n = 5 ) were dissected in PBS and resuspended in 2 ml of respiratory buffer ( 120 mM sucrose , 50 mM KCl , 20 mM Tris-HCl , 4 mM KH2PO4 , 2 mM MgCl2 , 1 mM EGTA , 0 . 01% digitonin , pH 7 . 2 ) . Oxygen consumption was measured at 25°C using an oxygraph chamber ( OROBOROS ) . Complex I-dependent respiration was assessed by adding the substrates proline ( 10 mM ) , pyruvate ( 10 mM ) , malate ( 5 mM ) and glutamate ( 5 mM ) . Succinate and glycerol-3-phosphate dehydrogenase activities were measured using 20 mM succinate ( SUCC ) and 15 mM glycerol-3-phosphate ( G3P ) , respectively . Mitochondrial quality of each sample was assessed by measuring the respiratory control rate ( RCR ) , using 1 mM ADP ( state 3 ) or 1 mM ADP and 2 . 5 µg/ml oligomycin ( pseudo state 4 ) . Permeabilized control mitochondria consistently had RCR values between 4 and 7 with complex I substrates . The respiration was uncoupled by the addition of 400 µM CCCP and the rotenone-sensitive flux was measured in the presence of 200 µM rotenone . Finally , the protein content was determined by the Bradford method ( BioRad ) in order to normalize the oxygen consumption flux to mitochondrial protein content . Flies ( n = 10 ) were snap frozen directly in liquid nitrogen and kept at -80°C . Acidic extraction ( PCA 7% ) was performed , samples were centrifuged ( 16000 g , 10 min ) , the supernatant was neutralized with 2N KOH , 10 mM MOPS and metabolites were quantified . ADP and ATP levels were assessed as previously described [43] , [44] . Briefly , ATP was quantified by using the ATPlite one step kit ( PerkinElmer ) . For ADP levels , samples were incubated for 10 min at 37°C in 75 mM KCl , 8 mM MgSO4 , 10 µg/ml pyruvate kinase and 2 mM phosphoenolpyruvate . Lactate and pyruvate concentrations were determined after 1 h incubation with horse radish peroxydase 5 U/ml , Amplex red 20 µM , 0 . 1 M phosphate , pH 7 . 2 , supplemented with lactate oxidase or pyruvate oxidase , followed by fluorimetric analysis ( ex:560 nm , em:590 nm ) , using an Infinite 200 Pro fluorimeter ( Tecan ) . For the preparation of mitochondria , third-instar larvae or flies were homogenized in ice-cold isolation buffer STE+BSA ( 250 mM sucrose , 5 mM Tris , 2 mM EGTA , 1% ( w/v ) BSA , pH 7 . 4 ) using a 15 ml Dounce homogenizer . Cellular debris were pelleted at 1000 g for 5 min and supernatants were transferred to new tubes . Mitochondria were washed two times and final mitochondrial pellets were resuspend in 1 ml STE buffer in the presence of 200 µg/ml emetine ( Sigma ) and 100 µg/ml cycloheximide ( Sigma ) to inhibit cytoplasmic translation . Protein concentrations were determined using the Bradford assay . In organello transcription assays were performed as described [45] using 200 µg mitochondria/sample and a modified transcription buffer ( 25 mM sucrose , 75 mM sorbitol , 100 mM KCl , 10 mM K2HPO4 , 50 µM EDTA , 5 mM MgCl2 , 1 mM ADP , 10 mM glutamate , 2 . 5 mM malate , 10 mM Tris-HCl ( pH 7 . 4 ) and 1% ( w/v ) BSA ) . In short , after labeling , mitochondrial RNA was isolated using Totally RNA kit ( Ambion ) . Mitochondrial RNA was fractionated on 1 . 2% agarose gels and blotted to Hybond-N+ membranes ( Amersham Biosciences ) . In vitro assays to study mitochondrial de novo translation with [35S]-methionine were performed as described [46] . Equal amounts of total mitochondrial protein were loaded on 15% SDS-PAGE gels . Gels were fixed in isopropanol-acetic solution , stained with Coomassie , destained in ethanol-acetic acid solution and treated with Amplify Solution ( GE Healthcare ) . Afterwards gels were dried and [35S]–methionine-labelled proteins were visualized by autoradiography . The mitochondrial translation profile was compared to previously published profiles in Schneider cell lines [47] , additionally ND2 and ATP6 were identified by endopeptidase fingerprinting in the second dimension ( data not shown ) [48] . An RNA circularization protocol was modified from [49] , [50] . Approximately 6 ng total mitochondrial RNA was circularized with 5 U T4 RNA ligase in 200 µl at 16°C for at least 16 h in manufacturer-supplied buffer ( NEB ) . The circularized RNA was precipitated with an equal volume of isopropanol , incubated at -20°C for at least 4 h , and centrifuged for 20 min at top speed in a bench top centrifuge . The entire precipitate was used for complementary DNA synthesis with gene specific primers using GeneAmp RNA PCR kit ( Applied Biosystems ) . PCR products were purified using ExoSAP-IT ( Affymetrix ) and sequenced . Selected PCR products were cloned into pCR-II ( Invitrogen ) and sequenced in order to confirm the results from direct sequencing . Primer sequences for RT-PCR and subsequent PCR are contained within Table S2 . For BN-PAGE , 75 µg of mitochondria were pelleted and lyzed in 50 µl ice-cold digitonin buffer ( 1% digitonin , 20 mM Tris pH 7 . 4 , 0 . 1 mM EDTA , 50 mM NaCl , 10% glycerol , 1 mM PMSF ) . After 15 min of incubation on ice , unsolubilized material was removed by centrifugation at 4°C . The supernatant was mixed with 5 µl of 10 x loading dye ( 5% ( w/v ) Coomassie Brilliant Blue G-250 , 100 mM Tris pH 7 , 500 mM 6-aminocaproic acid ) and loaded on 4–10% gradient BN-PAGE gels [51] , [52] . In gel complex I activity was determined by incubating the BN-PAGE gels in 2 mM Tris-HCl pH 7 . 4 , 0 . 1 mg/ml NADH ( Roche ) and 2 . 5 mg/ml iodonitrozolium ( Sigma ) . In gel complex IV activity was determined by incubating the BN-PAGE gels in 50 ml of 0 . 05 mM phosphate buffer pH 7 . 4 , 25 mg 3 . 3′-diamidobenzidine tetrahydrochloride ( DAB ) , 50 mg cytochrome c , 3 . 75 g sucrose and 1 mg catalase . All stainings were carried out at room temperature . Data were presented as mean ± SD . The Mann-Whitney test was used to analyze climbing index and the log-rank test was used to analyze lifespan . Unpaired t-test was used to analyze all other data statistically .
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The majority of the cellular energy currency ATP is formed in a tubular network , termed mitochondria , present within virtually all eukaryotic cells . The mitochondria are unique among cellular organelles in that they contain their own genome , which encodes critical proteins necessary for cellular energy production . However , the vast majority of mitochondrial proteins are encoded in the nucleus and imported into mitochondria . Gene expression thus needs to be coordinated between the two genomes to ensure efficient mitochondrial function and sufficient adaptation to different physiological demands . The regulation of the mitochondrial genome is poorly understood , with many of the basic regulators not yet being characterized . We used RNAi in the fruit fly to study the in vivo function of the bicoid stability factor ( BSF ) , previously thought to be a cytoplasmic and nuclear protein important for fly development . We show here that BSF is mainly localized to mitochondria , where it is essential for mtDNA gene expression , regulating the polyadenylation and maturation of specific mRNAs . Furthermore , BSF coordinates the translation and assembly of mitochondrial peptides in the inner mitochondrial membrane .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology"
] |
2011
|
The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster
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The ascomycete fungus Fusarium graminearum causes stalk rot in maize . We tracked this pathogen’s growth in wound-inoculated maize stalks using a fluorescence-labeled fungal isolate and observed that invasive hyphae grew intercellularly up to 24 h post inoculation , grew intra- and inter-cellularly between 36–48 h , and fully occupied invaded cells after 72 h . Using laser microdissection and microarray analysis , we profiled changes in global gene expression during pathogen growth inside pith tissues of maize stalk from 12 h to six days after inoculation and documented transcriptomic patterns that provide further insights into the infection process . Expression changes in transcripts encoding various plant cell wall degrading enzymes appeared to correlate with inter- and intracellular hyphal growth . Genes associated with 36 secondary metabolite biosynthesis clusters were expressed . Expression of several F . graminearum genes potentially involved in mobilization of the storage lipid triacylglycerol and phosphorus-free lipid biosynthesis were induced during early infection time points , and deletion of these genes caused reduction of virulence in maize stalk . Furthermore , we demonstrated that the F . graminearum betaine lipid synthase 1 ( BTA1 ) gene was necessary and sufficient for production of phosphorus-free membrane lipids , and that deletion of BTA1 interfered with F . graminearum’s ability to advance intercellularly . We conclude that F . graminearum produces phosphorus-free membrane lipids to adapt to a phosphate-limited extracellular microenvironment during early stages of its invasion of maize stalk .
The main stem ( i . e . stalk ) of maize ( Zea mays ) represents about 50% of the total plant dry biomass at the grain maturity stage [1] . Stalk rot is a major disease of maize worldwide [2] , and tends to be more common in higher yielding hybrids that produce larger ears [3] . The ascomycete fungal genus Fusarium is the most frequently reported causative agent of maize stalk rot diseases [3] . Fusarium graminearum ( previously also named Gibberella zeae ) , which can cause Gibberella stalk rot , is thought to possess the highest pathogenicity and aggressiveness among species responsible for stalk rot [4] . No specific resistance genes that confer immunity to this disease have been identified to date . A few quantitative trait loci for resistance have been reported , but the nature of the resistance still unknown [5 , 6] . The diferulic acid content of the cell walls of maize stalks has been linked to quantitative resistance by an inbred lines survey [4] . The molecular mechanisms underlying fungal pathogenesis during maize stalk infection are not clear , which limits progress toward effectively controlling the disease . The final symptom of Gibberella stalk rot is lodging ( i . e . breakage of the stalk below the ear ) , which can result in loss of harvestable yield . In the late stages of infection , small , round , black specks ( perithecia , the sexual structures of F . graminearum ) are formed on the surface of the stalk rind . The stalks become hollow tubes with only the rinds and vascular bundle strands remaining . F . graminearum may enter the host through the roots of seedlings or through the bases of the leaf sheaths of young plants , where spores ( sexual ascospores or asexual conidia ) may germinate and grow up into the stalk . Spores may also directly enter adult maize stems through wounds made by corn borers , hail , or mechanical injury [7] . How this fungus manages to overcome various physical barriers and the active defenses of the host and progressively accommodates itself inside the stalk is largely unknown . Upon F . graminearum infection , maize stalk produces kauralexins and zealexins , which can inhibit fungal growth in vitro [8–10] . The invasion of living tissues requires the fungus to be able to break barriers ( such as plant cell walls and plasma membranes ) , and to overcome active host defenses which often involve reactive oxygen species , anti-fungal proteins and small molecular weight metabolites like the phytoalexins mentioned above ) [11 , 12] . The F . graminearum genome has been sequenced [13] and the current annotation [14] reveals an arsenal of potential invasion-related factors including secreted plant cell wall degrading enzymes , proteases , secondary metabolites ( including mycotoxins ) biosynthesis gene clusters , and so on [15] . But how F . graminearum chooses from this arsenal to confront maize stalk defenses is yet to be discovered . Besides causing stalk rot of maize , F . graminearum also causes Fusarium head blight on wheat and barley , ear rot disease of maize , and seedling blight on maize and wheat [16–18] . It has been reported that F . graminearum forms a specialized infection structure on wheat florets [19] , then grows inside the florets , expands through the rachis , and travels down to colonize the stem finally forming sexual structures known as perithecia [20] . The pathogenesis of wheat head blight caused by F . graminearum has been intensively studied . One key event in wheat head blight infection is the production of the harmful trichothecene mycotoxin deoxynivalenol ( DON ) [21] . DON binds to the peptidyltransferase of ribosomes and inhibits protein synthesis [22] , allowing F . graminearum to pass through rachis and escape host cell wall thickening [23] . The outcomes of plant cell exposure to DON include hydrogen peroxide production , host programmed cell death and probably ethylene signaling [24–27] , with specific details remaining to be elucidated . Interestingly , although DON is produced during barley head blight infection , it is not a virulence factor for barley head infection [23] . It has been reported that low amounts of DON can be detected in maize stalks from fields with stalk rot diseases , ( much lower than the level detected in infected ears of maize [28] ) . The role of DON in maize stalk infection is still unknown . Other than DON production genes , numerous virulence genes have been identified in F . graminearum [29–31] . Because mutants of many virulence genes also showed severe defects in in vitro growth , these genes are considered as fungal essential genes . It is therefore more accepted to consider those dispensable for in vitro fungal growth but required for full virulence as pathogenicity genes . The number of pathogenicity genes using this definition is still limited , including genes encoding a secreted lipase FGL1 [32] , a cyclin-dependent kinase CDC2A [33] , two other type filamentous fungi-specific protein kinases ( Fg03146 and Fg04770 ) [34] , a Rab GTPase Rab2 [35] , four phosphatases [36] , transcription factors FGSG_01915 [37] and Tri6 [38] , and a secondary metabolite biosynthesis cluster gene FGSG_10992 [39] . Most of the proteins encoded by pathogenicity genes are predicted to be intracellular proteins of F . graminearum , and thus are not in direct contact with plant components . Only FGL1 is an extracellular enzyme , mediating release of polyunsaturated free fatty acids that inhibit plant callose synthase activity [40] . Most of these Fusarium head blight pathogenicity genes have not been tested in maize stalk infection , except for CDC2A whose deletion also caused reduction in maize stalk infection [33] . It certainly will be interesting to delineate details of pathogenicity conservation and divergence between wheat head blight infection and maize stalk infection , two very different diseases both caused by the same fungus . In the current study , we assessed fungal growth during maize stalk rot development at the cellular level , then used laser microdissection and microarray analysis to provide high-resolution gene expression profiles of F . graminearum transcripts over the course of infection at eight time points spanning from 12 h to 6 days after inoculation . A number of gene expression changes can be ascribed to invasive and/or survival strategies tailored to maize stalk infection . We also obtained evidence for non-phosphorus lipid accumulation during early infection . Using targeted gene knockout mutants , Using targeted gene knockout mutants , we show that a fungal betaine lipid synthase responsible for biosynthesis of non-phosphorus membrane lipids , two putative triacylglycerol lipases and three putative phosphatases are required for F . graminearum full virulence in maize stalk . We propose that F . graminearum employs a strategy to produce non-phosphorus membrane lipids in order to overcome growth-limiting phosphorus levels during intercellular growth in maize stalk .
To shed light on the process of F . graminearum proliferation inside maize stalks , we wounded the stalk of whole plants of maize roughly at the appearance of the tenth leaf , and inoculated plants with spores of a fluorescently ( AmCyan ) -tagged F . graminearum strain , which , other than its fluorescence , behaves like wild-type strains [39 , 41] . From 2 h to 2 weeks after inoculation , we harvested individual maize plants , split the inoculated internodes and observed fungal migration and the appearance of the stalk tissue in detail ( Fig 1A and Figure A in S1 Text ) . To the naked eye , the split internodes were almost symptomless for up to 36 h after inoculation ( hai ) . Stalk tissues at the wound site turned pale brown by 48 hai , and by 72 hai the brown color became darker and the brown area elongated above and below the site of the wound ( Fig 1B ) . We then observed the infection process by wide-field and confocal microscopy . By 12 hai , the fungal spore germinated , and then advanced inside stalk tissues in a hyphal form . In general , fungal hyphae grew longitudinally rather than transversely ( Fig 1C and 1D ) . There are three major groups of cells in maize stalks , fiber cells in the rind , pith parenchyma cells , and vascular bundles ( Figure B in S1 Text ) . Fungal hyphal progression in the pith precedes progression in the rind , but vascular bundles seemed to be avoided ( Figure C in S1 Text ) . Fig 1C and Figure C in S1 text show that at 12–18 hai , most hyphae in the pith , which were unbranched and originated from germinated spores at wound sites , were found in the intercellular space of pith tissue , up to five layers of parenchyma cells away from the wounded layer , while the maize pith tissue remained symptomless to the naked eye . At approximately 18 hai , some multiple unbranched hyphae grew in parallel with each other along the intercellular space . At 24–36 hai , the majority of hyphae were unbranched and advanced intercellularly , but some hyphae had become branched and entered intracellular space . The stalk tissues still appeared symptomless under white light; but under UV light , the plant cell walls of an area beyond the fungal invasion exhibited strong autofluorescence ( Figure D in S1 Text ) . At approximately 48 hai , more hyphae had penetrated maize cells , while the majority of hyphae were still intercellular . The infected pith tissues turned pale brown; the brown areas extended beyond the area hyphae had reached , and many vascular bundles far beyond the infected area turned brown . At approximately 72 hai , near the wounding site , some host parenchyma cell spaces were occupied by hyphae , while these parenchyma cell walls ( marked by strong autofluorescence ) still maintained a regular shape ( Figure C in S1 Text ) . The hyphae at the advancing frontline continued to be intercellular . The brown area of plant tissues darkened , and also expanded , still exceeding the area reached by the fungal hyphae . The lengths of lesions increased most rapidly at 48–72 hai , preceding the peak time of the hyphal front extension , which occurred at 72–96 hai ( Fig 2 ) . Interestingly , by around 96 hai , most maize cells in the brown zone exhibited debris-like structures ( Fig 3A ) . The structure could be weakly stained with 4’ , 6-diamino-2-phenylindole ( DAPI ) ( Figure E in S1 Text ) and we therefore suspected them to be deformed nuclei . It is worth noting that many maize cells that harbored deformed nuclei were not directly invaded by F . graminearum hyphae , but were a few cell layers away from those hyphae . At approximately 108 hai , the host cell walls within the infected area became deformed and partially disappeared ( Figure C in S1 Text ) . At approximately 132–144 hai , most of the infected cells near the wound site were full of hyphae , and the pith tissue had totally collapsed ( Figure C in S1 Text ) . The brown lesions further expanded to approximately 12 mm on either side of the wound site at 144 hai . After 360 hai , only the vascular strands remained intact , although they turned black , and the pith tissue in the center of the infected area had disintegrated . Condensed or deformed nuclei can be seen in cells that undergo programmed cell death [42] . We assessed the viability of host cells near to fungal hyphae using a NaCl treatment ( see Methods ) , and found that within 24 hai , among the maize cells that had been reached by hyphae , at least some still exhibited plasmolysis ( Fig 3B and Figure F in S1 Text ) , suggesting these cells still had their plasma membrane intact and may thus be alive; around 48–72 hai , we did not observe plasmolysis by any maize cells that directly interacting with fungal hyphae , but maize cells adjacent to hyphal-invaded cells exhibited plasmolysis ( Fig 3B and Figure F in S1 Text ) ; around 96 hai , only those cells that were beyond or at the edge of the brown zone were still capable of plasmolysis ( Fig 3C ) ; around 144 hai , only cells beyond the brown zone maintained plasmolytic ability . Our results indicate that , at the early intercellular invasion stage , the region of dead host cells was limited to those directly interacting with fungal hyphae . Later , the “killing zone” extended to surrounding cells without direct interaction ( Fig 3C ) . In summary ( Fig 2C ) , the proliferation of F . graminearum in maize stalk pith is at first exclusively a process of intercellular growth ( 12–18 hai ) . Then , intercellular growth continues along with the initiation of intracellular growth , in which the pathogen penetrates the surrounding parenchyma cells ( 24–48 hai ) and then occupies the invaded parenchyma cells ( 72–108 hai ) , ultimately destroying the host pith tissues except for the vascular bundles ( 132–144 hai ) . In the early time points ( 12–48 hai ) , the fungal growth route is clearly restricted by plant cell wall structures , and at later time points ( 72–144 hai ) there seems to be no plant restriction of fungal growth . Before 48 hai , the infected stalk tissue showed no symptoms visible to the unaided eye; after 72 hai , the brown lesion became pronounced ( Fig 2C ) . Also at early time points ( 12–48 hai ) hyphae grew between live host cells , while after 72 hai the hyphal front grew between dying/dead host cells with a greater speed of advance ( Fig 3B ) . To gain insight into how F . graminearum survives and proliferates inside the maize stalk , we charted the in planta fungal transcriptome dynamics along with disease development , using laser microdissection in combination with fungal microarray hybridization . Our previous experience studying cell behavior from cell type specific transcriptomes [39 , 43] has reinforced that the predictive power of laser microdissection-derived transcriptome data relies on the homogeneity of the harvested cells . Maize stalks are composed of various cell types ( Figure B in S1 Text ) , and F . graminearum progression in rind fiber cells or vascular bundles is different from the progression in pith parenchyma cells ( Figure C in S1 Text ) . To tackle a complex process in a simple way , we focused on F . graminearum infection in parenchyma cells , which constitute the major cell type in the pith of maize stalk . We used laser microdissection to capture hyphal samples infecting maize stalk parenchyma cells at eight representative time points , ( 12 , 18 , 36 , 48 , 72 , 108 , 132 , and 144 hai ) , carefully avoiding hyphae growing in rinds or vascular bundles ( Fig 4A ) . RNA derived from these samples was hybridized to the F . graminearum whole genome Affymetrix GeneChip [44] . We thus obtained in planta F . graminearum transcriptome data at eight time points during maize stalk infection . In vitro-cultured spores used for inoculation represented the initial time point ( 0 hai ) , and hyphae cultured in vitro for 72 h represented the in vitro growth stage . RNA from these samples was also hybridized to the F . graminearum GeneChip to obtain in vitro fungal transcriptomes for comparison . The much greater effort we invested in isolating fungal hyphae from infected stalks , rather than directly extracting RNA from bulk infected stalk , was rewarded by the quality of the data we obtained . At a very early infection stage , the fungal proportion of the infected tissue may be <0 . 1% , resulting in a very low signal-to-noise ratio in the transcriptomic data . In late infection stages after colonization , the fungal proportion could be much higher . A large range of fungal proportion between early and late infection stages makes it very difficult to chart true fungal expression changes over the course of the infection [45 , 46] . However , by using laser microdissection , we were able to isolate enriched fungal samples throughout the infection process , and this allowed us to directly compare gene expression changes at each stage . Unsupervised hierarchical clustering of F . graminearum transcriptomes showed that all the biological replicates at a given time point grouped together ( Fig 4B ) with high correlations ( Dataset A in S2 Text ) , indicating the high reproducibility of our microarray data . We randomly chose 16 genes for real-time PCR verification at seven time points ( spore , in vitro 72 h , 12 hai , 18 hai , 48 hai , 72 hai , and 132 hai ) , and results for 13 genes were highly consistent with the microarray data ( Figure G in S1 Text ) . Using the Significance Analysis of Microarrays ( SAM ) method [47] , a false discovery rate of 0 . 05 was used as the cutoff value for statistical significance , and a 2-fold change in expression was used as the cutoff for fold changes . Within these bounds , we identified that at each in planta time point , an average of 1 , 848 genes were significantly up-regulated and an average of 2 , 160 genes were significantly down-regulated , compared to the reference in vitro growth stage ( Fig 4D and Dataset B in S2 Text ) . The expression data at eight time points were obtained from the most synchronous materials we could collect . Considering the total of 4 , 575 genes that were significantly up-regulated in at least one in planta time point , we found 394 groups that clustered based on their expression patterns over the course of infection from 0 hai to 144 hai ( Dataset C in S2 Text ) . The heat maps in Fig 4E and Figure H in S1 Text show that 794 genes ( 17 . 4% of 4 , 575 genes ) were up-regulated in at least seven out of the eight in planta time points , 1108 genes ( 24 . 2% ) were up-regulated either in at least three out of the four early in planta time points ( 12 hai , 18 hai , 36 hai and 48 hai ) or in at least 3 of the four later in planta time points ( 72 hai , 108 hai , 132 hai and 144 hai ) ; and 1289 genes ( 28 . 2% of 4 , 575 genes ) were up-regulated in only one in planta time point or in only two to three adjacent in planta time points , which indicates stage-specific expression features . The genes in the three major types mentioned above constitute 70% of the 4 , 575 genes . Among the 4 , 575 differentially-expressed genes ( DEGs ) , 3 , 006 were not up-regulated in spores compared to in vitro grown hyphae ( Fig 4E and Dataset D in S2 Text ) , representing the group of genes that most likely function in hyphal growth in a maize stalk . Thereafter , we term these 3006 F . graminearum DEGs up-regulated in at least one time point during maize stalk infection but not up-regulated in the spore sample ( compared to in vitro hyphae ) as genes preferentially expressed during hyphal growth in maize stalk ( PEMS ) . Based on Munich Information Center for Protein Sequences ( MIPS ) FGDB Functional Catalogue ( FunCat ) annotation [14 , 48 , 49] , 5 , 147 F . graminearum genes are assigned to FunCat categories , 4 , 966 of the 13 , 346 genes presented on our microarray have assigned FunCat categories . A total of 163 FunCat categories are enriched in the 3 , 006 PEMS genes ( detailed lists provided in Dataset E in S2 Text ) . Most of the enriched categories are in C-compound and carbohydrate metabolism ( FunCat 1 . 05 ) , amino acid metabolism ( FunCat 1 . 01 ) , lipid , fatty acid and isoprenoid metabolism ( FunCat 1 . 06 ) , secondary metabolism ( FunCat 1 . 20 ) , and extracellular metabolism ( FunCat 1 . 25 ) . Regarding subcellular localization of encoded proteins , extracellular/secreted protein ( FunCat 70 . 27 ) are enriched at the 12–36 and 108–132 hai timepoints , while plasma membrane proteins ( FunCat 70 . 02 ) are enriched in the 108–144 hai DEGs . Considering the PEMS genes that are up-regulated in only one in planta time point , or in only two to three adjacent in planta time points ( Fig 4E ) , we can observe over-represented functional categories defined as the FunCat classification system . For example , among genes significantly up-regulated at 12–18 hai ( Fig 4E b ) , the enriched groups are metabolism of the cysteine—aromatic group ( FunCat 01 . 01 . 09 ) , phosphate metabolism ( FunCat 01 . 04 ) , C-compound and carbohydrate metabolism ( FunCat 01 . 05 ) , lipid , fatty acid and isoprenoid metabolism ( FunCat 01 . 06 ) , secondary metabolism ( FunCat 01 . 20 ) , cell cycle ( FunCat 10 . 03 ) , cellular transport ( FunCat 20 ) , transport routes ( FunCat 20 . 09 ) , cell rescue , defense and virulence ( FunCat 32 ) ( Dataset F in S2 Text ) . The enrichment of FunCat categories “C-compound and carbohydrate metabolism” and “extracellular metabolism” during maize infection implies the production of plant cell wall degrading enzymes . The F . graminearum genome encodes 134 putative carbohydrate-active enzymes [50] ( CAZymes , www . cazy . org ) with signal peptides that potentially degrade various plant cell wall components , including cellulose , hemicelluloses , pectin , lignin and cutin ( Dataset G in S2 Text ) . As expected , the aggregate ( i . e . total combined ) expression of these 134 putative plant cell wall degradation enzymes ( CWDEs ) was more than tripled during infection compared with in vitro grown F . graminearum ( Fig 5A ) . The expression of 98 of these CWDEs was significantly up-regulated in at least one time point in maize stalk infection . There were two peaks of elevated overall expression levels of these genes during maize stalk infection ( shown in Fig 5A and detailed in Dataset B in S2 Text ) ; the first peak was at 12 to 36 hai , the second peak was at 108 hai . Considering the cell wall components potentially targeted during infection , the overall expression levels of genes encoding enzymes targeting lignin , for example , were low . As Figure B in S1 Text shows , lignin accumulation ( as indicated by pink staining ) could be observed in cell walls in vascular bundles , but not in cell walls of pith parenchyma cells . The low expression of lignin digestion enzyme genes [51] may result in low-level synthesis of the enzymes , and may explain why F . graminearum does not frequently break into vascular bundles . Pectin main chain degradation enzymes reached very high levels at the early time points of infection when F . graminearum is primarily elongating intercellularly ( Fig 5B ) . This matches our expectations: pectin is the major component of the intercellular middle lamella and would therefore play a significant part in blocking intercellular advancement of F . graminearum , whereas endolytic pectinase activity of the fungus might allow hyphal passage . In general , the expression patterns of CWDE genes suggest that the expression of pathogen partition enzymes is fine-tuned to allow the pathogen to adapt to the changing conditions of the maize stalk environment . FunCat category 32 . 07 detoxification , a branch of the main category FunCat 32 cell rescue , defense and virulence , was significantly enriched in PEMS genes ( Dataset F in S2 Text ) . The expression of 167 out of the 391 F . graminearum genes annotated with the function “detoxification” was significantly increased during maize infection compared to that in in vitro grown hyphae . ( Fig 5C and Dataset H in S2 Text ) . It is interesting to note that the aggregate expression of three subgroups of detoxification-related genes ( i . e . genes encoding proteins related to detoxification by modification , degradation , and export ) peaked at different time points ( Fig 5C and Dataset H in S2 Text ) . Genes related to detoxification by modification ( FunCat 32 . 07 . 03 ) including those encoding proteins potentially with oxidoreductase and/or isomerase activity showed the peak expression at 36 hai; expression of genes involved in detoxification by export ( FunCat 32 . 07 . 05 ) including putative multidrug resistance proteins peaked at 132 hai , and genes involved in detoxification by degradation ( FunCat 32 . 07 . 09 ) such as putative epoxide hydrolase , reached their maximum expression at 144 hai . These observations suggest that the fungus may have different priorities in using detoxification approaches in different stages of infection . The FunCat category “secondary metabolism biosynthesis” ( SMB ) was enriched in PEMS genes . Two low molecular weight secondary metabolites have been identified as virulence factors of F . graminearum: the mycotoxin DON , a virulence factor in wheat [21]; and an extracellular siderophore , a conserved virulence factor in many pathogens whose role is to acquire nutrients or to protect from oxidative stress [52] . Notably , the extracellular siderophore triacetylfusarinine biosynthesis FG3_60 cluster genes , such as non-ribosomal peptide synthetase 6 ( FGSG_03747 ) [53] and a putative siderophore permease MIR1 ( FGSG_03744 ) [54] , were highly expressed from 12 to 108 hai during maize stalk infection ( Fig 5D and Dataset I in S2 Text ) . The expression data accord with the hypothesis that F . graminearum secretes siderophores during maize stalk infection . DON biosynthesis genes ( Fig 5E ) were not induced during maize stalk infection up to 6 days after inoculation ( dai ) . Furthermore , no significant DON accumulation was detected in 3 or 7 dai maize stalk tissues using an enzyme-linked immunosorbent assay ( Fig 5F ) . In addition , F . graminearum is capable of producing 10 other secondary metabolites , including terpenes , polyketides and nonribosomal peptides [55] . The expression of clusters responsible for producing carotenoid , malonichrome , ferricrocin , triacetylfusarinine , aurofusarin , orcinol and culmorin was detected in maize stalk infection ( Dataset I in S2 Text ) . For example , the carotenoid ( terpenoid pigment ) biosynthesis gene cluster was induced at 48 hai . The aurofusarin ( polyketide pigment ) biosynthesis cluster was induced at 12 hai and maintained at a high expression level at 18 hai and 48 hai ( Fig 5D ) . We also detected aurofusarin by mass spectrometry in infected maize stalk tissue . The expression of genes in clusters responsible for production of zearalenone , fusarielin or fusarin C was not significantly induced in maize stalk infection ( Dataset I in S2 Text ) . In fungi , most of the genes required for the biosynthesis of a particular secondary metabolite occur in a cluster [56] . A total of 67 SMB clusters , including 10 with known products and others with unknown products , have been previously reported in F . graminearum [55 , 57 , 58] . Thirty-six out of these clusters showed a co-expression pattern in maize stalk infection in our data . We also found 7 additional coexpressed gene clusters based on expression profiles of adjacent genes ( Dataset I in S2 Text ) . We observed that the aggregate ( i . e . combined total ) expression of 36 genes whose function is assigned to be in the metabolism of glycerophospholipids , essential components of all biological membranes , was actually lower during maize infection than in in vitro grown hyphae ( Dataset J in S2 Text ) . But membrane biogenesis is essential for fungal hyphal growth in all conditions , and transcripts significantly up-regulated at early time points in maize stalk infection included several annotated as being involved in lipid , fatty acid and isoprenoid metabolism ( Dataset F in S2 Text ) . So we explored whether this could be associated with active biosynthesis of membrane lipids other than glycerophospholipids . Fig 6A shows the expression of genes encoding enzymes responsible for reactions involving diacylglycerol ( DAG ) , which is a major precursor for the glycerophospholipids phosphatidylcholine ( PC ) and phosphatidylethanolamine ( PE ) . FGSG_09402 and FGSG_08706 , encode putative ethanolamine-phosphotransferases responsible for converting DAG to PC and PE . Both genes were expressed at lower level during maize infection than during germination in vitro . Particularly , the expression of FGSG_09402 at 12–18 hai , was <30% of the level in in vitro hyphae ( Dataset J in S2 Text ) . The expression of FGSG_11236 , which encodes a putative non-specific type phospholipase C ( PLC ) responsible for converting PC to DAG , was significantly increased at 12–18 hai , and increased more than 80-fold at 18 hai compared with its expression in in vitro hyphae . Triacylglycerol lipases ( TAGL ) are responsible for mobilizing triacylglycerol ( TAG ) , the major storage lipid in fungi , to DAG . F . graminearum genome contains four genes encoding putative TAGLs . Two of these ( FGSG_03846 and FGSG_03243 ) were significantly up-regulated in maize infection compared with in vitro . The above gene expression suggests an increase in DAG production and a reduction in DAG consumption , at 12–18 hai in maize infection . This prompted us to consider what is the next intermediate after DAG in membrane lipid synthesis by F . graminearum in maize stalk if it is not PC or PE . We noted differential expression of FGSG_00742 encoding a putative S-adenosylmethionine:diacylglycerol 3-amino-3-carboxypropyl transferase ( also called betaine lipid synthase , BTA1 ) . This enzyme is responsible for the conversion of DAG to diacylglyceryl-N , N , N-trimethylhomoserine ( DGTS ) . DGTS is a phosphorus-free lipid , but resembles the glycerophospholipid PC in structure and physical phase behavior [59] . The expression of FGSG_00742 at 18 hai was very significantly higher than that in in vitro growth , and more than 200-fold higher than in spores ( 0 hai ) . Almost no expression of BTA1 was detected during in vitro germination and hyphal growth ( at 0 , 2 , 8 , 24 , or 72 h ) . Real-time PCR experiments confirmed that expression of BTA1 and PLC was much higher in maize 18 hai than in vitro-grown hyphae ( Figure I in S1 Text ) . In addition to DAG , BTA1 uses S-adenosylmethionine ( SAM ) as a cosubstrate in the production of DGTS . The gene encoding the enzyme responsible for SAM production from methionine is predicted to be FGSG_00421 , which was expressed at high levels in most samples examined and exhibited a 1 . 7-fold increase ( at 18 hai ) over the level in spores ( Fig 6A ) . The expression of the methionine synthase MSY1 gene ( FGSG_10825 ) also increased approximately eightfold at 12 hai , and maintained a fourfold increase at 18 hai over the level in spores . Thus , the enzymes supplying the BTA1 substrate SAM had an elevated expression level at 18 hai , the point at which BTA1 expression reached its peak . We also checked the expression of genes encoding enzymes involved in glycerophospholipid metabolism that carry out reactions without net consumption or release of phosphorus . The expression of the gene encoding phospholipase D ( FGSG_01973 ) , an enzyme that digests membrane lipids but does not release phosphate groups , was lower at 12–18 hai than in spores and in vitro grown hyphae ( Fig 6A ) . Along with the observation that the expression of PLC and BTA1 , which encode enzymes converting phospholipid to phosphorus-free lipid , was higher at 18 hai than in spores and in vitro grown hyphae , these results hinted at a reduction in phosphorus content in membrane lipids composition at 18 hai in maize stalk infection . The FunCat category “phosphate metabolism” is enriched in DEGs up-regulated in maize 12–18 hai but not up-regulated in the spore sample ( compared to in vitro grown hyphae ) ( Dataset F in S2 Text ) . Several genes encoding putative phosphatases are preferentially expressed during hyphal growth in maize stalk ( Fig 6B ) . For example , FGSG_06610 encodes a putative protein with a conserved phosphodiesterase/alkaline phosphatase D ( PhoD ) domain , and high similarity to PhoD from the cyanobacterium Aphanothece halophytica ( overall 35% identity and 50% similarity; Figure J in S1 Text ) , an enzyme that is responsible for the release of free phosphate from organic compounds [60]; FGSG_03366 encodes a putative secreted protein with a conserved histidine phosphatase domain ( branch 2 ) found in histidine acid phosphatases and phytases and a His residue that is phosphorylated during the reaction ( similar to Pho12 in budding yeast ) ; FGSG_07678 , encodes a secretory acid phosphatase domain belonging to the aspartate-based protein phosphatase family . Our expression analyses suggested a hypothesis in which F . graminearum conserves phosphorus by producing phosphorus-free membrane lipids so as to use as little phosphorus as possible but still maintain hyphal growth during the early time points of maize stalk infection . We further propose these changes are a response to phosphorus starvation . Phosphorus starvation responses have been studied extensively in the fungus Saccharomyces cerevisiae [61–63] , and it was observed that the plasma membrane high-affinity phosphate importers PHO89 and PHO84 are induced at the transcriptional level in phosphorus-limited conditions [61 , 62] , and that PHO84 plays a role in sensing phosphate [63] . The PHO89 homologous genes in F . graminearum , FGSG_03172 and FGSG_02426 , were highly induced at 12 hai ( before 18 hai when BTA1 and PLC reached peak expression levels ) , while the expression of the gene homologous to PHO84 ( FGSG_01295 ) increased by 12 hai and continued to increase , peaking at 18 hai ( Fig 6B ) . In addition , RT-PCR analysis showed that expression of the genes encoding these phosphate transporters was induced when F . graminearum was grown in phosphorus-depleted medium , as was expression of BTA1 and PLC ( Fig 6C ) . These results are consistent with the idea that F . graminearum is confronting phosphate limited conditions 12–18 hai in maize stalk , at a time when it is mostly growing intercellularly in pith tissues surrounded by living maize cells . We then used inductively coupled plasma mass spectrometry ( ICP-MS ) to measure the phosphorus content in maize stalk , which was approximately 120 micrograms per gram fresh weight , equivalent to about 3 mM phosphorus ( Fig 6D ) , which is in the same range as in routine minimal medium ( 7 mM ) . However , because phosphorus is vital to plant growth , living plants tightly preserve their cellular phosphorus content , and the apoplastic concentration of phosphorus is thought to be low [64] . So we analyzed stalk apoplastic fluid of a mature maize plant , by first collecting the xylem sap from excised stalk tissue using hydraulic pressure , then collecting apoplastic fluid by vacuum infiltration and centrifugation . The residual stalk tissues were ground into powder to measure non-apoplastic phosphorus ( Figure K in S1 Text ) . Using ICP-MS , the measured phosphorus content in apoplastic fluid was below 3 micrograms per gram fresh weight , i . e . , much lower than the stalk tissue phosphorus content ( Fig 6D and Figure L in S1 Text ) . Furthermore , the measured volume of a 6-gram stalk tissue sample was about 8 cm3 , while the apoplastic space was about 20% of the total stalk volume , meaning that the phosphorus concentration in the stalk apoplastic space was 0 . 3 mM , about one-tenth of the intracellular concentration ( Fig 6D ) . This demonstrates that the phosphorus content is indeed low in the apoplastic space of maize stalk and intercellularly invading F . graminearum is faced with a phosphorus shortage . To further evaluate the involvement of the putative non-phosphorus membrane lipid production enzymes and phosphatases in F . graminearum infection of maize stalk , we generated deletion mutants for BTA1 , TAG lipases FGSG_03846 and FGSG_03243 , PLC , and phosphatases FGSG_06610 , FGSG_03366 , FGSG_07678 , FGSG_03402 , and FGSG_07783 ( Figure M in S1 Text ) . All nine gene deletion mutants grew similarly to the wild-type strain on routine medium , but six showed significant reductions in the size of lesions at 7 days after inoculation into maize stalk ( Fig 6E and 6F ) . Multiple independent mutants with FGSG_03846 or FGSG_03243 putative TAG lipase gene knockouts produced a 40% reduction in lesion size relative to the wild type , while deletion of PLC had no effect on lesion size . Lesions caused by mutants in FGSG_06610 , FGSG_03366 or FGSG_07678 were about 50% as large as wild-type lesions , but deletion of the other two putative phosphatase genes , FGSG_03402 and FGSG_07783 , did not significantly affect maize stalk infection . Of these nine genes , the deletion of BTA1 affected maize stalk infection most severely . When examined 7 days after inoculation , the lesions caused by two independent bta1 mutant strains were reduced to approximately one-third the size of lesions caused by the wild-type strain; complemented strains produced lesions that were similar in size to those caused by the wild-type strain ( Fig 6E ) . When infected maize stalks were left open to air to dry for two more days ( i . e . until 9 dai ) , it was apparent that fewer vascular bundles were exposed in lesions caused by the bta1 mutant than in lesions caused by the wild-type fungus ( Fig 7B ) . Therefore , BTA1 is important for the virulence of F . graminearum in maize stalk infection . To understand how the deletion of BTA1 attenuates F . graminearum colonization of maize stalk , we compared the stalk infection progress of mutant and wild-type fungus at 2 , 4 and 6 dai . Fig 7A shows that the mutant and wild-type exhibited differences as early as 2 dai; stalk inoculated with the wild-type strain had already turned brownish at the infection site , whereas stalk tissues inoculated with mutant strains were still of similar color to mock-inoculated stalks . This makes sense as F . graminearum BTA1 expression is induced as early as 18 hai in infection of maize stalks by the wild-type ( Fig 6A ) . By 4 dai , the stalk tissues infected by the mutant strain were brown , but the brown region was limited to 3 mm above and below the inoculation site on both up and down sides , while the brown region caused by wild-type strain usually reached 6 mm . By 6 dai , the differences in brown lesion sizes were more evident , and the debris-like structures observed in maize cells surrounding wild-type hyphae were not seen in maize cells surrounding bta1 mutant hyphae ( Fig 7C ) . In addition , hyphae were generally observed in the intracellular regions of lesions caused by bta1 mutants , while in lesions caused by the wild type , the hyphal front migrated intercellularly ( Fig 7C ) . When we examined the fungal progression as early as 1 dai , we found that the mutant hyphae were growing intracellularly , while most wild-type hyphae were growing intercellularly . This result indicates that the capacity for intercellular expansion is compromised in the bta1 mutant . If our hypothesis is correct that the bta1 mutant’s inability to cope with phosphorus-limitation compromises its virulence in maize stalks , then supplementation of phosphate during the early phase of invasion should restore its virulence . Phosphate supplementation applied via soil mix did not affect lesion size for either mutant or wild-type strains , but it may be difficult to affect apoplastic phosphate levels in this way . However , when we added a KH2PO4 solution to the infection site 8 h after inoculation of bta1 mutant spores , the lesion size observed at 7 dai significantly increased , and was similar to those inoculated by wild-type F . graminearum ( Fig 7D and Figure N in S1 Text ) . The results showed that , as little as 10 nmol exogenous phosphate restored the virulence of the bta1 mutant in maize stalk . We next assessed the viability of the BTA1 knockout strains on phosphorus depleted medium . bta1 mutant colonies grew on minimal medium and had similar expansion rates to the wild type ( Fig 7E ) , although they accumulated red pigmentation earlier than the wild type . Both the mutant and wild-type strains grew slowly on phosphate depleted medium than on complete medium . We suspected that phosphorus stored in the original spore persisted and enabled growth . Therefore , we transferred hyphae from phosphate-deprivation medium to fresh phosphate-deprivation medium for a second round of growth . Fig 7E shows that in this second round growth , the bta1 mutants barely grew hyphae , while the wild-type and complemented strains produced some new hyphae . These results demonstrate that without BTA1 , the growth of F . graminearum was impaired under extreme phosphate starvation in vitro . The putative BTA1 in F . graminearum is similar to BTA1 in Neurospora crassa ( overall identity 36% , Figure I in S1 Text ) . N . crassa BTA1 is responsible for synthesis of the phosphorus-free betaine lipid DGTS upon phosphate starvation [59] . BTA1 gene expression in F . graminearum is induced in response to phosphate starvation in vitro ( Fig 6C ) . We directly assessed whether F . graminearum BTA1 can , like its homolog in N . crassa , synthesize DGTS . The lipids extracted from F . graminearum hyphae grown on phosphate-deprived medium for 14 days were analyzed by thin layer chromatography ( TLC ) . Fig 8A and 8B shows that a spot migrating at a similar position to DGTS was detected from wild-type hyphae; this was confirmed to be DGTS by quadrupole time-of-flight liquid chromatography–mass spectrometry ( Q-TOF LC-MS ) . DGTS could not be detected in the extracts from bta1 mutants grown on either phosphate-replete or phosphate depleted medium ( Fig 8D and Figure I in S1 Text ) . BTA1 cDNA obtained from F . graminearum was fused with GST and expressed in Escherichia coli . DGTS production in this system was similarly detected by TLC and Q-TOF LC-MS ( Fig 8A and 8B and Figure I in S1 Text ) . Furthermore , we constructed a F . graminearum strain constitutively expressing BTA1 ( BTA1 OE; Fig 8C ) . DGTS could be detected in extracts from this strain grown on minimal medium ( Fig 8D and 8E ) . These results demonstrate that F . graminearum BTA1 is responsible for DGTS biosynthesis . We investigated the subcellular localization of F . graminearum BTA1 ( Fig 8F ) and detected it in the endoplasmic reticulum ( ER ) where it colocalized with an ER marker protein fused to YFP [65] . The BTA1 product of Chlamydomonas reinhardtii is also thought to localize to the ER [66] , and the ER is a major location for membrane lipid biosynthesis . BTA1 OE strains express BTA1 at the spore and germinated spore stages , i . e . earlier than in the wild type , which induces BTA1 expression at 18 hai . BTA1 OE strains caused even bigger lesions than the wild-type strain in maize stalks ( Fig 8G and Figure N in S1 Text ) , suggesting that the ability to conserve phosphate through BTA1 activity at an earlier stage of growth can actually enhance F . graminearum virulence in the phosphorus-limited microenvironment of maize stalks .
In this study , we performed in-depth anatomical studies and laser microdissection-assisted fungal transcriptomic profiling on the development of an important disease , maize stalk rot caused by F . graminearum ( Figs 1–4 ) . These data provided many hypotheses regarding the mechanisms used by F . graminearum to confront host environments , including cell wall degradation , detoxification , and the generation of diverse secondary metabolites ( Fig 9 ) . Most excitingly , following up on our observation of changes in the expression of membrane lipid metabolism pathways , we showed experimentally that F . graminearum uses betaine lipid synthase ( BTA1 ) to produce non-phosphorus DGTS ( instead of PC ) for use in membrane lipids during early time points of maize stalk infection , and we have correlated this change with enhanced ability to overcome phosphate limitation in the apoplast of maize stalk tissue ( Figs 6–8 ) . Therefore the non-phosphorus membrane lipid DGTS and the enzyme BTA1 might be potential targets for future design of F . graminearum resistance in maize . Our results reveal a previously unreported stress for apoplastic pathogens inside healthy maize stalks , i . e . , phosphate limitation . Even when the whole maize plant is not phosphate-limited , the apoplastic phosphorus level is low ( Fig 6D ) . Pathogens that take an extracellular route in early infection have to confront this stress . In our wounding inoculation system , F . graminearum spores started to germinate around 8 hai . In planta profiling hinted that , shortly after germination ( at 12 hai ) , intercellular F . graminearum may , in a common phosphate starvation response , produce three putative high-affinity phosphate transporters ( Fig 6B ) to fetch environmental phosphate . However , as most host cells have intact plasma membranes ( based on our plasmolysis results—Fig 3B ) and the host extracellular space is phosphorus limited ( Fig 6D ) , the fungus still may not obtain enough phosphate by the use of transporters to support rapid hyphal growth . We propose that a more specialized strategy is used by F . graminearum around 18 hai , allowing more sparing use of phosphorus-containing PC to support a vigorous intercellular advance . Without this capability ( as suggested by the BTA1 knockout mutant ) , F . graminearum may still make progress by intracellular growth , but with only very slow fungal expansion ( Fig 7C ) . Between 36 and 48 h after infection , we observed a reduction in BTA1 expression , which is consistent with the onset of intracellular growth and the concomitant liberation of phosphate from host cells that have lost membrane integrity . Later on , although hyphae at the front still travel intercellularly , the surrounding cells have already lost plasma membrane integrity ( Fig 3B ) and it is possible that cellular phosphate leaks into the intercellular space and thus is available to the fungus . Using BTA1 to survive phosphate starvation has also been reported in non-pathogens , such as N . crassa [59] . It has been reported that DGTS accumulation is under the control of the transcriptional activator NUC-1 . During maize infection , FGSG_01438 , a homolog of NUC-1 , is also expressed in F . graminearum , although its expression does not increase . As Figure O in S1 Text shows , the bta1 promoter ( 1 kb immediately upstream of the start codon ) contains several sequences that are very similar to phosphate starvation response elements conserved among fungi [67] . Our results highlight the importance of adaptation to a challenging microenvironment during the early phases of host infection . Many fungal pathogens , including several maize stalk rot pathogens , possess the BTA1 gene ( Figure P in S1 Text ) . Maize is known for locking up phosphate in the form of phytate ( also called inositol hexakisphosphate ) . Phosphorus starvation might be a common hurdle to maize stalk fungal pathogens . The maize anthracnose stalk rot fungus Colletotrichum graminicola increased expression of genes encoding putative secreted phytases when it infected maize stalk [45 , 68] , suggesting C . graminicola may gain access to phosphate from phytate . Our results lead to the prediction that the membrane of fungal hyphae around 18 hai in maize stalk contains more DGTS than hyphae grown in vitro . Although DGTS resembles PC in many ways , it is not cleaved by lipase PLC or PLD , while PC can be cleaved by PLC and PLD to release fatty acids , which might be used as signaling molecules in communication between the plant host and the fungal pathogen [69] . Given that fatty acid-derived signals play roles in plant defense [70] , the changes of fungal membrane lipids possibly have effects on plant defense . These are speculations requiring further exploration . F . graminearum also causes wheat head blight disease . Among the pathogenic genes reported in wheat head blight infection , only the cyclin-dependent kinase CDC2A has been reported to also function in maize stalk infection [33] . Among other pathogenic genes reported in wheat head blight infection , we found that the expression of FGL1 [32] , a putative protein kinase Fg04770 [34] , Rab2 [35] , three putative phosphatases [36] and the putative transcription factor FGSG_01915 [37] was significantly increased expression during maize stalk infection compared to in vitro growth ( Dataset K in S2 Text ) . This suggests these genes might also participate in maize stalk rot infection . The mycotoxin DON is required for wheat head blight infection [21] , but DON biosynthesis genes except Tri101 and Tri16 were not induced during maize stalk infection up to 144 hai ( Fig 5E ) . Trichothecene 3-O-acetyltransferase ( Tri101 ) catalyzes the conversion of toxic trichothecenes to less-toxic products; and has been proposed as a metabolic self-protection mechanism in F . graminearum [71] . Furthermore , the metabolite DON could not be detected in infected maize stalks up to 7 days post inoculation . Our results suggest the role of DON in early infection of maize stalk might not be as important as it is in wheat head infection . Several reports describe F . graminearum transcriptomes during head blight development in cereals using RNA extracted from bulk infected plant tissues and the same design of GeneChip as in this study [44 , 72 , 73] . Lysoe and colleagues studied F . graminearum gene expression at 24 , 48 , 72 , 96 , 144 and 192 hai during wheat head infection [72] , and compared with F . graminearum gene expression at 24 , 48 , 72 , 96 and 144 hai during barley head infection , as well as during in vitro growth on three types of media [44] . Because of sampling method differences , direct global normalization of transcriptomes from our work and those from wheat or barley head blight infection may not deliver meaningful conclusions . However , Lysoe and colleagues generated three lists of F . graminearum genes based on their expression: 1 . 404 probe sets ( corresponding to 355 genes ) named “exclusively” in wheat , are those whose expression can be detected in wheat head infection but not detected in barley head infection or during growth in media; 2 . 113 probe sets named “exclusively” in barley , are those whose expression can be detected in barely head infection but not in wheat or in media; 3 . 395 probe sets ( corresponding to 369 genes ) named “both in wheat and barley” whose expression was detected both in wheat and barley head infection but not in media . Comparing these gene lists derived from wheat and barley head blight infection to our list of genes with significantly increased expression during maize stalk infection than in vitro grown hyphae may provide insights into common and different pathogenesis strategies in wheat/barely head blight and maize stalk rot above the level of individual genes . For example , among the 404 and 113 probe sets detected exclusively in wheat and barley , respectively [72] , 82 and nine , respectively , were also up-regulated in at least one time point in maize stalk infection; while in the 369 genes expressed in both wheat and barley , 323 were also significantly up-regulated in at least one time point in maize stalk infection ( Figure Q in S1 Text and Dataset L in S2 Text ) . These 323 common genes could be candidate core pathogenesis genes for F . graminearum infection in various hosts . Dataset K in S2 Text also provides detail on the expression in maize stalk of previously reported SMB related genes [72] . Lysoe et al . [72] further reported that the 355 “exclusively” expressed in wheat genes are enriched in 18 FunCat groups including allantoin and allantoate transport , degradation of polysaccharides and ester compounds , and so on . We identified 163 enriched FunCat groups in 3006 genes that were significantly up-regulated in maize stalk infection but not up-regulated in spores ( relative to in vitro grown hyphae ) . Dataset M in S2 Text provides detailed comparisons between [72] and our work . Two-thirds of the 18 functional groups enriched in wheat infection are also enriched in maize stalk infection . For example , the functional group predicted to be involved in FunCat 20 . 01 . 23 allantoin and allantoate transport is also enriched during maize infection with genes significantly up-regulated at 108 , 132 and 144 hai . For another example , FunCat 01 . 25 extracellular metabolism has been identified as enriched in both wheat head infection and maize stalk infection , our study pointed out that more branches of FunCat 01 . 25 extracellular metabolism are enriched at 12–18 hai in maize stalk infection , including 01 . 25 . 07 exogenous polysaccharide degradation , extracellular polysaccharide degradation , 01 . 25 . 09 extracellular lignin degradation ( Dataset M in S2 Text ) . Lysoe et al . [72] also reported that in the 395 probe sets expressed in both wheat and barley , the over-represented functional categories were carbohydrate metabolism , extracellular ester and polysaccharide degradation , polysaccharide binding , disease , virulence and defense and secondary metabolism . These functional categories are also enriched in genes up-regulated during maize stalk infection . A Fungal Fruit Body Lectin ( FFBL , FGSG_07558 ) protein has been identified as a fungal effector that causes plant cell death in Arabidopsis [74] . We found its expression increased around the late colonization phase of maize stalk infection by F . graminearum ( after 108 hai; Dataset K in S2 Text ) , suggesting this protein might also function in late-stage maize stalk infection . The biotrophic basidiomycete pathogen Ustilago maydis uses both common core effectors and organ-specific effectors to colonize its host [75 , 76] . We previously used a similar approach to that in this work to profile F . graminearum expression in a stage-specific manner during infection of coleoptiles of wheat seedlings at 16 , 40 and 64 hai [38] . Global comparison of dynamic transcriptomes provides a chance to comprehensively illustrate the overlapping but distinct infection strategies of F . graminearum during the infection of the coleoptiles of wheat seedlings and the pith tissues of mature maize stems . Comparisons suggest that , in wheat coleoptiles and maize stalks , F . graminearum may use similar cell surface molecules such as eight-cysteine-containing fungal extracellular membrane ( CFEM ) proteins , putative fungal cell surface proteins containing a [SG]-P-C-[KR]-P motif , glycosylphosphatidylinositol-anchored proteins and G protein-coupled receptors ( GPCRs ) , as well as secreted peptidases ( Figure R , S in S1 Text , Dataset N in S2 Text ) . This is consistent with the result that F . graminearum strains with CFEM1 deleted exhibited reduced virulence in both wheat coleoptiles [39] and maize stalk infections , while a complemented stain resumed full virulence ( Figure W in S1 Text ) . However , the use of secondary metabolite biosynthesis clusters , detoxification genes and membrane lipid remodeling enzyme genes was different between infection of maize stalk and wheat coleoptile ( Figure T , U , V in S1 Text ) , with essentially more diverse and higher expression in maize stalk infection than in wheat coleoptile infection . This indicates that F . graminearum might produce more secondary metabolites ( and produce them earlier ) during maize stalk infection than during wheat coleoptile infection . Furthermore , we also examined the virulence of our bta1 mutants during wheat coleoptile infection and found no significant difference in lesion size between the mutant and wild-type fungus ( Figure X in S1 Text ) , consistent with the result that BTA1 expression was lower during wheat coleoptile infection than during maize stalk infection ( Dataset J in S2 Text ) . Figure L in S1 Text shows that , as in maize stalk apoplast , the phosphorus content in wheat coleoptiles apoplast is low . It is possible that F . graminearum in wheat progresses more quickly to intracellular growth than in maize or that it utilizes other strategies to conserved phosphorus that do not involve BTA1 .
The F . graminearum strain AmCyanPH-1 [41] was previously generated by our lab from the sequenced strain PH-1 ( NRRL 31084 , [13] ) , and is isogenic to PH-1 in all respects except for constitutively expressing fluorescent protein AmCyan ( Figure D in S1 Text ) . AmCyanPH-1 conidia were produced in liquid mung bean broth ( 40 g mung beans per 1 L H2O ) [39 , 77] and resuspended in sterile water for inoculating maize plants within 2 h . Maize ( Zea mays ssp . mays L . ) cultivar B73 plants [78] , which are susceptible to F . graminearum causing Gibberella stalk rot [79] , were cultivated in a phytotron at 22–26°C with 65% relative humidity and a 14 h photoperiod for 8 weeks until the tenth leaf appeared . Maize plants were inoculated by punching a hole in the stem at the second or third internode above the soil line using a sterile micropipet tip ( 10 mm hole depth ) , followed by injection of 20 μL macroconidia suspension at a concentration of 106/mL . Mock-inoculated maize stalks treated with water served as the control . The plants were kept growing with the wounds being covered by sterile gauze to maintain moisture and avoid contamination from other organisms . At a given time point spanning 12 to 360 hai as indicated , the stalks of three plants were cut down , and the inoculated internodes were split longitudinally for symptom measurements . Each time point was repeated at least five times ( independent experiments ) . The longitudinal length of brown infected areas was measured as the lesion size at the indicated time ( illustrated in Figure D in S1 Text ) using ImageJ software . The average distance from the wounding line to upside/downside front of the brown area was measured as half lesion size ( illustrated in Fig 2A ) . Half of the split internodes were further sliced longitudinally and/or transversely for microscopy ( as illustrated in Figure B in S1 Text ) ; the other half was saved for laser microdissection ( see below ) . An Olympus BX51 microscope equipped with a green fluorescent protein filter set ( 450- to 480-nm excitation; 515-nm emission ) and a differential interference contrast ( DIC ) module was used to take wide-field images , and the hyphal advance distances were measured based on images of longitudinal slices ( as illustrated in Fig 2A ) . Confocal images were acquired on an Olympus Fv10i with two emission-collecting windows operating simultaneously . Excitation/emission wavelengths were 405 nm/460 to 500 nm for AmCyan and 405 nm/570 to 670 nm for plant cell wall autofluorescence . For visualizing the plant secondary cell wall component lignin , longitudinal or transverse slices of mock-inoculated internodes were immersed in a phloroglucinol–HCl solution [80] for 5 min and pictured under a bright field . The intensity of phloroglucinol staining reflects the lignin content . For visualization of host cell nuclei , longitudinal slices of the infected internode were stained with 4’ , 6-diamino-2-phenylindole ( DAPI ) solution ( 1 μg/mL with 0 . 2% [v/v] Tween 20 ) for 5 min before observation under a UV channel . For testing host cell viability and/or plasma membrane intactness , cross sections of infected internodes were treated with 1 M NaCl for 10 min . Microscopic images of the same area were taken before and after treatment for plasmolysis assessment . Pith tissues of the other half split internodes of mock-inoculated and F . graminearum-inoculated maize stalks at 12 , 18 , 36 , 48 , 72 , 108 , 132 , and 144 hours after inoculation ( hai ) were fixed in acetone and embedded in paraffin as described [39] using a vacuum infiltration time of up to 30 min . Then , 12 μm-thick sections were obtained using a rotary microtome ( Leica RM2235 , Leica Biosystems Nussloch ) . A Veritas Microdissection Instrument ( Acturus Bioscience ) at the SIPPE core facility was used for LCM . The target fungal mycelia fluorescence-tagged with AmCyan were captured using the following settings: spot size 15–20 μm , power 95–100 mW , and pulse duration 3–4 ms . Targeted fungal hyphae were those invasively growing in pith tissues , not in rind or vascular bundle . In obtaining 72–144 hai samples , aerial hyphae in disintegrated tissues close to the wound site were also avoided . Approximately 500 , 000 μm2 of tissue was obtained for each sample . Two or three independent biological samples were obtained for each time point . Approximately 10 ng total RNA in 11 μL volume was extracted from each laser captured sample using a Picopure RNA Isolation Kit ( Arcturus Bioscience ) , and 1μL of the total RNA was used for quality assessment with an Agilent RNA 6000 Pico Kit and a Bioanalyzer 2100 instrument ( Agilent Biotechnologies ) . Only those total RNAs with a 28S:18S ribosomal RNA peak height ratio >1 were kept for RNA amplification . Approximately 10 μg of biotinylated complementary RNA ( cRNA ) was generated from each total RNA sample using an Affymetrix Two-Cycle Target Labeling Assay Kit ( Affymetrix ) combined with a MEGAscript High Yield Transcription Kit ( Ambion AM1333 ) , as previously described [39] . Labeled cRNAs were hybridized to the Affymetrix F . graminearum genome GeneChip with ~14 , 000 F . graminearum genes presented [44] . Chip hybridization , washes , and chip reading were carried out with cRNA at ShanghaiBio Corporation according to the Affymetrix Expression Analysis Technical Manual . Three independent biological replicates were obtained for the time points 12 , 36 , 72 and 132 hai . Two independent biological replicates were obtained for mock inoculated sample and the time points 18 , 48 , 108 and 144 hai . Three biological replicates for spore suspension ( 0 hai ) and three biological replicates for in vitro grown hyphae ( 72 h ) were also obtained at the time of this work , as previously reported [39] . Original CEL files were normalized by Robust Multichip Analysis ( RMA ) as described in [39] . Correlation coefficients between biological replicates ( see Dataset A in S2 Text ) for LCM-derived samples were approximately 0 . 95 ( ranging from 0 . 93 to 0 . 99 ) based on RMA-normalized data . A presence/absence test was conducted with the detection quality P value < 0 . 004 . A gene was considered to be present if the P value was < 0 . 004 , or absent if the P value was > 0 . 065 , in at least two biological replicates . To identify differentially expressed genes compared to in vitro grown hyphae , we used the web-enabled and cross-platform Significance Analysis of Microarrays ( SAM ) software package via Shiny [47] ( http://statweb . stanford . edu/~tibs/SAM/ ) . A false discovery rate of 0 . 05 was used as the cutoff value for statistical significance , and a two-fold change in expression was used as the cutoff for fold changes . A total of 13 , 429 probe sets represent 13 , 429 unigenes from all 18 , 069 probe sets [39] . Eighty-three probe sets that were called present in mock-inoculated maize stalk samples were excluded to avoid cross-hybridization from maize stalk cell RNA in the laser-captured samples . Thus , 13 , 346 unigenes were chosen for subsequent enrichment and function analysis . Expression patterns in maize infection were identified based on the ratio of expression at eight time points during maize infection ( 12 , 18 , 36 , 48 , 72 , 108 , 132 , and 144 hai ) and in the spore ( 0 hai ) versus in vitro grown hyphae ( 72 h ) . Results are shown in Dataset C in S2 Text . To compare in vitro germination data with in planta expression data , CEL files of F . graminearum gene expression profiles obtained during conidia germination stages [81] were downloaded from Plexdb ( http://www . plexdb . org/ ) , and RMA normalization was performed on 45 combined microarray data sets at 13 time points ( in vitro at 0 , 2 , 8 , 24 , and 72 h and in maize at 12 , 18 , 36 , 48 , 72 , 108 , 132 , and 144 hai ) . FunCat [48] annotations for F . graminearum genes were downloaded from FGDB ( [49] ) . Out of 13 , 346 unigenes , 4965 had FunCat annotations . Using the χ2 test and P < 0 . 05 as the threshold for significance level , enriched FunCat families in maize stalk infection were delineated . Genes involved in cell wall degradation ( Dataset B in S2 Text ) were manually annotated with reference to the Carbohydrate-Active Enzymes ( CAZy; www . cazy . org ) annotation for F . graminearum genes by Ma et al . ( 2010 ) [58] . SMB cluster genes ( Dataset I in S2 Text ) were obtained from [39 , 55 , 58] . Gene expression heatmap matrices were generated using the “heatmap” function of the amap package in the bioconductor software of R language software version 2 . 15 . 3 according to correlation coefficients calculated by a Pearson correlation algorithm . Principal component analysis was performed using library “scatterplot3d” in the “pca” package . To compare transcription during maize stalk infection to wheat and barley head blight infection , gene lists from Supplemental Tables 1–5 in [72] were integrated with various gene lists generated in our work ( see Datasets L , M , and G-J in S2 Text for results ) . The total RNA extracted from laser-captured samples was amplified to generate antisense RNAs using a TargetAmp two-round Aminoallyl-aRNA Amplification Kit ( Epicentre Biotechnologies ) and processed with an RNeasy MinElute Cleanup Kit ( Qiagen ) . The cDNAs were synthesized using reverse transcriptase M-MLV ( TaKaRa Biotechnology ) . Quantitative PCR experiments were conducted on a Bio-Rad MyiQ single-color realtime PCR detection system . The reaction mixture contained 5 μL of template ( ~20 ng ) , 10 μL of 2× SYBR green premix , 0 . 4 μL of forward primer ( 10 μM ) , 0 . 4 μL of reverse primer ( 10 μM ) , and 4 . 2 μL of nucleotide-free water . The reference gene was β-tubulin ( FGSG_09530 ) . Primer sequences are listed in Dataset O in S2 Text . Reaction efficiency was calculated from standard curves using a serial template dilution for each pair of primers . For specificity , only primers that generated a single peak in the melting curve were used ( Figure Y in S1 Text ) . Each experiment included at least two biological replicates for each pair of primers and was repeated twice . BTA1 and other knockout mutants were generated using the split marker recombination method [82] . For complementation assays , a fragment containing the gene and promoter region was cloned into a vector containing a neomycin resistance cassette . The resulting constructs were transformed into protoplasts of the mutants . Mutants were verified as shown in Figure M in S1 Text . For subcellular localization analysis , BTA1 ( FGSG_00742 ) cDNA was in-frame fused with mRFP in a hygromycin resistance containing vector , driven by the F . graminearum constitutive expression promoter EF1-A ( FGSG_08811 , translation elongation factor 1 alpha ) . An ER marker protein [65] fused with YFP , was inserted after the promoter EF1-A in a vector which confers neomycin resistance . To generate F . graminearum strain constitutively expressing BTA1 , BTA1 cDNA was inserted into a hygromycin resistance containing vector under the control of the EF1-A promoter . Dataset O in S2 Text lists all the primer sequences . For heterologous expression , Fg-Bta1 cDNA harboring a C-terminal GST tag was cloned into vector pGEX-4T-3 ( GE Healthcare ) using a CloneExpress II One Step Cloning Kit ( Vazyme ) . The resultant vector was transformed into E . coli BL21 cells; cells harboring plasmids with or without BTA1 were grown in 2 mL overnight cultures , and then were inoculated into 200 mL of Luria-Bertani medium containing appropriate antibiotics . After growth to an optical density of about 0 . 6 at 600 nm , 0 . 2 mM isopropyl-D-thiogalactoside ( IPTG ) was added . Cultures were incubated at 18°C for 20 h and centrifuged to harvest for membrane lipid extraction . To determine the polar lipid composition , F . graminearum hyphae grown on phosphate limited media for 14 days and E . coli harboring the BTA1 expression vector grown after IPTG induction for 20 h , respectively , were harvested ( ~40 mg ) . Lipids were extracted according to [83] , and then spotted on thin layer chromatography plates ( TLC Silica gel 60 F254; Merck ) , resolved with chloroform-acetone-methanol-acetic acid-water in the ratio 10/4/2/2/1 , and visualized with iodine vapor . The spots corresponding to a DGTS standard sample were scraped from the TLC plates , eluted with chloroform , and then concentrated under a stream of nitrogen . The lipid extracts were dissolved in CHCl3/methanol/50 mM sodium acetate in water in the ratio 300/665/35 . 1 , 2-dipalmitoyl-sn-glycero-3-O-4'- ( N , N , N-trimethyl ) -homoserine ( Avanti 857464 ) was used as external standard DGTS . Samples were further analyzed using the Agilent G6520A accurate-mass Q-TOF LC-MS system with an Eclipse Plus C18 column ( 4 . 6 × 250 mm , 5 μm ) . The injected volume was 10 μL with a flow rate of 0 . 3 mL/min . A was water + 20 mM NH4OAc . B was methanol , and the gradient profile was from 0 min 65% to 15 min 85% , 25 min 100% , 28 min 100% , 40 min 65% . The mass range was 400 to 1500; nebulizer pressure 40 psig; drying gas N2 350°C , 9 L/min; ESI Vcap 3500 Vl fragmentor 214 V; skimmer 65 V; and Oct RF Vpp 750 V . DGTS was analyzed as [M+CH3COO]− ions , had a UV peak at ~25 min retention time , and its corresponding MS peak appeared in the extracted ion chromatograph with a mass-to-charge ratio 770 . 610 , ( DGTS formula: C42H81NO7 , calculated accurate-mass for M+CH3COO−: 770 . 6152; measured mass: 770 . 610; error: 6 . 75 ppm ) . DGTS was further confirmed by matching measured versus predicted isotopic distributions of masses 770 . 61 , 771 . 61 and 772 . 61 ( Fig 8 ) . For virulence assays in maize stalks , fully grown maize plants were inoculated as described above in the session of maize stalk infection and microscopic observation , with indicated strains . At 7 dpi , the stalks were split longitudinally and the symptoms were documented by photography . The extent of the lesion area was quantified using ImageJ software version 1 . 46 ( http://rsbweb . nih . gov/ij/index . html ) . Statistical significance was determined by a Student’s t-test ( P < 0 . 05 ) as implemented in GraphPad Prism 5 software . At least five internodes were inoculated at a time; inoculations were repeated five times for each fungal strain . KH2PO4 was added to distilled water to final concentrations of 0 . 1 M , 0 . 01 M and 0 . 001 M , respectively , at pH 6 . 8 . At 8 h after inoculation by bta1 mutant M1 , 10 μL KH2PO4 solution were added into the wound site of maize stalk using a sterile micropipet tip . Stalk internodes of 8-week-old maize B73 plants were fractioned into xylem sap , apoplastic fluid and stalk tissues ( Figure K in S1 Text ) . The xylem sap collection method was modified from previous reports [84 , 85] . After the root system was removed , stem internode sections about 6 cm in length ( roughly 6 g ) were used . A 5-mL syringe filled with sterile distilled water was jointed to the lower end of the stem and the juncture sealed tightly with parafilm . Hydraulic pressure was applied slowly using the water-filled syringe and sap was collected in a tube . After removing the xylem sap , the 6 cm-long internodes were cut equally into two parts . One-half was used for apoplastic fluid collection using the method described in [86] . The half maize stalk was cut into flakes and put in a beaker with 100 mL water . The beaker was placed in a desiccator jar and vacuumed for about 30 min . After removing excess water , the flakes were put into a filtering centrifuge tube and centrifuge at 3000 × g for 10 min at 4°C . After centrifugation , about 1 mL of apoplastic fluid was extruded from the stalk . The other half internode was ground into powder in liquid nitrogen to obtain the total fluid of maize stalk as a control . The same vacuum-centrifugation method as above was used to collect apoplastic fluid from wheat coleoptiles . Phosphorus content was analyzed by inductively coupled plasma mass spectrometry using a PerkinElmer ELAN DRC-e ICP-MS at the Shanghai Institute of Plant Physiology and Ecology Core Facility for Mass Spectrometry , according to [87] and [88] . Tissue extracts were digested overnight in concentrated nitric acid at room temperature and diluted with deionized water to a final volume of 7 mL . Aqueous standards at 125 , 250 , 500 , and 1000 ppb phosphorus , purchased from Shanghai Institute of Measurement and Testing Technology ( Shanghai , China ) were used to generate a calibration curve . Phosphorus content was determined as a mono-isotopic element at mass 30 . 9938 atomic mass unit . DON concentration was measured by ELISA using the RIDASCREEN FAST DON Kit ( R-Biopharm AG , Germany ) [89]; its determination limit is 0 . 2 ppm . A calibration curve was established using 0 , 0 . 222 , 0 . 666 , 2 and 6 ppm DON . For sample preparation , 1 g of maize tissue was ground and extracted in 20 mL of distilled water . After shaking vigorously for 3 min , the solution was filtered through a Whatman No . 1 filter and the filtrate was used for further analysis following the kit manufacturer’s protocol . Finally , after addition of stop solution , the absorbance at 450 nm was measured on an Eon Microplate Spectrophotometers ( BioTek , USA ) . Experiments were repeated twice . XM_380918 . 1 for FGSG_00742 ( BTA1 ) , XM_382253 . 1 for FGSG_02077 ( CFEM1 ) , XM_011327926 . 1 for FGSG_06610 ( PhoD ) , XM_011324063 . 1 for FGSG_03366 ( PHO12 ) , XM_011329171 . 1 for FGSG_07678 ( PHO 610 ) , XM_011324021 . 1 for FGSG_03402 ( PHO11 ) , XM_391412 . 1 for FGSG_11236 ( PLC ) , XM_011323534 . 1 for FGSG_03846 ( TAGL ) , XM_011324206 . 1 for FGSG_03243 ( TAGL ) , XM_011329299 . 1 for FGSG_07783 , and XP_389706 . 1 for FGSG_09530 ( tubulin ) . The microarray data have been deposited in NCBI's Gene Expression Omnibus [90] and are accessible through GEO Series accession number GSE53854 .
|
Gibberella stalk rot of maize is an economically important crop disease caused by the filamentous fungus Fusarium graminearum ( previously also called Gibberella zeae ) . Stalk rots develop in the main stem of mature maize plants . Detailed anatomical studies of the complete infection process in maize stalk have not previously been reported at the cellular level . Using a fluorescently-tagged strain of F . graminearum , we observed that the fungus initially grows between live maize cells ( intercellular growth ) and only later penetrates host cells , also killing nearby plant cells . Using laser microdissection , we selectively retrieved fungal biomass from infected maize stalks and obtained high-resolution gene expression profiles for the fungal-enriched samples at 0 , 12 , 18 , 36 , 48 , 72 , 108 , 132 and 144 hours after inoculation ( hai ) . Many of the gene expression patterns are consistent with microscopically observed fungal growth at the corresponding stage of infection . For example , enzymes with the potential to break pectin primary chains are most highly expressed at 12–36 hai when F . graminearum is observed to grow , for the most part , intercellularly , for which breakdown of pectin , a major intercellular component , is required . We propose a previously unreported strategy for the successful colonization of maize stalk by F . graminearum that involves the enzyme betaine lipid synthase 1: the pathogen produces phosphorus-free membrane lipids which allows it to maintain an aggressive intercellular advance in the phosphorus depleted environment of the maize apoplast before it kills nearby plant cells and releases their cellular nutrients .
|
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"Abstract",
"Introduction",
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"Discussion",
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2016
|
Cellular Tracking and Gene Profiling of Fusarium graminearum during Maize Stalk Rot Disease Development Elucidates Its Strategies in Confronting Phosphorus Limitation in the Host Apoplast
|
Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria . Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics , by allowing them to evolve and adapt to these hostile conditions . Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA . We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase . The SOS response controls a series of genes responsible for DNA damage repair , which can lead to recombination and mutagenesis . In this manuscript , we show that conjugative transfer of ssDNA induces the bacterial SOS stress response , unless an anti-SOS factor is present to alleviate this response . We also show that integron integrases are up-regulated during this process , resulting in increased cassette rearrangements . Moreover , the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer , even abortive , can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell . Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria .
Free-living bacteria commonly face changing environments and must cope with varying conditions . These adaptive strategies involve temporary physiological responses through various groups of genes gathered in regulons that are induced or repressed according to the surrounding conditions . This is the case for the quorum sensing regulon [1] , [2] , the stringent response and catabolite repression systems , which allow adjustment of gene expression according to the growth conditions [3]–[5] . In other instances , the only adaptive solution requires a genetic change , and bacteria have developed mechanisms that favour genome modifications either by transiently increasing their mutation rates , inducing re-arrangements , or lateral ( horizontal ) gene transfer ( HGT ) . One of the better known responses of this kind is the trigger of the SOS regulon , which controls DNA repair and recombination genes [6] . SOS is a bacterial stress response induced when an abnormal rate of single stranded DNA ( ssDNA ) is present in the cell . ssDNA is the substrate for RecA polymerization . The formation of a ssDNA/RecA nucleofilament stimulates auto-proteolysis of the LexA repressor , leading to de-repression of genes composing the SOS regulon . The SOS response is triggered by the accumulation of ssDNA , for example when cells try to replicate damaged DNA , after UV irradiation or treatment with antibiotics ( fluoroquinolones , β-lactams ) or mitomycin C ( MMC ) , a DNA cross-linking agent . In addition to these endogenous sources , ssDNA is also produced by several mechanisms of exogenous DNA uptake involved in lateral gene transfer , namely by conjugation , transformation and occasionally transduction . Conjugation is indeed one mechanism of lateral transfer that leads to the transient occurrence of ssDNA in the recipient cell [7] , [8] . The presence of anti-SOS factors in some conjugative plasmids , such as the psiB gene of R64drd and R100-1 [9] , suggests that conjugative DNA transfer can induce SOS . In plasmid R100-1 , psiB ( plasmidic SOS inhibition ) was shown to be transiently expressed during the first 20 to 40 minutes of conjugation [10] , [11] from a ssDNA promoter [12] , and inhibited the bacterial SOS response [13] , [14] . Plasmids carrying psiB do not all express it at levels sufficient to alleviate SOS , as seems to be the case in F plasmids for instance [9] , [10] , [13] , [15] , [16] . Conjugation is a widespread mechanism in the intestinal tract of host animals where there is a high concentration of bacterial populations [17]–[22] ) . Lateral gene transfer plays a large role in the evolution of genomes and emergence of new functions , such as antibiotic resistance , virulence and metabolic activities in bacterial species [23] . Bacteria can also possess other internal adaptive genetic resources . Vibrio cholerae carries a superintegron ( SI ) , that can be used as a reservoir of silent genes that can be mobilized when needed . Integrons are natural gene expression systems allowing the integration of an ORF by site-specific recombination , transforming it into a functional gene [24] . Multi-resistant integrons ( RI ) have been isolated on mobile elements responsible for the assembly and rapid propagation of multiple antibiotic resistances in Gram-negative bacteria through association with conjugative plasmids [25] , [26] . An integron is characterized by an intI gene , coding a site specific recombinase from the tyrosine recombinase family , and an adjacent primary recombination site attI [27] . The IntI integrase allows the integration of a circular promoterless gene cassette carrying a recombination site , attC , by driving recombination between attI and attC [28] . The integrated gene cassettes are expressed from the Pc promoter located upstream of the attI site in the integron platform [29] . The discovery of integrons in the chromosome of environmental strains of bacteria , and among these the superintegrons ( SI ) mentioned above , has led to the extension of their role from the “simple” acquisition of resistance genes to a wider role in the adaptation of bacteria to different environments [30] . The dynamics of cassette recombination and the regulation of integrase expression are poorly understood . Recently , it was shown that intI is regulated by the bacterial SOS response [31] . Since SOS is now known to induce both the RI and V . cholerae integrase expression , an important issue is to understand when and where cassette recombination takes place and how the integrase inducing SOS response is activated . Our objective was to determine if conjugative ssDNA transfer can trigger the SOS response , and to which extent this affects intI expression and cassette recombination . SOS induction in promiscuous environments can prepare bacteria to face the many threats they can encounter there . In order to understand regulatory networks existing between conjugation and its effect on integron content and cassette expression , we first addressed if conjugation induces SOS using reporter fusions in V . cholerae and E . coli . After quantifying expression from the V . cholerae intIA promoter using GFP fusions , we adopted a genetic approach to test integrase-dependent site-specific recombination in vivo . We show that conjugative plasmid transfer generally induces the SOS response and up-regulates integrase expression , triggering cassette recombination . However , this is not the case when an anti-SOS factor ( psiB ) is expressed , as seen for some narrow host range conjugative plasmids isolated from Enterobacteria . We further show that this anti-SOS function prevents up-regulation of the SOS regulon in a host-specific manner after conjugation . We demonstrate that conjugative transfer is sufficient to trigger integron cassette recombination in recipient cells . This study outlines the connections between conjugative lateral DNA transfer , bacterial stress response and recombination of gene cassettes in integrons , and provides new insights into the development of the antibiotic resistance within a population .
During conjugation , plasmid DNA enters the host cell in a single stranded fashion [7] , [8] . In order to test whether conjugation induces the SOS response in the recipient cell , we used reporter E . coli and V . cholerae strains carrying sfiA::lacZ ( 7651 ) and recN::lacZ ( 7453 ) β-galactosidase fusions , respectively . sfiA ( cell division in E . coli ) and recN ( recombinational repair ) genes belong to the SOS regulon of E . coli . We also identified a LexA binding box upstream of recN in V . cholerae . We confirmed that induction of SOS in these strains results in expression of the β-galactosidase ( β-gal ) enzyme ( not shown ) . Table 1 summarizes the conjugative plasmids belonging to several incompatibility groups we used in this study [32] . The donor ( DH5α ) strain was recA- and ΔlacZ . The conjugation rates of these plasmids were first measured at various time points after donor and recipient cells were mixed ( Figure 1A ) . In E . coli , all plasmids conjugate approximately at the same rate so that nearly all recipients have received a plasmid after 60 min of conjugation . In V . cholerae transfer rates vary considerably , only 1 in 105 cells have received a plasmid after 4h of mating with R6Kdrd and R388 , while RP4 has a transfer rate similar to that of E . coli ( 10−1 to 1 ) . Neither R64drd nor R100-1 replicate in V . cholerae . In order to address whether R100-1 actually transfers from E . coli into V . cholerae , we used pSU19-oriTF plasmids containing the oriTF ( 72 bp ) of plasmid F . Plasmid F does not replicate in V . cholerae and oriTF is 98% identical to oriTR100 . The high oriTF transfer rate observed at 1h of mating confirms that plasmids F and R100-1 ( and presumably R64drd ) can indeed transfer into V . cholerae and that the lack of R100/R64 transconjugants is due to their inability to establish themselves in this bacterium . SOS induction linked to conjugation was measured in the total recipient population by counting the actual number of recipient cells plated on selective medium instead of using OD units ( Materials and Methods ) , to obtained an induction value per potential recipient cell . Mating was interrupted at various time points ( t0 , t40 , t60 , t120 , t180 , t240 ) and β-gal activity was measured in both E . coli and V . cholerae recipients ( Figure 1B ) . The results are represented on the graph as the induction ratios at times t0 , t60 and t240 over the induction at t0 . When the recipient strain was mixed with empty donor , no SOS induction was observed . A peak of SOS induction in E . coli was detected after 40 min to 60 min of mating with a conjugation proficient donor , 1 . 7 fold induction for RP4 and R6Kdrd and 2 . 3 fold for R388 . The induction peak was also observed in V . cholerae ( 2 . 3 fold for R6Kdrd , 2 . 7 fold for RP4 and 3 . 4 fold for R388 ) . To verify that the β-gal activity was due to the SOS induction , we deleted the recA gene in the recipient E . coli strain . No induction of β-gal activity was observed in the ΔrecA strain after conjugation with RP4 , R6Kdrd and R388 . This confirms that the β-gal induction observed in recA+ strain indeed reflects the SOS induction by RP4 , R6Kdrd and R388 . As described above , β-gal induction peaks between t40 and t60 minute of mating . The induction then decreases to reach the level shown at t240 , forming bell shaped curves ( data not shown ) . This induction pattern reflects the SOS induction in an asynchronous population of bacteria . It can be explained by the fact that plasmids RP4 , R6Kdrd and R388 replicate in recipient cell . Once mating has started and as time goes by , there tends to be less plasmidless recipient cells . Indeed , entry of the plasmid DNA induces SOS , the incoming plasmidic ssDNA then replicates in the conjugant cell and the entry exclusion systems prevents entry of another plasmid [33] , [34] . However , cells continue to divide so that the population of kanamycin resistant ( kanR ) host cells increases . Accordingly , even when the transfer rate remains constant ( especially for low rated plasmids ) , the increase in the number of kanR cells can explain the drop of activity per recipient in the curve . The SOS response is expected to return to normal once all the cells have acquired the plasmid . Since all cells in the recipient population have not received a conjugating DNA at the time of the β-gal assay , we calculated the SOS induction per conjugant , i . e . per recipient cell that has actually undergone DNA uptake ( Materials and Methods ) . The results are represented as ratios over t0 in Figure 1C . As expected , the induction signal is amplified when one takes into account the conjugation rate for each plasmid . This amplification of several orders of magnitude is likely to be an effect of unsuccessful conjugation: SOS is induced by incoming DNA , that is not always converted into a replicating plasmid . The induction profiles , however , are compatible with Figure 1B: R388 and R6Kdrd strongly induce SOS , RP4 also shows a high induction , however it is lower than the former two plasmids . Once again , no ( or very little ) induction was observed in E . coli ΔrecA strain , confirming that conjugation induces RecA-dependent SOS response . SOS induction by RP4 is weaker in both E . coli and V . cholerae , compared to induction during conjugation with R388 or R6Kdrd . We did not find any particular feature in the DNA sequence , or gene order of RP4 that could explain this observation . However , one can imagine that the higher transfer rate during RP4 conjugation is coupled with an early expression of entry exclusion systems , resulting in a quick decrease of ssDNA levels and repression of the SOS response . To check if lower SOS induction was specific of RP4 , we decided to test 2 other plasmids ( Rs-a and RIP113 ) belonging to different incompatibility groups , at t60 - the peak for SOS induction observed for the plasmids mentioned earlier . Rs-a ( IncW ) induced SOS in E . coli and V . cholerae ( Figure 1C and data not shown ) , confirming that SOS induction can be triggered by this plasmid . Interestingly , Rs-a conjugates at a rate of 10−1 and yields an intermediate induction level ( like RP4 ) . Even though we have a small sample of plasmids , SOS induction during mating seems to inversely correlate with conjugation rate ( at 1h of mating ) or replication of plasmids , except for R6Kdrd . Further study is needed to verify this observation . On the other hand , RIP113 ( IncN ) induced SOS in V . cholerae only ( Figure S2 ) . An increasing number of non-replicative conjugative elements , generally named ICE , have been described in bacteria . One of the best studied is the SXT element discovered in V . cholerae [35]–[37] . We addressed if conjugative transfer of an SXT element integrated in the chromosome of E . coli [38] to V . cholerae also induces the SOS response . We observed a similar induction profile as for the conjugative plasmids with a peak of induction measured at t210 ( Figure 1B ) . The delay can likely be explained by the very low transfer rate ( 10−6 after 6 six hours of mating ) . Our results show that plasmids lacking the psiB gene ( here RP4 , R388 , R6Kdrd and Rs-a ) induce SOS upon conjugation into the recipient cell . On the other hand , RIP113 ( IncN ) induced SOS in V . cholerae only ( Figure S2 ) , thus behaving like R64drd and R100-1 plasmids . R64drd and R100-1 plasmids do not induce ( or very poorly ) the SOS response in E . coli ( Figure 1B and 1C ) . This was expected as these plasmids carry a psiB anti-SOS gene . Plasmid RIP113 behaves like R64drd and R100-1 in terms of SOS induction in E . coli . We thus suspected RIP113 to carry a psiB gene as R64drd and R100 plasmids . This was confirmed by PCR amplification with psiB-specific primers ( data not shown ) . This finding is supported by another IncN plasmid which has been sequenced: the R64 plasmid carries a gene named stbA ( locus R46_027 ) , presenting 42% DNA sequence identity with psiBF . We observed a strong induction of SOS by the same 3 plasmids in V . cholerae ( Figure 1C ) , suggesting that the psiB gene is either not expressed in V . cholerae or that its product is not active in this species ( R64drd and R100-1 do not replicate in V . cholerae , thus no activity per conjugant could be calculated ) . Moreover , SOS induction is continuously high for R64drd and R100-1 plasmids after ∼60 min , whereas SOS induction declined after 60 min for RP4 , R6Kdrd and R388 , as mentioned above . We were unable to delete psiB from R64drd and thus could not check if in its absence SOS induction would be restored in E . coli . The reason for the unsuccessful cloning attempts could be the presence of several genes ( such as ssb coding the single strand binding protein , anti-restriction gene ardA , or flm/hok ) in the same region where ORFs and regulatory regions overlap [9] , [11] , [39]–[41] , such that deletion of psiB could have unpredicted consequences on plasmid transfer and replication . Instead , psiB from R64drd was cloned and over-expressed from a pBAD plasmid , under the control of the arabinose inducible promoter . SOS induction after mitomycin C ( MMC ) treatment was measured in E . coli sfiA::lacZ and V . cholerae recN::lacZ containing either empty pBAD or pBAD-PsiB+ plasmids . As previously published [31] , MMC treatment induced SOS in E . coli and V . cholerae ( Figure 2 ) . SOS induction was strongly reduced in E . coli when PsiB was expressed from pBAD ( 6 fold induction instead of 11 . 6 fold , Figure 2A ) whereas SOS induction was insensitive to PsiB expression in V . cholerae ( ∼60 fold induction with and without PsiB over-expression , Figure 2B ) . These results show that the psiBR64drd ( and presumably the psiBR100-1 which presents 85% identity to psiBR64 ) is expressed during conjugation in E . coli and inhibits the SOS response , whereas in V . cholerae , psiBR64drd/R100-1 has no or very little anti-SOS activity , allowing R64drd and R100-1 transfer to induce SOS . The fact that R64 and R100-1 are narrow host range enterobacterial plasmids [40] , [42] and do not replicate in V . cholerae , can explain the continuous induction we observe . Entering plasmid DNA is not replicated and new rounds of conjugation can carry on , resulting in continuous re-induction of SOS . The psiB anti-SOS function is found in several narrow host range plasmids belonging to the IncFI , IncFII , IncI1 , IncK and IncN incompatibility groups [9] ( and results obtained by blasting PsiB on GenBank plasmid sequences ) . These plasmids replicate in bacteria from the genera Enterobacter , Escherichia , Salmonella and Klebsiella . Bagdasarian and colleagues have suggested that PsiB could interact with RecA to inhibit its ability to induce SOS [16] . It was recently shown that PsiB binds to RecA in solution [43] . PsiB would then inhibit SOS by preventing RecA nucleofilament formation on ssDNA . Since PsiB from R64 , R100-1 and RIP113 plasmids does not inhibit the SOS response in V . cholerae when over-expressed , we hypothesized that PsiB would be deficient in interacting with RecAVch . Our β-gal tests show that when expressed in V . cholerae together with RecAEco , PsiB reduces the SOS response from 60 fold to 24 fold induction ( Figure 2B ) . Consistently , when co-expressed with RecAVch in an E . coli ΔrecA strain , PsiB does not alleviate SOS ( Figure 2A , note that RecAVch is active in E . coli ) . Finally , expression of RecAEco in the E . coli ΔrecA strain complements SOS induction alleviation by PsiB . Altogether , these data suggest that PsiB is functional only in bacterial species where its carrier plasmids normally reside ( here E . coli ) , thus antagonising RecA in a species-specific manner . On the other hand , we showed that the RIP113 plasmid isolated from Salmonella , an enterobacterium , also carries the psiB gene and behaves like R64drd and R100-1 in inhibiting SOS in E . coli but not in V . cholerae . Unlike these two plasmids , RIP113 replicates in V . cholerae but since it was isolated in Salmonella , and to our knowledge IncN plasmids have not been described in V . cholerae so far , we considered that V . cholerae is not one of its usual hosts . To our knowledge PsiB is present only in narrow host range plasmids . We conclude that PsiB functions in a species-specific manner . It was recently shown that the integron integrase is regulated by the SOS response [31] . We showed above that conjugational DNA transfer induces SOS . We then addressed whether conjugation affects V . cholerae IntIA SI integrase expression levels . To do this , we constructed a V . cholerae reporter strain containing a translational fusion between intIA and gfp ( 7093::p4640 ) , and used flow cytometry to determine the fraction of cells where the integrase-GFP fusion was induced . As expected , no induction was observed in the ΔrecA control strain ( Figure 3 ) . In the recA+ strain we observe no induction after conjugation with RP4 and R6Kdrd ( Figure 3 ) . Alternatively , the integrase expression increased 2 . 8 fold when the strain is conjugated with R388 , and 5 . 3 and 6 . 2 fold with R64drd and R100-1 , respectively . In β-gal SOS induction tests shown earlier , RP4 and R6Kdrd also yielded a lower induction in total population graphs ( Figure 1B ) . Note that β-gal induction reflects the recN promoter , which is more strongly expressed than the intI promoter . Our results imply that the SOS induction during RP4/R6Kdrd conjugation may not reach sufficiently high levels to induce the integrase reporter used in flow cytometry experiments . Finally we tested mating of E . coli carrying an SXT element with V . cholerae . SXT transfer is induced through induction of SOS when the donor is treated with MMC [37] . Transfer of the SXT element into V . cholerae increased intIA promoter activity 12 fold compared to a plasmidless control and was 2 fold higher than uninduced cells ( i . e . without MMC treatment of donor ) . We have shown that conjugation induces SOS in the recipient bacteria and flow cytometry analysis clearly shows that the integron integrase is induced during conjugation in V . cholerae . In a first set of experiments , we wanted to test if the SOS induction leads to a higher activity of the integrase promoter in E . coli , using the class 1 integrase IntI1 . We developed an experimental strategy in an E . coli strain that contains an insertion in the dapA gene ( 7949 ) . This strain is unable to synthesize DAP ( 2 , 6-diaminopimelic acid ) , and as a result is not viable without DAP supplemented in the medium . The insertion in dapA is flanked by two specific recombination sites , attI and attC . Integrase expression causes site-specific recombination and excision of the synthetic cassette , restoring a functional dapA gene and allowing the strain to grow on DAP-free medium ( Figure 4A ) . We transformed in this dapA- strain a multi-copy plasmid ( p7755 ) carrying the intI1 gene under the control of its natural SOS regulated promoter . The recombination rate due to integrase expression is calculated as the ratio of the number of cells growing in the absence of DAP over the total number of cells . Figure 4B shows the cassette excision rate in E . coli 7949 p7755 after conjugation with different conjugative plasmids . In the absence of a conjugative plasmid in the donor cell , the spontaneous excision rate is about 10−5 , which reflects the stringency of the intI promoter . Conjugation with R6Kdrd and R388 increases excision rate to 10−3 and 10−2 respectively , whereas conjugation with R64drd does not increase significantly beyond the basal recombination level . RP4 yields an intermediate level of DAP+ cells , which is compatible with its intermediate SOS induction level in E . coli . These results are consistent with SOS induction results in E . coli , and as expected , there is a correlation between SOS induction and integrase induced cassette recombination . To confirm that cassette recombination is due to integrase expression , we performed the same experiment in strain 7949 lacking the integrase carrying plasmid p7755 , and no cassette excision was observed ( <10−8 ) . We conclude that conjugation with psiB deficient plasmids in E . coli induces the expression of the integrase from the intI1 promoter , and thus triggers cassette recombination . In the cassette excision experiment described above , we used a multicopy plasmid expressing the intI1 integrase in E . coli . Since conjugation induces the SOS response and in turn expression of the integron integrase in V . cholerae , we addressed in a second set of experiments whether conjugation in wild type V . cholerae can trigger recombination events in the superintegron . The V . cholerae SI carries a promoterless catB cassette that is not expressed in V . cholerae laboratory strain N16961 because it is located 7 cassettes ( approximately 5000 bp ) downstream of the Pc promoter [44] . When expressed , the catB gene confers resistance to chloramphenicol ( Cm ) . We tested if conjugation can spontaneously yield Cm-resistant ( Cm-R ) V . cholerae cells , i . e . if IntIA is induced and recombines the catB cassette to a location allowing its expression ( Figure 5A ) . Our results show that when the donor strain does not carry any conjugative plasmid , the rate of CmR cells is about 7 . 10−11 ( Figure 5B ) . Consistent with the intIA induction results , conjugation with RP4 and R6Kdrd did not increase this frequency ( 6 . 10−11 ) . Conjugation with R388 , R64drd and R100-1 increased the CmR cfu appearance rate 28 fold , 280 fold and 140 fold , respectively . To verify that this increase was dependent on SOS , we deleted recA in the recipient strain and found that the conjugative plasmids yielded a rate of CmR lower than 10−11 for all plasmids ( no colony observed ) . To determine if these events corresponded to IntIA mediated cassette rearrangement , we performed a PCR analysis with primers in the Pc promoter and at the beginning of the catB cassette . In the wild type strain , this PCR amplifies a band of about 5000 bp . In the CmR colonies , the PCR amplified a band of 1432 bp ( Figure S2A ) . Sequencing confirmed that the catB cassette had been relocated closer to the Pc promoter . catB was now present in second position , after the first cassette , compatible with an excision and integration in the first attC site downstream of attI . We know that IntIA can promote recombination between two attC sites [45] . The first cassette coding for a hypothetical protein downstream of attI may be important for viability under laboratory conditions . Alternatively , there may be a strong promoter in this first cassette , allowing a better expression of the catB cassette that could be insufficiently expressed from any other location under our selective conditions ( involving high Cm concentrations ) . In order to determine if cassettes between the Pc promoter and catB gene were deleted after rearrangement – i . e . if catB moved because cassettes were deleted or because it was re-integrated – we performed PCR analysis with several oligonucleotides amplifying cassettes located between attI and catB in V . cholerae N16961 . We found that these cassettes were still present in the genome of the Cm-resistant clones ( data not shown ) , showing that they were not deleted , and indicating that catB was relocated by recombination events . To further address if other cassette rearrangements had occurred in those cells after conjugation , we isolated the genomic DNA ( gDNA ) from three CmR colonies obtained after conjugation . We digested gDNA with AccI , which has 35 restriction sites evenly distributed within the SI . We then hybridized with a mix of 10 probes complementary to 17 cassettes in the SI . The southern blot ( Figure S2B ) clearly shows that 2 of the 3 colonies have different hybridization profiles compared to the original N16961 gDNA , confirming that several cassettes ( other than catB ) have moved within the SI after conjugation induced SOS . We conclude that conjugation with strong SOS inducing plasmids , R388 , R64drd and R100-1 , increases IntIA expression levels and promotes cassette rearrangements at a 100 fold to 1000 fold higher rate than under non stressful conditions . Although conjugation of R6Kdrd strongly induced the SOS response , it did not have any effect on intIA expression and cassette recombination in our experimental setup . Even though it is possible that R6Kdrd encodes an uncharacterized function able to specifically prevent the IntI expression during the SOS induction , we think this observation is most likely due to an insufficient sensitivity of our setup .
It has been shown that during inter-species Hfr conjugation , SOS is induced in the host cell [46] , [47] . It was proposed that the low level of homology prevents rapid recombination of incoming DNA into the chromosome and thus dramatically enhances the SOS induction . This suggests that SOS induction levels may reflect the ability of RecA to find homologous DNA and initiate strand exchange [48] . In the case of plasmid conjugation , there is no homology with the bacterial chromosome , explaining the very high SOS induction levels we observe . Moreover , SOS induction proceeds as a wave . At the early stages of SOS induction , the concentration of LexA decreases as it is self-cleaved , then LexA synthesis is induced at later stages , and if ssDNA does not persist , the SOS induction level gradually decreases . This is what happens when RP4 , R388 and R6Kdrd conjugate into E . coli cells ( and RP4 in V . cholerae ) , and explains the bell-shaped induction curves we obtained . After 1h , nearly all the recipient cells are conjugants and no new conjugation is initiated because of plasmidic entry exclusion systems [49] . As conjugation or establishment rates are very low for R388 and R6Kdrd in V . cholerae , plasmidless host cells are always present in the total population so we observe a plateau reflecting new rounds of conjugation during the course of the experiment . The fact that R64drd and R100-1 are the strongest inducers in V . cholerae could thus be due to the inability of these plasmids to synthesize the complementary DNA strand and establish themselves in V . cholerae , increasing the prevalence of ssDNA accessible to RecA binding . Moreover , this strongly suggests that abortive conjugation induces SOS , which explains the fact that we are able to detect SOS induction in the total population despite the lack of transconjugants in V . cholerae . This is consistent with data showing very high induction values when we calculate the SOS induction in conjugants only . The plateau of induction in the whole population points to a “permanent conjugation state” where ssDNA enters the host cell , induces SOS , but does not replicated , and a new round of conjugation begins . Another interesting observation is that SOS induction does not seem to prevent conjugation . Indeed , the conjugation rate for all plasmids ( in E . coli for instance ) is approximately the same after 2h of mating even though they induce SOS differently at the beginning of mating . To test this point , we used recipient cells already induced for SOS by pre-incubation with MMC , and these cells yielded the same conjugation rates in E . coli for a given plasmid regardless of the SOS induction level ( Figure S1 ) . SOS induction is due to RecA binding to ssDNA . We have shown above that plasmids R64drd , R100-1 and RIP113 that carry the anti-SOS psiB gene do not induce SOS when the recipient cell is E . coli . PsiB has been shown to interact with RecAEco in vitro [43] and in vivo ( Figure 2 ) , preventing it from binding to ssDNA and inducing the SOS response . Even though RecAVch and RecAEco show 79% protein identity , our data suggests that PsiB is impaired in its interaction with RecAVch in vivo ( Figure 2 ) , explaining why PsiB does not strongly reduce the SOS induction in V . cholerae as it does in E . coli . The presence of the psiB anti-SOS function in narrow host range plasmids such as R64 and R100 suggests that the dissemination strategies of narrow and broad host range plasmids could be distinct . Induction of the SOS response can be potentially detrimental to the host cell because of the induction of mutagenic polymerases or cell division arrest ( like E . coli sfiA ) [50] . Thus , it is tempting to speculate that narrow host range plasmids use their anti-SOS gene as a furtive strategy to hide from their customary host and thus prevent the host cell from being stressed and change its own or the incoming plasmid DNA . Note that by narrow host range plasmids , we mean plasmids that only replicate in a restricted number of bacteria ( such as R64 ) but also plasmids that are found only in a few kinds of hosts in nature , even though they are able to replicate in others , such as the RIP113 originally in Salmonella . One consequence of SOS induction during conjugative DNA transfer is the triggering of integron cassette recombination . Conjugation with strong SOS inducer plasmids R388 and R6Kdrd in E . coli increases expression of IntI1 from its SOS regulated natural promoter leading to an increased RecA-dependent cassette excision rate , whereas plasmids R64drd and R100-1 that do not induce SOS in E . coli do not trigger cassette recombination in our E . coli cassette recombination assay . These results highlight the existence of a link between conjugation and site-specific recombination , leading to genome evolution . We also showed that conjugation triggers cassette recombination in the natural context of the SI carried in wild type V . cholerae . Plasmids R388 , R64drd and R100-1 strongly induce SOS ( and intIA ) in V . cholerae and significantly increases the cassette recombination rate . Our results highlight the link between conjugative HGT and genome evolvability in V . cholerae . Since conjugation induces integrase activity , one can consider conjugative plasmids as both vehicles for cassette dissemination and cassette shuffling for those already present in the SI . Indeed , some plasmids such as R388 [51] and R64 [52] carry an RI platform that can acquire new cassettes and transmit them to a new host by conjugation . It was shown that R388 can incorporate the catB cassette from the V . cholerae SI and transfer it to other bacteria [44] . Here we observed the displacement of a cat cassette catalyzed by the V . cholerae IntIA in its natural context . Conjugation can thus bring new cassettes but also favour their integration into the host chromosomal integron by inducing SOS . Cassette recombination upon conjugation could be a widespread mechanism , since conjugation is a naturally occurring phenomenon in highly concentrated bacterial environments , such as the host intestinal tract ( see for example [17]–[19] , [21] ) , biofilms [53]–[55] forming in the aquatic environment where V . cholerae grows , or even on medical equipment in hospitals [56] . Moreover , no mutation has been found in the bacterial chromosome that can prevent the uptake of conjugative DNA , meaning that bacteria cannot avoid being used as recipient cells [57] . By inducing the SOS response , incoming DNA triggers its own recombination not only through integrase induction but also homologous recombination , promoting genomic rearrangements . Another important effect of SOS induction is the derepression of genes implicated in the transfer of integrating conjugative elements ( ICEs ) , such as SXT from V . cholerae , which is a ∼100 kb ICE that transfers and integrates into the recipient bacterial genome , conferring resistance to several antibiotics [37] . Moreover , different ICEs are able to combine and create their own diversity in a RecA-dependent manner via homologous recombination [35] , [36] , and also , as observed here for SXT transfer in V . cholerae , by inducing SOS following transfer . Thus , SOS induction leads to genetic diversification of these mobile elements and to their transfer to surrounding bacteria , spreading antibiotic resistance genes , among others . Conjugation induced SOS is thus one of the mechanisms allowing bacteria to evolve in their natural niches , creating the diversity that allows them to adapt to new environments and survive . Under conditions where SOS is prevented ( by bacterial means such as the PsiB system or exogenously ) , cassette recombination is decreased to experimentally undetectable levels , showing that SOS induction plays an important role in adaptation , and can be used by broad host range plasmids to adapt to a new host . Consistently , narrow host range plasmids that do not need to adapt to a new host , express an SOS inhibitor to maintain the integrity of the plasmid DNA and host genome . This connection between host range and SOS induction needs to be expanded to a larger range of plasmids to determine its general character . A significant association between laterally transferred genes and gene rearrangements was already suggested in [58] , which is consistent with our data , when we consider that SOS induction plays a major role in gene rearrangements . Remarkably , induction of SOS considerably enhances genome duplications and mutagenesis [59] . Further work is needed to test whether other HGT mechanisms , such as transformation , also induces SOS; considering that many bacterial species , like V . cholerae for instance , are naturally competent [60] . It would be interesting to investigate if SOS is induced in the gut of the host animal . If this were the case , inhibiting the bacterial SOS response would become an ideal target to prevent the acquisition of antibiotic resistance genes , and could be used in combination with antibiotics for the treatment of infections .
Overnight cultures of donor and recipient cells were diluted 100× in LB and grown until OD∼0 . 5 . Donor and recipient cells were then mixed in 1∶1 ratio on 0 . 045µm conjugation filters on LB plates preheated at 37°C . At each time point , a filter was resuspended in 5ml LB and dilutions were plated on selective plates to count ( i ) conjugants and ( ii ) total number of recipients . For details , see Text S1 . β-gal tests were performed on these cultures as described ( [61] and Text S1 ) . According to the Miller formula:and we calculated:andwhere is the basal expression per cell when SOS is not induced . For details , see Text S1 . SOS induction tests using MMC were performed as published [31] . The same conjugation assay was performed overnight for the flow cytometry experiments . For each experiment , 100000 events were counted on the FACS-Calibur device . For details , see Text S1 and Figure S3 . Described in the Results section . For details , see Text S1 . The recipient strain was V . cholerae N16961 ( Cm sensitive 5µg/ml Cm ) . The donor strain was DH5α or a dap- derivative ( Π1 ) for counter selection of Cm-R plasmids . Conjugations were performed as described for 4h . Filters were resuspended , centrifuged and the pellet was plated on LB medium containing 25µg/ml Cm . PCR screenings were performed using oligonucleotides cat2/i4 and the GoTaq polymerase . Oligonucleotides 896 to 905 were used to verify the presence of other cassettes .
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Bacteria exchange DNA in their natural environments . The process called conjugation consists of DNA transfer by cell contact from one bacterium to another . Conjugative circular plasmids have been identified as shuttles and reservoirs for adaptive genes . It is now established that such lateral gene transfer plays an essential role , especially for the antibiotic resistance development and dissemination among bacteria . Moreover , integrons , platforms of mobile gene cassettes , have been instrumental in this phenomenon , through their successful association with conjugative resistance plasmids . We demonstrate in this study that the conjugative transfer of plasmids triggers a bacterial stress response—the SOS response—in recipient cells and can impact the cassette content of integrons . The SOS response is already known to induce various genome modifications . Human and animal pathogens cohabit with environmental bacteria , in niches which will favor DNA exchange . SOS induction during conjugation is thus most probably able to impact a wide range of genomes . Bacterial SOS response could then be a suitable target for co-treatment of infections in order to prevent exchange of antibiotic resistance/adaptation genes .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"cell",
"biology/microbial",
"physiology",
"and",
"metabolism",
"genetics",
"and",
"genomics/microbial",
"evolution",
"and",
"genomics",
"genetics",
"and",
"genomics/gene",
"expression",
"microbiology/microbial",
"evolution",
"and",
"genomics",
"genetics",
"and",
"genomics/chromosome",
"biology",
"cell",
"biology/microbial",
"growth",
"and",
"development",
"biochemistry/replication",
"and",
"repair",
"biochemistry/transcription",
"and",
"translation",
"microbiology/microbial",
"physiology",
"and",
"metabolism",
"microbiology/cellular",
"microbiology",
"and",
"pathogenesis",
"cell",
"biology/gene",
"expression"
] |
2010
|
Conjugative DNA Transfer Induces the Bacterial SOS Response and Promotes Antibiotic Resistance Development through Integron Activation
|
Antibodies developed for research and clinical applications may exhibit suboptimal stability , expressibility , or affinity . Existing optimization strategies focus on surface mutations , whereas natural affinity maturation also introduces mutations in the antibody core , simultaneously improving stability and affinity . To systematically map the mutational tolerance of an antibody variable fragment ( Fv ) , we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface , within the antibody core . Rosetta design combined enhancing mutations , yielding a variant with tenfold higher affinity and substantially improved stability . To make this approach broadly accessible , we developed AbLIFT , an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains ( http://AbLIFT . weizmann . ac . il ) . We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1 . Strikingly , the designs improved stability , affinity , and expression yields . The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design .
High-affinity natural antibodies are generated through an iterative process of mutation and selection for antigen binding known as affinity maturation . Affinity maturation also selects antibodies that exhibit higher stability and expressibility [1] , both of which are essential parameters in the development of antibodies into research or medical tools [2] . In recent decades , synthetic antibody repertoires have been widely adopted in antibody discovery and optimization , providing greater control over the selection process than animal immunization . In this approach , a library of antibody variable fragments ( Fv ) is displayed , for instance on yeast cells , and screened to select high-affinity binders or to improve the affinity of existing antibodies [3] . These methods are powerful [4 , 5] , but a large fraction of high-affinity antibodies isolated from synthetic repertoires exhibits impaired stability [6] . Impaired stability can limit expression yields and increase aggregation propensity [7] , resulting in high production costs [8] , fast antibody clearance from circulation and adverse immune responses in patients [9] . Thus , the tradeoff between antibody stability ( including solubility and expressibility ) and affinity can delay and even block the development of antibodies in research and medicine [10] . General methods to improve antibody stability while maintaining or even increasing affinity are therefore urgently needed to reduce the attrition rate in antibody development pipelines [11] . To boost antibody stability and affinity , computational design methods have been developed . These have focused on the Fv complementarity-determining regions ( CDRs ) , which are typically in direct contact with the antigen . Some methods , for instance , improved electrostatic complementarity with the antigen [12] or eliminated hydrophobic surface patches [13–18] . Natural and laboratory affinity maturation , by contrast , introduce mutations in both the CDRs and the antibody core [1 , 5] . Core mutations may improve antibody stability by eliminating packing defects , and they may enhance affinity by preorganizing the antigen-binding site [1] . Although mutations in the core may contribute less to affinity than ones in the CDRs , they are more likely to retain the intricate structure of the antigen-binding site and are therefore likely to be compatible with affinity-enhancing mutations in the CDRs that were obtained through other optimization strategies . The antibody core , however , is a large and densely packed region , complicating the design of improved variants [5 , 19] . For instance , we recently presented and validated an automated computational strategy , called PROSS [20] , for protein-stability design . Similar to other stability design algorithms [21] , however , PROSS only rarely introduces core mutations and does not improve binding affinity [22] . Reliable prediction of mutational effects in the antibody core and especially successful design of networks of interacting multipoint core mutations has , therefore , remained a challenge [21 , 23] . Recently , deep mutational scanning has been successfully applied to study the mutational tolerance of antibodies and other binders [24–30] . In this approach , amino acid positions in the binder are systematically mutated to all of the natural amino acid identities; the mutants are pooled into one library containing all single-point mutations; populations of binders are selected from this library using in vitro display and high-throughput screening; and the selected and unselected populations are subjected to deep-sequencing analysis to infer which mutations are enriched relative to the starting binder , thus systematically identifying affinity-enhancing mutations . Deep mutational scanning has been very successfully used to guide protein design and engineering of improved binders [24 , 26 , 31 , 32] but has not yet been exploited to improve protein-design methodology itself . The large improvements in the reliability and breadth of detection of affinity-enhancing mutations through deep mutational scanning inspired us to revisit the challenge of accurately predicting the effects of core mutations on antibody affinity and stability . Deep mutational scanning guided us in uncovering a cluster of core positions at the light-heavy chain ( vL-vH ) interface where many affinity-enhancing mutations occurred . We then used these systematic data to establish general rules for computational design of antibody Fvs with improved vL-vH interactions; we implemented these rules as an automated method , called AbLIFT and made it available through a web server ( http://AbLIFT . weizmann . ac . il ) . AbLIFT designs exhibited striking gains in affinity , stability , and expressibility in two unrelated antibodies that target the human disease markers Vascular-Epidermal Growth Factor ( VEGF ) and the enzyme Quiescin Sulfhydryl Oxidase 1 ( QSOX1 ) .
To study the mutational tolerance of an antibody Fv , we selected 135 positions in the anti-lysozyme antibody D44 . 1 [33] for deep mutational scanning ( S1A Fig ) . The positions encompassed most of the CDRs , the vL-vH interface and additional peripheral positions ( Fig 1A ) . D44 . 1 and each point mutant were genetically encoded as single-chain variable fragments ( scFv ) in which the heavy chain was fused to the light chain via a flexible linker , and the genes were transformed into yeast cells for binding and expression screens by yeast display [3] . Following incubation with hen egg-white lysozyme , the top 15% binders were selected from this library , and the same library was also subjected to low-stringency selection for expression levels to provide a baseline . The plasmids containing scFv-encoding genes from both selections were purified , amplified by PCR , and subjected to deep sequencing , resulting in 8 million high-quality reads [32] . We then determined the enrichment of each mutant relative to D44 . 1 as the ratio between populations selected for binding and expression ( Fig 1B ) . We found affinity-enhancing mutations at 34 positions , mostly within the CDRs , as expected ( Fig 1C ) . We also noticed a surprisingly large cluster of eight positions at the vL-vH interface where affinity-enhancing mutations occurred , although they were not in direct contact with the antigen ( Fig 1D and 1E ) . This cluster of affinity-enhancing mutations in the vL-vH interface is intriguing for four reasons: ( 1 ) the vL-vH interface mediates the assembly of the Fv from the two antibody chains , and mutations in this region have the potential to also enhance stability through improved Fv assembly [1]; ( 2 ) the genetic pairing of light and heavy chains during germline antibody generation is a random process which may result in suboptimal vL-vH interfaces , flexibility in the antigen-binding site [34] , and therefore in lower antigen affinity [35]; ( 3 ) this region is distant from the mutational hotspots in the CDRs and may not be fully optimized in the course of natural affinity maturation [36]; and ( 4 ) antibody-engineering procedures , such as humanization or CDR grafting may inadvertently compromise the structural integrity of this region by mispairing CDRs and frameworks [37] . Based on these considerations , we hypothesized that the vL-vH interface may be especially amenable to the design of multipoint mutants that simultaneously improve stability and affinity in both natural and engineered antibodies . Combining mutations in densely packed protein cores , such as the vL-vH interface , is challenging , however , because inadvertently introduced voids , steric overlaps , or mispaired polar amino acid side-chains can lead to protein instability , misfolding , and aggregation [21 , 23] . We , therefore , asked whether the mutational-tolerance map could guide Rosetta design in finding improved multipoint mutants at the vL-vH interface . In preliminary calculations starting from the lysozyme-bound D44 . 1 structure ( PDB entry: 1MLC ) , we restricted Rosetta combinatorial design to the eight positions and 38 identities ( including the wild type identities ) at these positions that showed at least threefold enrichment relative to D44 . 1 according to the mutational-tolerance map ( Fig 1B and 1E ) . The resulting design , however , comprised only three conservative mutations , suggesting that the dense packing and backbone rigidity at the vL-vH interface restricted sequence optimization . We , therefore , repeated design calculations but this time excluded the wild type identities at the eight positions , forcing the design of an optimal combination of mutations only from those that were substantially enriched in deep mutational scanning . We iterated sequence design and backbone and sidechain minimization to promote the acceptance of even radical mutations yielding design D44 . 1des with eight mutations . As a preliminary qualitative test , we analyzed D44 . 1des binding to lysozyme and to seven of the eight single-point mutations formatted as scFvs using yeast display [3] . As expected , each of the point mutations improved the apparent binding affinity relative to D44 . 1; and yet , the multipoint D44 . 1des exhibited a substantial improvement in apparent affinity compared to the single-point mutations ( S1B Fig ) . To determine what molecular factors might underlie higher affinity in D44 . 1des , we expressed the design as an antigen-binding fragment ( Fab ) and determined its structure by X-ray crystallography in the absence of lysozyme ( S1 Table ) . Despite eight core mutations , the overall agreement between D44 . 1des and the bound structure of D44 . 1 , which served as the starting point for designing D44 . 1des , was excellent ( S2 Fig ) : The two structures deviated by <1 Å backbone root-mean-square deviation ( rmsd ) and in side-chain residues comprising the lysozyme-binding site . The mutations apparently improved various molecular aspects of the vL-vH interface including interface packing , solvation , and backbone rigidity ( Fig 2A ) . Next , we tested lysozyme binding by D44 . 1 and D44 . 1des ( both expressed and purified as Fab ) using surface-plasmon resonance ( SPR ) . D44 . 1des exhibited nearly tenfold improvement in affinity ( KD of 15 versus 135 nM for D44 . 1des and D44 . 1 , respectively ) , with a 25-fold slower off-rate ( 8 * 10−4 s-1 ) ( Fig 2B ) . D44 . 1des also exhibited improved thermal denaturation and aggregation resistance ( Fig 2C and 2D ) . We also compared the molecular structure of D44 . 1des to the unbound structure of D44 . 1 ( PDB entry: 1MLB ) . The main difference between the two structures was localized to the backbone conformation of CDR H2: Whereas H2 in the unbound structure of D44 . 1 adopts a conformation that would sterically overlap with lysozyme in the bound structure , the H2 backbone of D44 . 1des moves away from this position such that , even in the unbound state , the design is sterically compatible with lysozyme binding . The H2 backbone conformation of D44 . 1des is not identical but is similar to the H2 conformation in the bound D44 . 1 structure and also to the conformation observed in the unbound structure of the high-affinity anti-lysozyme antibody F10 . 6 . 6 ( PDB entry: 1P2C ) ( Fig 2E ) . Although it is possible that the observed conformational differences among the structures are due to differences in crystallographic conditions , we note that the mutation Trp47HTyr in D44 . 1des is incompatible with the observed H2 conformation in the unbound state of D44 . 1 and may induce the observed change in the design’s backbone conformation . Hence , the structure-based analysis suggested that the design of the vL-vH interface based on the bound antibody structure might increase the compatibility of the CDR backbones for the ligand while simultaneously improving stability . The successful optimization of antibody affinity and stability encouraged us to fully automate the design procedure , eliminating the requirement for experimental deep mutational scanning . We , therefore , sought a general computational strategy that would predict which mutations in the vL-vH interface were likely to enhance affinity and stability , with the goal of developing a general computational procedure for mutational-tolerance mapping . To achieve this goal , we exploited the large experimental dataset of the D44 . 1 mutational tolerance map , comprising 2 , 294 point mutations , for training . At each of the mutated D44 . 1 Fv positions , we used Rosetta to compute the changes to native-state energy due to each of the 19 amino acid mutations ( ΔΔG ) . Using a multiple-sequence alignment of homologous Fvs , we additionally computed each point mutation’s evolutionary-conservation score , as represented in a Position-Specific Scoring Matrix ( PSSM ) [38] . These two computed parameters provide complementary predictions of mutational tolerance: the former predicts the impact of a mutation on native-state stability and the latter discriminates between evolutionarily tolerated mutations and those that have been purged by evolution . The use of these two parameters has recently led to substantial improvement in design accuracy in binder and enzyme design challenges in our laboratory [20–22 , 38–43] . We specifically used these two parameters because they can be computed for any antibody given an accurate experimental or model structure , allowing us , in principle , to compute mutational tolerance maps for any antibody Fv . We systematically screened different combinations of ΔΔG and PSSM thresholds to determine which combination optimally discriminates enhancing from deleterious mutations as observed in the experimental mutational-tolerance map of D44 . 1 . We defined the prediction true-positive rate ( TPR ) as the proportion of correctly predicted affinity-enhancing mutations ( >1 . 5-fold enrichment according to deep mutational scanning ) and the true-negative rate ( TNR ) as the proportion of correctly predicted deleterious ones ( enrichment ratio <1 ) . The resulting phase space of ( PSSM , ΔΔG ) thresholds revealed an expected tradeoff , wherein high TNR came at the cost of low TPR , and vice versa ( Fig 3A ) . The likelihood of obtaining a multipoint mutant without a single deleterious mutation can be roughly approximated by TNRn , where n is the number of mutations . Given the large size of the vL-vH interface ( 20–30 positions [44] ) , we aimed for a large maximum number of mutations in each multipoint mutant ( n = 10 ) and therefore selected a stringent cutoff TNR = 94% , providing a rough estimate that 50% of designs with ten mutations would not contain a single deleterious mutation ( Fig 3B ) . At this high TNR , the TPR is only 40% , reflecting the challenging tradeoff in the design of multipoint variants . We anticipate that in certain applications , such as in the design of improved antibodies for therapeutic application , a smaller number of mutations may be preferred . In such cases , a lower TNR and therefore a higher TPR may be implemented , and Fig 3C provides a guide for choosing different ( PSSM , ΔΔG ) thresholds . Instructions for computing a mutation-tolerance map based on any structure of an antibody Fv are available as Supplemental Data , and the AbLIFT web server enables user control of these parameters . We next sought to develop a general and fully automated design protocol for improving molecular interactions across the vL-vH interface . AbLIFT starts by computing a mutational-tolerance map at the vL-vH interface using the approach described above; it then exhaustively enumerates the multipoint combinations of tolerated mutations; ranks them by energy , and selects low-energy variants for experimental testing . This algorithm resembles our recently described FuncLib method for designing functionally diverse enzyme repertoires [41] , with the key differences that AbLIFT is applied to the core of obligatory binding surfaces rather than to solvent-exposed surfaces and most importantly , AbLIFT does not require an initial design round of protein stabilization . To validate AbLIFT , we chose two antibodies as subjects for design: the synthetic antibody G6 , which targets human Vascular-Endothelial Growth Factor ( VEGF ) [45] , and an engineered variant of the 492 . 1 antibody , designated h492 . 1 , which targets human Quiescin Sulfhydryl Oxidase 1 ( QSOX1 ) . QSOX1 is a multi-domain disulfide-catalyst that is overproduced in tumors [46] and is a potential drug target [47 , 48] . These antibodies are unrelated to D44 . 1 or to one another and are the products of protein engineering . G6 is widely used in animal studies and resulted from a phage-displayed synthetic Fab library of the light chain with a heavy chain sequence of an anti-mVEGF antibody ( KD approximately 1 nM ) [49] . The h492 . 1 antibody was obtained by fusing the variable domains from the high-affinity ( KD approximately 1 nM ) QSOX1-inhibiting murine antibody 492 . 1 onto a human IgG scaffold . Following this fusion , h492 . 1 could not be expressed to detectable levels in a recombinant cultured human cell system , frustrating its further development . Thus , with these two targets , we sought to test the ability of AbLIFT to optimize high-affinity antibodies that resulted from conventional antibody-engineering procedures , whether well-behaved ones ( G6 ) or ones that showed low ( or no ) detectable expression levels ( h492 . 1 ) . The computed mutational-tolerance map of G6 ( starting from its bound structure , PDB entry 2FJG ) at 30 vL-vH interface positions defined 26 affinity-enhancing mutations at 11 positions . To achieve significant improvement of vL-vH interface packing , we sought to design multipoint mutants with 4–10 mutations relative to G6 , resulting in a tolerated sequence space of 203 , 835 unique multipoint mutants . All multipoint mutants were modeled in Rosetta , including by a backbone and side-chain minimization step , which is essential for enabling cavity-filling small-to-large mutations [50 , 51] , and the models were then ranked by energy . 53% of the mutants ( >100 , 000 ) exhibited energies as favorable as or better than the G6-bound antibody . Therefore , although the exhaustive enumeration of this large number of mutants is computationally demanding ( approximately 6 , 000-CPU hours ) , the very large number of potentially improved designs makes a compelling case for exhaustive enumeration and ranking of variants within the tolerated sequence space . Furthermore , the computed mutational-tolerance map focuses exhaustive enumeration on a subset of stable multipoint mutants within the vast hypothetical sequence space of mutants at the vL-vH interface ( 2030 = 1039 unique sequences ) , >99% of which are predicted to have reduced stability compared to the parental antibody ( S3 Fig ) . We clustered the designs , eliminating ones that had fewer than four mutations relative to one another and selected the 18 lowest-energy ones for experimental testing . The designs were formatted as scFvs , and their binding signals relative to the G6 antibody were first qualitatively measured at 8 nM VEGF concentration using yeast display [3] ( Fig 4A ) . Encouragingly , seven designs ( approximately 40% ) showed comparable or higher binding signal at this concentration . The best two designs , G6des1 and G6des13 , were expressed as Fabs . When subjected to Ni-NTA purification , G6 exhibited multiple bands , indicative of sample heterogeneity , whereas , remarkably , both designs eluted mostly in the size expected for a Fab ( S4A and S4B Fig ) [52] . Next , the designs’ affinities for VEGF were determined using SPR ( Fig 4B ) . Both designs improved binding on-rate , and G6des13 also improved the off-rate , resulting in fivefold improvement in KD relative to G6 . Both designs also exhibited substantial improvements in thermal stability and the temperature of aggregation onset ( 19° C and 10° C , respectively ) ( Fig 4C and 4D ) . We examined the model structure of G6des13 , which comprised six mutations at the vL-vH interface relative to G6 , finding that the mutations were likely to improve the interface through backbone rigidification and the introduction of a new buried polar hydrogen-bond network ( Fig 4E ) . Such cooperative interaction networks do not typically arise in conventional antibody affinity-maturation processes ( either in nature or the laboratory ) , which select mutations in a stepwise manner and are therefore biased towards additive rather than cooperative multipoint mutations . Introducing accurate new polar interaction networks is also a fundamental challenge for computational design [53 , 54] and the use of evolutionary constraints during design has recently been shown to overcome this challenge [42] . We next tested the stability and expressibility of the VEGF designs formatted as full-length IgGs . We expressed G6 , G6des1 , and G6des13 in HEK293 cells and found that the designs exhibited nearly an order of magnitude higher expression level than G6 ( Fig 4F ) . We next measured the relative stabilities of G6 and G6des13 using native mass spectrometry [55] under reducing conditions by titrating the collision energy ( Fig 4G ) . We found that G6 IgG disassembly started at lower collision energy compared to G6des13 , indicative of the design’s higher stability ( S5 Fig ) . We , therefore , concluded that AbLIFT could substantially improve expressibility , stability , and affinity , regardless of whether the antibody was formatted as Fab or IgG . We applied the same computational strategy to h492 . 1 , in which the Fv was derived from a murine antibody and the constant regions were derived from human IgG1 . Since h492 . 1 failed to show detectable expression in HEK293 cell cultures , we started the computational design from the structure of the murine 492 . 1 parental antibody in complex with QSOX1 ( PDB entry: 4IJ3 ) [47] . We selected the 20 lowest-energy , sequence-clustered AbLIFT designs , fused them to human IgG1 constant domains and subjected them to HEK293-expression screening from crude cell lysate supernatant . Dot-blot analysis showed detectable expression levels for all 20 designs , in clear contrast with the lack of detectable expression for h492 . 1 ( Fig 5A ) . We further quantitated expression levels using Western blot , revealing substantial variation in the expression levels among the designs ( Fig 5B ) . In parallel , we examined the levels of QSOX1 inhibition by the 20 designs , finding that 50% showed high levels of QSOX1 inhibition ( S6 Fig ) . Based on activities and expression levels , we selected h492 . 1des3 and h492 . 1des18 for further analysis . These designs were purified and added to QSOX1 activity assays to test for inhibition . h492 . 1des18 showed comparable inhibitory levels to the murine parent antibody when provided at equimolar amounts to a typical physiological concentration of QSOX1 ( 25 nM ) as found in human serum ( Fig 5C ) [56] . This analysis demonstrated that h492 . 1des18 almost completely recovered the activity of the parental antibody while gaining high expression levels ( approximately 75 mg/L supernatant ) . Structural analysis indicated that this design improved packing at the vL-vH interface ( Fig 5D ) , demonstrating that in some cases optimizing this region could have a dramatic effect on the expression levels of engineered antibodies . Finally , we asked whether there were any sequence features in common among the designs ( S3 and S4 Tables ) . Strikingly , position 43L ( Chothia numbering ) was mutated to Pro in D44 . 1des and in >60% of the G6 and h492 . 1 designs . Position 43L is located in a tight turn that connects two neighboring β strands , away from the CDRs , but Pro is not the consensus identity at this position ( Ala and Ser are preferred ) . Furthermore , mutations at this position may have an important effect on the rigid-body angle formed by the variable light and heavy domains [44 , 57] , and it is , therefore , unlikely that this mutation would universally improve antibody stability and affinity . Other than this mutation to Pro , we did not observe common sequence features among the designs . Overlapping but non-identical sets of positions were varied in each of the three case studies presented here , and the mutations at aligned positions were dissimilar . We , therefore , concluded that the designs improved interactions across the vL-vH interface through a variety of mechanisms that depended on the specific molecular structure of the parental antibodies .
Our study demonstrates that improved interactions across the vL-vH interface may result in substantial optimization of a range of essential parameters for antibody development , including expressibility , stability , and affinity . The automated AbLIFT strategy enables the design of cooperative networks of multipoint mutations in the antibody core that are likely to be inaccessible to experimental affinity maturation processes since these latter methods select mutations in a stepwise manner . Since AbLIFT impacts the antibody core and does not alter the structure of the antigen-binding site , the designed mutations cooperate with surface mutations identified through conventional antibody-engineering processes to further increase affinity and stability . AbLIFT may be particularly beneficial in antibodies , such as G6 and h492 . 1 , which were the product of antibody-engineering approaches that might compromise antibody structural integrity , resulting in reduced affinity or stability . Moreover , antibody structure-prediction methods now often produce atomically accurate models at the vL-vH interface ( though still not at the CDR H3 ) [58–60] , suggesting that by restricting design to the framework regions , AbLIFT may in some cases enable antibody optimisation even in the absence of an experimental structure . We note , however , that AbLIFT considers only phylogenetic information and molecular energetics and disregards immunogenicity , which may be an important consideration in antibodies developed for clinical use . To address this concern , the AbLIFT web server enables complete control over the design sequence space and can be used to eliminate mutations with immunogenic potential . The surprisingly broad ability of vL-vH design to optimize antibody properties is consistent with the Colman interface-adaptor hypothesis , according to which the formation of the Fv from two chains renders it flexible [34] . According to this hypothesis , Fv flexibility is likely to be an adaptive property selected by evolution to broaden molecular recognition by each individual antibody to a range of antigens through induced fit or conformational selection [61] , thereby solving the conundrum of how a large but finite antibody repertoire could recognize a potentially infinite range of antigens [62] . Flexibility , however , might come at a cost , since an Fv that exhibits flexible vL-vH pairing may occupy multiple molecular states that compete with the binding-competent state , thus lowering antigen-binding affinity . Flexibility may moreover result in misfolding or transient dissociation of the two variable chains , resulting in terminal aggregation or degradation by the cellular proteostasis machinery , thereby lowering expression yields . In extreme cases , poorly defined packing at the vL-vH interface can lead to substantial rearrangements of the antibody variable domain during binding [63] , and such rearrangements could lower antigen-binding affinity and specificity . Therefore , while the interface-adaptor hypothesis neatly explains why flexibility at the vL-vH interface is advantageous in early steps of antibody selection , broad specificity and marginal vL-vH interface stability become liabilities in later stages of antibody development into research or therapeutic tools . We anticipate that AbLIFT will have a wide scope to automatically and reliably improve stability , solubility , expressibility , affinity , and structural integrity in numerous antibodies in which these important properties are compromised .
Forward and reverse primers with the degenerate codon NNS were generated for all 135 positions on the Fv of D44 . 1 , essentially as described [64] . Primers were ordered from Sigma ( Sigma-Aldrich , Rehovot , Israel ) and were used to introduce all possible amino acids per position by QuickChange mutagenesis [65] . Next , the PCR product of each position was transformed into yeast ( EBY100 cells ) and plated on SD-Trp as described [66] . Briefly , plates with more than 400 colonies were scraped with 1 ml SDCAA , 50 μl was added to 5 ml SDCAA tube and cells were then grown at 30°C overnight . The point mutants were split into six libraries , corresponding to positions that were at most 130 bp apart from one another to enable deep mutational scanning using 150 bp reads . Yeast-display experiments were conducted essentially as described [3] . Briefly , yeast cells were grown in selective medium SDCAA overnight at 30°C . The cells were then resuspended in 10 ml induction medium and incubated at 20°C for 20 h . 107 cells were then used for yeast-cell surface display experiments: cells were subjected to primary antibody ( mouse monoclonal IgG1 anti-c-Myc ( 9E10 ) sc-40 , Santa Cruz Biotechnology ) for expression monitoring and biotinylated ligand at 90 nM lysozyme ( GeneTex ) in PBS-F for 30 min at room temperature . The cells then underwent a second staining with fluorescently labeled secondary antibody ( AlexaFluor488—goat-anti-mouse IgG1 ( Life Technologies ) for scFv labeling , Streptavidin-APC ( SouthernBiotech ) for ligand labeling ) for 10 min at 4°C . Next , the cell fluorescence was measured and cells were collected under sorting conditions for expression and top 15% binders . The selection gates were calibrated using the wild-type scFv D44 . 1 and these gates were subsequently applied to the library constructs . Following fluorescence-activated cell sorting ( FACS ) , cells were grown in SDCAA for 1–2 days and plasmids were extracted using Zymoprep Yeast Plasmid Miniprep II kit ( Zymo Research ) . 18 designs of improved binding affinity antibodies against VEGF and the wild-type G6 Fvs were ordered from Twist Biosciences as scFvs . These , as described above , were amplified by PCR and cloned into pCTCon2 using homologous recombination in yeast [66] . The plasmids were extracted by Zymoprep kit II , transformed into bacteria for sequence validation and verified clones were transformed to yeast for display [3] . The wild-type and designed antibodies were tested for binding by flow cytometry with 8 nM biotinylated VEGF ( Recombinant Human VEGF 165 , Biotinylated Protein R&D systems ) . To connect the DNA adaptors for deep sequencing , the plasmids extracted from the libraries were amplified using Phusion High-Fidelity DNA Polymerase ( ThermoFisher ) in a two-step PCR protocol . PCR 1: ( barcode: CTCTTTCCCTACACGACGCTCTTCCGATCT ) >forward ( seg1 ) :<barcode>AGGGTCGGCTAGCCATATG >forward ( seg2 ) :<barcode>GGATCGAATGGGTTAAACAACGT >forward ( seg3 ) :<barcode>ACACCTCCTCTAACACCGC >forward ( seg4 ) :<barcode>CTGGTGGCGGTGGCTC >forward ( seg5 ) :<barcode>GCCGTGCGTCTCAGTCTATT >forward ( seg6 ) : <barcode>CCATCTCGTTTCTCCGGC >reverse: CTGGAGTTCAGACGTGTGCTCTTCCGATCTGGATCGAATGGGTTAAACAACGT The PCR product for each population ( expressed and top 15% of binders for each of the six libraries ) was cleaned using Agencourt AMPure XP ( Beckman Coulter , Inc . ) and 1 μl from a 1:10 dilution was taken to the next PCR step for index labeling using KAPA Hifi DNA-polymerase ( Kapa Biosystems , London , England ) : >forward: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC >reverse: CAAGCAGAAGACGGCATACGAGAT<index>GTGACTGGAGTTCAGACGTGTGC Top 15%—index: CAATAGTC Expressed—index: TTGAGCCT All the primers were ordered as PAGE-purified oligos . The concentration of the PCR product was measured using Qu-bit assay ( Life Technologies , Grand Island , New York ) . DNA samples were run on an Illumina MiSeq using 150-bp paired-end kits . The FASTQ sequence files were obtained for each run , and customized scripts were used to generate the selection heat maps from the data as previously described [64] . Briefly , the script starts by translating the DNA sequence to amino acid sequence; eliminates sequences that harbor more than one amino acid mutation relative to wild type and also sequences that failed the QC test; counts each variant in each population; and eliminates variants with fewer than 100 counts in the reference population ( to reduce statistical uncertainty ) . To derive the mutational landscapes we compute the frequency Pi , j of each mutant relative to wild-type in the selected and reference pools , where i is the position and j is the substitution , relative to wild-type: Pi , j=counti , jcountwild−type ( Eq 1 ) where count is the number of reads for each mutant . The selection coefficients are then computed as the ratio: Si , j= ( Pi , j ) selected ( Pi , j ) reference ( Eq 2 ) where selected refers to the top 15% binding population and reference refers to the reference population ( Expression ) . The resulting Si , j values are then transformed to −ln enrichment values: −ln ( Si , j ) ( Eq 3 ) All Rosetta design simulations used git version fb77c732b4f08b6c30572a2ef7760ad3bb4535ca of the Rosetta biomolecular modeling software , which is freely available to academics at http://www . rosettacommons . org . Position-Specific Scoring Matrices ( PSSM ) for designed antibodies against VEGF ( PDB: 2FJG ) and against QSOX1 ( PDB: 4IJ3 ) were collected as described in ref . [38] and are distributed with the Rosetta release . RosettaScripts [67] and command lines are available in Supplemental Data . As in the AbDesign method [38] , separate PSSMs were generated for CDRs 3 and for CDRs 1 , 2 and the framework by aligning structurally similar antibodies in the PDB and selecting only sequences that did not exhibit gaps relative to the query sequence; furthermore , a strict cutoff of ≤ 0 . 5 Å backbone-carbonyl rmsd was used to eliminate structurally divergent sequences . Thus , the PSSMs were only based on structural considerations and not on sequence homology or source organism . We refined each bound PDB structure by four iterations of side-chain packing and side-chain and backbone minimization , saving the minimum-energy structure . Computational mutation scanning was applied to the refined structure using the FilterScan filter in Rosetta [24] . At every position , each allowed mutation ( that is , every amino acid identity with PSSM score ≥-1 ) was modeled singly against the background of the refined structure . Protein side chains within 8 Å of the modeled mutation were repacked , and side-chain and constrained backbone minimization were used to accommodate the mutation . The energy difference between the refined structure and the optimized configuration of the single-point mutant was calculated using the talaris2014 energy function [68] . The energy threshold used to define the tolerated mutation space was +1 R . e . u . We next enumerated all possible combinations of mutations against VEGF ( 203 , 835 models ) and against QSOX1 ( 491 , 235 models ) , modeled them in Rosetta and relaxed them by sidechain packing and sidechain , backbone and rigid-body minimization with harmonic backbone coordinate restraints . Designs were ranked based on their energy and the top 18 designs differing by 4–10 mutations relative to one another ( VEGF ) ( S2 Table ) and the top 20 designs differing by 3–14 mutations relative to one another ( QSOX1 ) ( S3 Table ) were selected for experimental characterization . The web-server implements several improvements relative to the method used to design the G6 and h492 . 1 variants [41] . In the AbLIFT web-server , the multiple-sequence alignment used to construct the PSSM is first filtered to eliminate all loops and secondary-structure elements that exhibit any gaps relative to the query sequence . Furthermore , the web-server implements more accurate atomistic scoring and enables greater user control: it uses the recent Rosetta energy function ref15 [69] with improved electrostatics and solvation potentials relative to the previous Rosetta energy function talaris and allows the user to manually modify the tolerated sequence space ( for instance , based on prior experimental data or to eliminate potential immunogenic sequence signatures ) . Accordingly , ΔΔG and PSSM cutoffs may be different from those used to in the designs described in the paper , and the web server provides user control over these parameters . The design and wild-type were transformed into RH2 . 2 plasmid for expression as Fabs , where the heavy chain was N-terminally His-tagged and the light chain was expressed as a separate protein . Both chains contain a secretion sequence for direction to the periplasmic space , where they fold and dimerize . Restriction-free cloning was done using Kapa HiFi Hotstart Readymix ( Kapa Biosystems ) according to the manufacturer’s protocol . Cells were induced with 1 mM IPTG at OD600 = 0 . 6 , transferred to 20°C , and harvested after 20 h . The cells were then resuspended in buffer A [20 mM phosphate buffer pH 6 . 2 , 150 mM NaCl] and sonicated . The supernatant was harvested by centrifugation ( 20 , 000 × g , 1 h ) , filtered , and loaded on HiTrap TALON crude 1 ml column ( GE Healthcare ) . Then it was washed with 15–20 bed volumes of buffer A , and then eluted with buffer B [20 mM phosphate buffer pH 6 . 2 , 150 mM NaCl , 150 mM imidazole] . Imidazole was removed from the eluate by dialysis against Buffer C [20 mM Hepes buffer pH 7 , 150 mM NaCl] ( 1:400 ) . The sample was then concentrated ( Amicon Ultra-15 Centrifugal Filter; Merck ) and purified by gel filtration in buffer C over a HiLoad 16/600 Superdex 200 pg column . Antibodies were expressed in suspension-HEK293F cells , grown in FreeStyle medium ( Gibco ) , in a shaking incubator ( 115 rpm ) , at 37°C , in a controlled environment of 8% CO2 . The variable regions of the different heavy and light chains were cloned separately , upstream of IgG1 human Ab scaffolds , into p3BNC plasmids . Transfections were done using linear 40 kDa polyethyleneimine ( PEI ) ( Polysciences ) at 3 mg of PEI per 1 mg of plasmid DNA per 1 L of culture , at a cell density of 1 million cells/ml . Growth media were collected after 5–7 days and separated from cells by centrifugation at 600 x g . Media were then supplemented with 0 . 02% ( wt/vol ) sodium azide and 0 . 1 mM PMSF and further clarified by centrifugation at 16 , 840 x g for 30 min . Adherent HEK293T cells were cotransfected with genes encoding the light and heavy chain Fabs ( heavy chain fused to C-terminal His tag ) in p3BNC plasmids using linear PEI as a transfection reagent ( 12 . 5 μg/12 . 5 μg/50 μg , respectively , per 15-cm plate ) . Seventy-two hours post-transfection , the medium containing the secreted protein was collected ( ~250 ml ) . The filtered medium was concentrated to ~200 ml using a diafiltration device ( QuixStand Benchtop System; GE Healthcare ) . The medium composition was exchanged to buffer A [50 mM Tris pH 8 and 150 mM NaCl] using the same device . This was loaded on a HisTrap HP 5 ml column ( GE Healthcare ) . Washed with 15 bed volumes of 20 mM Tris pH 8 , 150 mM NaCl and 10mM imidazole and was eluted with 20 mM Tris pH 8 , 150 mM NaCl and 250 mM imidazole . Imidazole was removed from the eluate by dialysis against Buffer A ( 1:400 ) . The sample was then concentrated ( Amicon Ultra-15 Centrifugal Filter; Merck ) and purified by gel filtration in buffer A over a HiLoad 16/600 Superdex 200 pg column . The apparent melting temperature of the antibodies was determined by Prometheus NT . Plex instrument ( NanoTemper Technologies ) , a label-free method . Fabs obtained from secreted Fab production in adherent cells ( D44 . 1 , G6 , G6des1 , G6des13 ) and from production in suspension ( D44 . 1des ) were diluted to 0 . 2 mg/ml ( in 20 mM Hepes pH 7 and 50mM NaCl for anti-lysozyme antibodies and in 20 mM Hepes pH 7 . 5 , 150 mM NaCl for anti VEGF antibodies ) . The temperature was ramped from 25°C to 100°C at 0 . 05°C/s , and both Tm and aggregation-onset temperature were measured . Surface plasmon resonance experiments on the anti-lysozyme ( D44 . 1 and D44 . 1des expressed in bacteria ) and anti-VEGF antibodies ( G6 , G6des1 and G6des13 expressed in adherent cells ) were carried out on a Biacore T200 instrument ( GE Healthcare ) at 25°C with HBS-N EP+ [10 mM Hepes , 150 mM NaCl , 3 mM EDTA , 0 . 005% vol/vol surfactant P20 ( pH 7 . 4 ) ] . For binding analysis , 1 , 000–1 , 600 response units ( RU ) of Fab were captured on a CM5 sensor chip . Samples of different protein concentrations were injected over the surface at a flow rate of 30 μL/min for 240 s , and the chip was washed with buffer for 2 , 000 s . If necessary , surface regeneration was performed with 30 s injection of 50 mM NaOH ( D44 . 1des ) or 10 mM NaOH ( VEGF antibodies ) at a flow rate of 30 μL/min . One flow cell contained no ligand and was used as a reference . The acquired data were analyzed using the device’s software , and kinetic fits were performed . HEK293T cells were seeded on a 24-well plate pre-coated with poly-L-lysine at 120 , 000 cells/well . The next day , cells were transfected with 1 μg DNA mixture consisting of 200 ng pLXN plasmid encoding Luciferase , 400 ng of a plasmid encoding the light chains and 400 ng of a plasmid encoding the heavy chains of the designated antibodies . Each transfection was carried in 100 μl DMEM in which 2 μg of linear 40 , 000 Da PEI ( Polysciences ) per μg of DNA were mixed . The transfection mixture was added to cells , for a total volume of 400 μl DMEM per well . 4 hours after transfection , cells were washed and fresh 1 ml DMEM with 1% penicillin and streptomycin , glutamine and non-essential amino acids was applied . 72 hours post-transfection supernatant was separated from cells and the cells were resuspended in 500 μl PBS . A sample of 100 μl from the suspended cells from each well was transferred to 96-well white plates ( Nunc ) with 100 μl of Bright-Glo reagent ( Promega ) to quantify the level of luciferase as a proxy for the transfection efficiency . Adjusted volumes of supernatants based on the luciferase levels were loaded on a gradient gel ( Bio-Rad ) and run according to manufacturer's instructions . Semi-dry blotting was performed to a nitrocellulose membrane followed by blocking in 5% milk powder in TBST ( 0 . 1% Tween 20 , 20 mM Tris pH 8 . 0 , 150 mM sodium chloride ) buffer for 30 min at room temperature . Donkey anti-human IgG conjugated to HRP ( Abcam ) was used to detect the human IgG scaffold for 1 h at room temperature . Following IgG production in suspension ( as described above ) , clarified media were aliquoted , snap frozen in liquid nitrogen and stored at -80°C . On the day of the measurements , samples were thawed and buffer exchanged into 1 M ammonium acetate , pH 7 , using Micro Bio-Spin 6 Columns ( Bio-Rad ) . To break all disulfide bonds , antibodies were then reduced for 4 h at 37°C in the presence of 20 mM TCEP , followed by two consecutive buffer exchanges into 1 M and 150 mM ammonium acetate , respectively . Nanoelectrospray ionization ( nano-ESI ) MS experiments were performed on a modified Q-Exactive Plus Orbitrap EMR ( Thermo Fisher Scientific , Bremen , Germany ) [70] . All spectra are shown without smoothing . The instrument was calibrated externally , using cesium iodide . Typically , an aliquot of 2 μl protein solution was loaded into a gold-coated nano-ESI capillary prepared in-house , as previously described [71] , and sprayed into the instrument . Conditions within the mass spectrometer were adjusted to preserve noncovalent interactions . The source was operated in positive mode , the capillary voltage was set to 1 . 7 kV , the capillary temperature was 180°C and argon was used as the collision gas in the higher-energy collision-induced dissociation ( HCD ) cell . MS spectra were recorded at a resolution of 10 , 000 and HCD voltage was set to 50 V , at trapping gas pressure setting of 3 . 9 , which corresponds to HV pressure of 1 . 04 x 10−4 mbar and UHV pressure of 2 . 35 x 10−10 mbar . Bent flatapole DC bias and axial gradient were set to 2 V and 25 V , respectively . Antibody stability was monitored by tandem MS ( MS/MS ) , at different HCD voltages . The 23+ charge state of the G6 and G6des13 antibodies was isolated in the quadrupole , with an isolation window of 20 m/z , and the transmitted ions were subjected to collision-induced dissociation in the HCD cell , at a gradient of accelerating voltages ranging between 50–200 V . The relative abundance of the full IgG’s and the dissociated light chains , recorded at the different HCD voltages , was determined by measuring their peak heights . The total intensity of the light chains was calculated as the sum of intensities of their corresponding charge states . In each experimental condition , the total intensities of all the measured species were summed and referenced as 100% intensity . The relative intensity of each species was then calculated as a percentage of the total intensity . The stability assay was performed six times . Error bars represent standard deviation . The coding sequences for variable domains of antibody 492 . 1 were fused to human antibody constant regions [72] . Mutations were introduced by site-directed mutagenesis into the resulting hybrid antibody expression plasmids according to published procedures [73] . Plasmids were transfected into suspension-adapted suspension-HEK 293F cells . The day before transfection , cells were split to 0 . 7 x 106 cells/ml . For parallel expression of the parent hybrid antibody and the 20 variants , transfections were performed using 0 . 5 μg of each plasmid ( heavy and light Ab chains ) mixed with 3 μg PEI Max reagent ( Polysciences Inc . ) and incubated 20 min in 24-well tissue culture trays prior to addition of 1 ml cells per well . Plates were then agitated vigorously in a tissue culture incubator/shaker to prevent cell settling . After 4 days , cultures were transferred to microfuge tubes , and cells were pelleted by centrifugation at 500 x g for 10 min . Supernatants were transferred to fresh microfuge tubes , from which aliquots were taken for quantification of antibody expression and activity . For purification of selected Ab designs , transfections were done in 40 ml volumes , and plasmid and PEI Max amounts were scaled up accordingly . Cultures were grown for 6 days , and Ab was purified from the supernatant by protein G affinity chromatography ( GE Healthcare ) . Relative antibody concentrations were determined from culture supernatants by dot and Western blotting . Blotting was conducted in triplicate for each of two biological replicates . For dot blots , 2 μl of each supernatant was spotted onto nitrocellulose membranes . Membranes were then covered with a blocking solution of PBS containing 0 . 1% Tween ( PBS-T ) and 5% bovine serum albumin ( BSA ) and gently agitated for 1 h at room temperature . For western blots , 10 μl of each supernatant was applied with non-reducing gel loading buffer to 10% SDS polyacrylamide gels . After electrophoresis , proteins were transferred to nitrocellulose , and the membranes were incubated in PBS-T with 5% BSA under gentle agitation . For both dot and Western blots , horseradish peroxidase-conjugated antibody recognizing human Fc was added to the blocking solution after the first hour , and incubation/shaking was continued for another 45 min . The membrane was then washed three times for 5 min each with PBS-T , and the blot was developed using SuperSignal West Pico ( ThermoFisher ) chemiluminescent substrate . Dot and band intensities were recorded on a ChemiDoc XRS+ system ( Bio-Rad ) . QSOX1 inhibition assays were conducted by using 5 , 5-dithio-bis-2-nitrobenzoic acid ( DTNB ) to quantify the remaining dithiothreitol ( DTT ) after incubation with purified recombinant QSOX1 and HEK293 culture supernatants or purified antibody . Culture supernatants ( 25 μl ) were mixed in a clear , flat-bottom , 96-well plate with 12 . 5 μl of 40 nM QSOX1 , and reactions were initiated by injection of 12 . 5 μl 600 μM DTT ( final concentrations 10 nM QSOX1 and 150 μM DTT ) . Reactions were stopped after 30 min by adding 150 μl 500 μM DTNB , and absorbance at 412 nm was measured after 5 min in a Tecan microplate reader . Purified antibody variants were quantified by absorbance at 280 nm after dilution into 6 M guanidine dissolved in PBS , using an extinction coefficient of 187 , 000 M-1cm-1 . Purified antibodies ( 12 . 5 μl ) at concentrations of 40 nM , 100 nM , and 200 nM were mixed in a 96-well plate with 12 . 5 μl 100 nM QSOX1 , and reactions were initiated by injection of 25 μl 600 μM DTT ( final concentrations 25 nM QSOX1 , 300 μM DTT , and 10 , 25 , or 50 nM antibody ) . Reactions were stopped after 20 min by adding 150 μl 500 μM DTNB , and absorbance at 412 nm was measured after 5 min . Background-subtracted absorbance readings were normalized relative to the uninhibited and fully inhibited reactions ( the latter mimicked by leaving QSOX1 out of the reaction ) , and results were plotted in Fig 5C as the relative inhibitory activity .
|
Antibodies are highly important in research , biotechnology , and medical applications . Despite their great utility , however , many antibodies exhibit suboptimal stability and affinity , raising production costs and limiting their practical usefulness . To tackle this general limitation , we used deep mutational scanning to characterize the effects of mutations in an antibody variable fragment on its antigen-binding affinity . Surprisingly , many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface . We , therefore , developed an automated method , called AbLIFT ( http://AbLIFT . weizmann . ac . il ) to optimize this interface through design . Two unrelated antibodies were tested and showed improvements in expression levels , stability , and antigen-binding affinity . Since AbLIFT requires testing of only a few dozen specific designs , it may dramatically accelerate the development of promising antibodies into useful research and clinical tools .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
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2019
|
Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces
|
Messenger RNAs ( mRNAs ) often contain binding sites for multiple , different microRNAs ( miRNAs ) . However , the biological significance of this feature is unclear , since such co-targeting miRNAs could function coordinately , independently , or redundantly with one another . Here , we show that two co-transcribed Drosophila miRNAs , let-7 and miR-125 , non-redundantly regulate a common target , the transcription factor Chronologically Inappropriate Morphogenesis ( Chinmo ) . We first characterize novel adult phenotypes associated with loss of both let-7 and miR-125 , which are derived from a common , polycistronic transcript that also encodes a third miRNA , miR-100 . Consistent with the coordinate upregulation of all three miRNAs in aging flies , these phenotypes include brain degeneration and shortened lifespan . However , transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction . In contrast , let-7 is predominantly responsible for regulating chinmo during nervous system formation . These results indicate that let-7 and miR-125 function during two distinct stages , development and adulthood , rather than acting at the same time . These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system . Thus , this work not only establishes a key role for the highly conserved miR-125 in aging . It also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target , raising the possibility that such biphasic control may be a general feature of clustered miRNAs .
RNA-mediated post-transcriptional mechanisms regulate the accumulation and homeostasis of proteins not only during animal development but also during adulthood [1–3] . These mechanisms include regulation by microRNAs ( miRNAs ) , a class of small non-coding RNAs that usually silence messenger RNAs ( mRNAs ) by binding to partially complementary sequences frequently found in the target 3’ untranslated ( 3’UTR ) sequence [4] . Some miRNAs are known to affect lifespan by post-transcriptionally silencing mRNAs that play critical , beneficial roles at early stages of the life cycle but are deleterious when expressed inappropriately at later stages [1 , 2 , 5–7] . For example , loss of C . elegans lin-4 , the first miRNA to be functionally characterized for its role in lifespan , leads to shortened lifespan due to the persistence of its target , lin-14 [2 , 8] . Similarly , the adult onset of Drosophila miR-34 promotes longevity and maintains neuronal homeostasis by repressing Eip74EF , a transcription factor required for progression through earlier life stages [1 , 3] . Although loss of other miRNAs like Drosophila miR-1000 lead to shortened lifespan [3] , the complete repertoire of miRNAs that regulate aging processes remains uncharacterized [9] . Understanding the role of miRNAs in the adult nervous system is particularly relevant to aging , since the nervous system is a key coordinator of age-related changes in overall organismal physiology [1 , 10 , 11] . For example , the ablation of specific neurons in both worms and flies extends lifespan [12 , 13] . In addition , conserved mechanisms that regulate organismal aging , including insulin signaling and mitochondrial function , modulate the pathology of neurodegenerative disease models [14–18] . Since premature loss of miRNAs has been linked to defective neuronal function and survival as well as the accumulation of disease related proteins , miRNA regulatory networks likely constitute an important component of the normal aging process in the brain [3 , 19–21] . Thus , exploring the functional roles of miRNAs and their mRNA targets in the adult brain is necessary to understand the mechanisms involved in the onset and progression of late onset neurodegenerative diseases . Multiple miRNAs are frequently predicted to regulate the same mRNA indicating that miRNA activity within tissues such as the nervous system is coordinated . Bioinformatic analyses estimate that greater than 70% of targeted human mRNAs and between 30 to 50% of targeted Drosophila mRNAs have sites for two or more miRNAs [22–24] . The Drosophila predictions are likely underestimates of the frequency of co-targeting in the nervous system , since they were generated prior to the discovery of dozens of Drosophila miRNAs as well as the 3’UTR extensions of numerous neural mRNAs [25–27] . Recent analyses have found that co-targeting is particularly prevalent for clustered miRNAs , which are likely to be co-transcribed and therefore co-expressed [28] . Based on reporter assays showing a positive correlation between the number of miRNA sites in a 3’UTR and the degree of its repression [29 , 30] , the current model suggests that miRNA activity is additive and predicts that spatially overlapping combinations of miRNAs–presumably including those that are co-transcribed–lead to greater target repression [31 , 32] . However , there are very few published investigations that have tested this model directly by delineating the individual activities of multiple co-targeting miRNAs . Here , we re-evaluate this model by distinguishing the effects of two co-transcribed neural miRNAs , let-7 and miR-125 , on a common target mRNA during development and adulthood .
The let-7-Complex ( let-7-C ) locus in Drosophila encodes an evolutionarily conserved cluster of three co-transcribed miRNAs: miR-100 , let-7 and miR-125 , the orthologue of C . elegans lin-4 [33 , 34] . Although the levels of processed let-7 are known to increase with age in testes and ovaries [35 , 36] , the relative expression levels of all three miRNAs have not been characterized in aging flies . To address this , we performed Northern blot and quantitative reverse transcription polymerase chain reaction ( qRT-PCR ) analyses of whole animals ( Fig 1A and 1B , left ) . These analyses revealed an age-dependent increase in all three let-7-C miRNAs in both adult males and females , suggesting a role for this miRNA cluster in aging-related processes . To characterize the role of this age-dependent increase in let-7-C miRNAs , we analyzed a let-7-C hypomorphic ( let-7-Chyp ) strain in which let-7-C miRNAs were expressed during development [37] but not maintained during adulthood ( Fig 1B , right ) . This hypomorphic strain was trans-heterozygous for two let-7-C null alleles but also harbored a single copy of a minimal let-7-C rescuing transgene that contained regulatory elements required for onset of pri-let-7-C ( let-7-Cp3 . 3kb::cDNA ) during development but lacked elements needed for its post-developmental maintenance . Consistent with our previous analysis [37] , young let-7-Chyp mutant males expressed reduced levels of miR-100 ( 9 . 2±4 . 6% of control ) , let-7 ( 22±10 . 2% of control ) and miR-125 ( 33±7 . 2% of control ) that decreased further as the adults aged ( Fig 1B ) . We therefore performed survival analysis of these let-7-Chyp mutant males and found that they died prematurely relative to control males ( Fig 1C , compare black and red curves; w1118: median survival 74d , maximum lifespan 98d; let-7-Chyp: median survival 36d , maximum lifespan 56d ) . Prompted by this reduced viability , we assayed the let-7-Chyp strain for additional functional and morphological age-dependent phenotypes . Young let-7-Chyp mutants climbed normally , indicating that the levels of let-7-C miRNAs they express during metamorphosis and early adulthood is sufficient for general adult function . However , aged let-7-Chyp mutants displayed a steep reduction in this ability ( Fig 1D ) . These results indicated that persistent expression of one or more of the three let-7-C miRNAs specifically during adulthood was required for normal adult healthspan . Given the neural expression of let-7-C miRNAs [33 , 37 , 38] , we next looked for age-associated deterioration in brain morphology . Brain degeneration has been anatomically characterized by an age-dependent increase in the number of scattered vacuoles that mark cells undergoing necrotic cell death [39] . Sections of 40-day old control and let-7-Chyp brains revealed a sharp increase in vacuole number in mutant brains ( Fig 1E–1G ) . As with the climbing defect described above , this phenotype had an adult onset since the brains of young mutant flies contained hardly any vacuoles ( 0 vacuoles in w1118 , 0 . 6 ± 0 . 9 vacuoles in let-7-Chyp , n = 5 ) . Importantly , a let-7-C transgene that substantially restored miR-100 levels ( 59 . 4±11 . 1% of control ) , let-7 levels ( 50 . 3±24% of control ) and miR-125 levels ( 108±28% ) in 3-day old adults ( let-7-Chyp rescue in Fig 1B ) rescued the lifespan and age-dependent climbing defects as well as the brain deterioration of let-7-Chyp mutants ( Fig 1C , 1D and 1G ) . Since our qRT-PCR analysis indicated that rescued let-7-Chyp mutants express a constant level of let-7-C miRNAs during adulthood ( Fig 1B ) , we inferred that the age-dependent increase in let-7-C miRNAs detected in wildtype adults was not absolutely required for their pro-survival and neuroprotective roles . Taken together , these results confirmed a role for let-7-C miRNAs in the aging processes that occur in the brain . In order to distinguish the roles of the three let-7-C miRNAs , we generated a set of rescuing transgenes with either miR-100 , let-7 or miR-125 deleted . These transgenes were inserted into identical chromosomal locations using phiC31-mediated integration [40] and crossed into a trans-heterozygous let-7-C null background , yielding strains we referred to as ΔmiR-100 , Δlet-7 and ΔmiR-125 single mutants , respectively ( see S1 Fig for our crossing scheme that ensured that single mutant strains were otherwise as close to identical as possible ) . Unlike previously generated strains with P-element rescue transgenes [33] , differences between these single mutants could be attributed to loss of an individual miRNA rather than to position effects . Quantitative RT-PCR analysis of miR-100 , let-7 and miR-125 confirmed the absence of miRNA expression in each of the deletion lines ( Fig 2A ) . However , this analysis also revealed cross-regulatory relationships between the three miRNAs: loss of let-7 resulted in reduced levels of both miR-100 ( 0 . 29 fold relative to control ) and miR-125 ( 0 . 35 fold relative to control ) , while loss of miR-100 and miR-125 resulted in increased levels of let-7 ( 2 . 5 fold relative to control ) and miR-100 ( 2 . 5 fold relative to control ) , respectively ( Fig 2A ) . To assess the cause of these changes , we turned to a cell culture assay in which we could quantify the activity of each miRNA in cells transfected with altered let-7-C versions . MiRNA activity was quantified as the fold repression in luciferase levels produced by previously validated “sensors” for each let-7-C miRNA [38] . Individual sensors were co-transfected along with UAS-let-7-C cDNA constructs into Kc-167 cells that do not ordinarily express let-7-C miRNAs [37] . First , confirming the effect of let-7 deletion on miR-100 and miR-125 levels , we found that miR-100 and miR-125 activity reporters were less repressed in cells transfected with a let-7-C cDNA lacking the let-7 hairpin ( miR-100: 5 . 6 ± 0 . 56 fold repression in Δlet-7 compared to 10 . 48 ± 1 . 7 in wild type; miR-125: 8 . 3 ± 0 . 66 fold in Δlet-7 compared to 13 . 8 ± 1 . 97 fold in wild type ) . Then , to test whether this effect was due to the absence of mature let-7 or some other cause ( e . g . altered RNA conformation of the Δlet-7 primary transcript that reduced miR-100 and miR-125 processing ) , we generated a chimeric UAS let-7-C cDNA construct in which the Drosophila let-7 hairpin was replaced with the human let-7-a2 hairpin that encoded the same mature let-7 but has a different hairpin structure . While the human let-7-a2 hairpin restored the let-7 mediated repression of its sensor , it did not restore miR-100 and miR-125 mediated repression ( Fig 2B , construct 5 ) . These data indicated that processed let-7 miRNA did not directly regulate the processing of miR-100 or miR-125 . Instead , we favor a model where the rate of let-7 processing has an effect on the rate of miR-100 and miR-125 processing , a model consistent with processing of other polycistronic microRNAs [41] . We note that Truscott et al . also recently found evidence for cross-regulatory interaction between let-7-C miRNAs [41] , although their results were slightly different—deletion of let-7 and miR-100 but not let-7 alone reduced miR-125 levels—probably due to technical differences in the constructs used . We also evaluated ΔmiR-100 or ΔmiR-125 let-7-C cDNA constructs in this cell culture assay , but detected no enhancement in miRNA activity ( Fig 2B ) , suggesting that the changes in miRNA levels detected in tissue ( Fig 2A ) may not be functionally significant . Taken together , these results indicated that the set of ΔmiR-100 , Δlet-7 and ΔmiR-125 strains described above would allow the dissection of the individual contributions of the three miRNAs since neighboring miRNAs continued to be expressed when individual miRNAs were deleted , albeit at altered levels in some cases . We then used the ΔmiR-100 , Δlet-7 and ΔmiR-125 single mutant lines to analyze the consequences of deleting each miRNA on age-associated brain degeneration and behavioral defects . Δlet-7 and ΔmiR-125 single mutant flies displayed significantly reduced longevity compared to control or ΔmiR-100 flies ( Fig 2C ) . In addition , while young Δlet-7 and ΔmiR-125 mutants had normal climbing behavior and brain morphology , a significant decrease in climbing ability as well as a marked increase in vacuole number was observed in both mutants with age ( Fig 2E and 2F ) . The vacuoles in both Δlet-7 and ΔmiR-125 mutants appeared to be scattered throughout the central brain region and some enrichment was also seen in the retina ( S2 Fig ) . These data indicated that loss of either let-7 or miR-125 but not miR-100 caused behavioral and morphological changes that were normally seen in much older flies and were indicative of rapid aging of the brain . Given that loss of let-7 and miR-125 triggered physiological processes involved in aging , we tested whether inhibition of individual let-7-C miRNAs enhanced the neurodegeneration of a disease model of fragile X-associated tremor/ataxia syndrome ( FXTAS ) [42] . FXTAS is a late onset human neurodegenerative disease that is characterized by the presence of ubiquitin positive nuclear inclusions containing RNAs with expanded CGG repeats ( rCGG ) in neurons and astrocytes [43] . Ectopic expression of transcripts with artificial expansion of these repeats in the fly retina causes a pathology similar to human FXTAS , including photoreceptor degeneration and disorganization of the ommatidia [42] . Using miRNA “sponge” constructs designed to individually inhibit miR-100 , let-7 , or miR-125 ( miR-100SP , let-7SP , or miR-125SP ) , we tested whether loss of any of these miRNAs’ activities enhanced the retinal degeneration in the FXTAS model . We found that driving let-7SP or miR-125SP but not miR-100SP specifically in the eye throughout development and adulthood resulted in significant enhancement of the rCGG phenotype ( S3 Fig ) . This result indicated that , in addition to their role in modulating lifespan , let-7 and miR-125 promoted disease pathogenesis while miR-100 did not . To pinpoint the specific stage during which let-7 and miR-125 activity were involved in FXTAS disease pathogenesis , we utilized a temperature sensitive allele of Gal80 ( tubP-Gal80ts ) . This approach allowed temporal control of both the UAS-rCGG90 transgene as well as the UAS-miRNA sponges in the eye , since animals at 29°C express UAS transgenes but animals at 18°C do not [44] . We reared strains to control expression in three ways: no expression ( 18→18 ) , constant expression ( 29→29 ) , or expression during development but not adulthood ( 29→18 ) ( Fig 3A–3L ) . As expected , constant expression of either let-7SP or miR-125SP enhanced the rCGG90 phenotype whereas no expression did not ( Fig 3A–3F ) . However , let-7SP and miR-125SP behaved differently from one another in the 29→18 regimen: let-7SP enhanced rCGG90 retinal degeneration while miR-125SP did not ( compare Fig 3G–3I ) . This result , along with the observation that let-7SP animals reared at 29→18 looked no worse than those reared at 29→29 , suggested that let-7’s main contribution to disease progression occurred during development . Conversely , miR-125SP animals reared at 29→18 looked no worse than those reared at 18→18 , suggesting that the phenotypes displayed by those reared at 29→29 was a specific consequence of adult miR-125SP expression . The reciprocal experiment involving adult-only transgene expression was not informative because none of the 18→29 animals displayed a phenotype , even when aged up to 20 days , perhaps because the underlying rCGG90 phenotype was at least partially of developmental origin . These data indicated that let-7 and mir-125 functioned during distinct temporal periods to effect disease progression , and that the retinal degeneration caused by a decline in let-7 activity was of a developmental origin while that caused by inhibition in miR-125 activity was due to degeneration of adult brains . Since miRNAs function by repressing target mRNAs , we investigated whether the age-associated Δlet-7 and ΔmiR-125 phenotypes were due to the elevated expression of chronologically inappropriate morphogenesis ( chinmo ) , the only verified target of both let-7 and miR-125 in flies [38] . Chinmo is a transcription factor that controls neuronal fate in a dosage-sensitive manner . In the mushroom body lineages in the central brain , for example , Chinmo is expressed at high levels early in development to promote early born cell fates and its post-transcriptional downregulation leads to the production of later born fates [45] . The chinmo 3’UTR contains multiple let-7 and miR-125 binding sites and is regulated by let-7-C miRNAs during development [38] . To address whether the adult phenotypes of Δlet-7 and ΔmiR-125 mutants were due to elevated Chinmo , we reduced the dosage of chinmo in these mutants by either removing one copy of chinmo using a null chinmo1 mutation [45] or by knocking down chinmo using a RNAi transgene that we verified in vivo ( S4A–S4L Fig ) . Lowering chinmo levels dramatically suppressed the premature death ( Fig 2D ) , climbing defects ( Fig 2E ) , and brain necrosis ( Fig 2F and S4M Fig ) of ΔmiR-125 mutants but not Δlet-7 mutants . This result indicated that elevated Chinmo was responsible for ΔmiR-125 phenotypes but that other factors were responsible for Δlet-7 phenotypes . This distinction also indicated that the Δlet-7 phenotypes described above ( reduced longevity , climbing and neurodegeneration ) were not solely due to the reduction in miR-125 levels observed in these mutants ( Fig 2A ) and implied the de-repression of other , currently unidentified , mRNAs . To test whether ectopic Chinmo was sufficient to cause the neurodegenerative phenotypes associated with loss of miR-125 , we ectopically expressed a chinmo transgene in adult brains using an inducible neural GAL4 driver ( Fig 4A ) . This forced expression resulted in a drastic reduction in both lifespan ( Fig 4B ) and climbing ability ( Fig 4C ) along with a dramatic increase in brain vacuole numbers in 20d aged flies ( Fig 4D and 4E ) . Interestingly over- expression of Chinmo in neurons showed an increased localization of vacuoles in the lamina and central brain regions , indicating that neurons in the lamina region were more sensitive to de-regulation of Chinmo ( Fig 4D and 4E and S2E Fig ) . These experiments confirmed that deregulated expression of Chinmo in adult neurons results in premature neurodegeneration in adult flies and supported the genetic suppression evidence above that silencing of this protein by miR-125 was critical for maintaining neuronal integrity and viability in adult flies . Messenger RNAs frequently contain binding sites for multiple miRNAs , which may repress common targets in an additive manner [31] . However , our analysis raised the possibility that the chinmo mRNA might be regulated by miR-125 but not let-7 in adult brains despite containing verified functional binding sites for let-7 [38] . Intriguingly , these experiments were supported by immunostaining of Chinmo in Δlet-7 and ΔmiR-125 adult brains . The degree of de-repression of Chinmo was more widespread and starkly higher in ΔmiR-125 brains when compared to Δlet-7 ( S5D Fig ) . In contrast , Δlet-7 mutant brains displayed a much weaker immunostaining signal of Chinmo ( S5C Fig ) . To distinguish the contributions of let-7 and miR-125 on chinmo repression , we compared the levels of Chinmo protein in immunostained brains of control , Δlet-7 , ΔmiR-125 , and let-7-C null adults . Shortly after let-7-C activation at 24 hours after puparium formation ( APF ) , elimination of both miRNAs resulted in much higher levels of Chinmo than loss of either miRNA alone ( Fig 5A–5I ) , indicating that both let-7 and miR-125 contributed to chinmo repression at this time-point . Three days later , however , let-7 played the predominant role in silencing chinmo , since Δlet-7 mutant brains expressed 86 . 7 ± 4 . 8 arbitrary units ( AU ) of Chinmo while ΔmiR-125 mutant brains expressed only 43 . 3 ± 5 . 2 AUs of Chinmo ( Fig 5A–5D and Fig 5I ) . A complete reversal in the relative contributions of let-7 and miR-125 occurred during the pupal-to-adult transition: Δlet-7 mutant adult brains expressed 38 . 5 ± 10 . 3 AUs while ΔmiR-125 mutant brains expressed 72 . 9 ± 9 . 2 AUs , indicating that miR-125 was primarily responsible for silencing chinmo in adults ( Fig 5E–5I and S5A–S5D Fig ) . To support this data , we also quantified the levels of chinmo mRNA in late pupal and adult Δlet-7 and ΔmiR-125 mutant heads , since miRNA regulation is known to cause mRNA destabilization . Consistent with our quantification of Chinmo protein levels , loss of let-7 affected chinmo mRNA levels in late pupae but not adults , while loss of miR-125 affected chinmo mRNA levels in adults but not pupae ( Fig 5J ) . Together , these data indicated that let-7 and miR-125 predominantly regulated chinmo during development and adulthood , respectively . Since the derepression of Chinmo during development was higher in Δlet-7 than ΔmiR-125 mutants , we tested whether chinmo-dependent developmental defects were more severe in Δlet-7 than ΔmiR-125 single mutants . To examine this possibility , we investigated the relative roles of let-7 and miR-125 in fate transitions in MB neuronal temporal identity . The MB is composed of four subtypes of neurons that are generated in a sequential manner ( γ → α’/β’ → pioneer α/β → α/β ) by neuroblasts [45–47] . High levels of Chinmo specify early born cell fates ( γ , α’/β’ ) , while low levels of Chinmo specify later born cell fates ( pioneer α/β , α/β ) . Altered dosages of chinmo lead to changes in the total numbers of these various neuronal classes so that , for example , elevated chinmo is associated with a smaller population of pioneer α/β neurons [38 , 45] . Therefore , to evaluate the relative contributions of let-7 and miR-125 chinmo regulation during neural development , we counted the number of pioneer α/β neurons in adult brains . As expected , Δlet-7 mutants displayed greater reduction in pioneer α/β neuron number than ΔmiR-125 mutants and , furthermore , Δlet-7 , miR-125 double mutants showed a reduction similar to Δlet-7 single mutants ( Fig 5K ) . Thus , regulation of chinmo was more dependent on let-7 than miR-125 during the larval-to-adult transition but was more dependent on miR-125 in aging adults . To assess whether let-7 and miR-125 had different strengths of repression that might contribute to their differential activities , we examined the expression of luciferase reporters containing a previously characterized 1 . 4kb 3’UTR fragment of chinmo that harbors six let-7 and four miR-125 binding sites that are conserved between Drosophila species [38] . Overexpression of the entire let-7-C primary transcript in Kc-167 cells repressed the wild type reporter 17-fold ( Fig 5L ) . In contrast the fold repression of mutants lacking let-7 binding sites or miR-125 sites or both was reduced 6 . 9- , 14 . 0- and 4 . 8-fold , respectively . While these data indicated that let-7 was a stronger repressor of chinmo than miR-125 , it was not clear whether the greater decrease in fold repression was due to a greater number of let-7 sites . To circumvent this issue , we designed and quantified the degree of repression of a luciferase reporter that contained a single verified miRNA binding site for let-7 and a single miR-125 site that had comparable base pairing characteristics ( S6 Fig ) . Ectopic expression of let-7-C miRNAs resulted in a 12-fold repression of the wild type reporter while deletion of the let-7 seed sequence , the miR-125 seed sequence or both sequences resulted in a 2 . 9- , 4 . 6- and 0 . 7-fold repression of luciferase activity respectively ( Fig 5M ) . Together these data confirmed that both let-7 and miR-125 were capable of silencing the luciferase sensor individually but maximum repression was achieved when both sites were functional and that let-7 was a stronger post-transcriptional repressor than miR-125 . To investigate the basis for the sequential repression of Chinmo by let-7 and miR-125 , we first compared the rate of let-7 and miR-125 production in the developing and adult nervous system . To do so , we quantified the ratio of processed miRNA to precursor miRNA for let-7 and miR-125 in staged nervous system samples ( Fig 6A ) . The let-7/pre-let-7 ratio was significantly higher than the miR-125/pre-miR-125 prior to 72h APF . In contrast , the miR-125/pre-miR-125 ratios were higher than the let-7/pre-let-7 ratios after 72h APF and into adulthood ( Fig 6A ) . The temporal dynamics of let-7 and miR-125 production in the nervous system correlated with their relative roles in chinmo regulation , suggesting that the basis for their sequential activity may involve differential processing and/or turnover . To determine the basis for this switch in the relative expression of let-7 and miR-125 , we first investigated the possibility that let-7 and miR-125 might be differentially processed . To do so , we again took advantage of the Kc-167 embryonic cell line . As mentioned above , the let-7-C locus is not ordinarily transcribed in this cell line , but it is activated in response to the Drosophila steroid hormone 20-hydroxyecdysone ( 20E ) [37] . Kc-167 cells were treated with 20E for 24h to induce primary let-7-C transcript and the levels of let-7 and miR-125 were monitored at different time intervals after washing off the steroid hormone . To measure the relative rates of processed let-7 and miR-125 production , we performed qRT-PCR on 20E-treated Kc-167 samples using a standard curve to extrapolate absolute miRNA levels . Forty-eight hours after the 20E treatment , we detected 1024±440 copies of let-7 per nanogram ( ng ) of total RNA but only 3±1 copies of miR-125 per ng of total RNA , indicating that let-7 was processed more efficiently than miR-125 ( Fig 6B ) . Incubation of cells for 120 hours after the 20E pulse resulted in a 3–4 fold increase in the copy number of let-7 ( 3872±1365 copies/ng of total RNA ) and a ~30 fold increase in the copy number of miR-125 ( 101±24 copies/ng of total RNA ) . The greater increase in the miR-125 copy number at later time points suggested that processed miR-125 persisted longer than let-7 . In order to identify the key steps in miRNA biogenesis that contributed to the inefficient processing of miR-125 , we performed in vitro Drosha and Dicer processing assays ( Fig 6C and 6D ) . We first examined the rate of generation of precursor miRNAs ( pre-miRNA ) from longer primary miRNA ( pri-miRNA ) transcripts by the Drosha-Pasha complex . In initial experiments , we found that pri-miR-125 processing was extremely inefficient ( S7A Fig ) . Therefore , we compared the processing of pri-let-7 to the processing of chimeric constructs in which either the pri-let-7 terminal loop or its stem-base were replaced with the pri-miR-125 loop ( pri-let-7miR-125L ) or pri-miR-125 stem base ( pri-let-7miR-125B ) , respectively ( Fig 6B and S7B Fig ) . The rates of processing of these three transcripts were examined by incubating with Drosha-Pasha complexes immunoprecipitated from Kc-167 cells ( S7B Fig ) . Substituting either the terminal loop or stem base of pri-miR-125 in pri-let-7 resulted in a dramatic reduction in Drosha processing . While 15% of the unmodified let-7 primary transcript was processed within 30 minutes , only 5% of pri-let-7miR-125L was cleaved by Drosha . Incubation with Drosha-Pasha complex for 120 minutes increased the percentage of precursor to 30% and 7% for pri-let-7 and pri-let-7miR-125L , respectively . However , substituting the stem-base of pri-miR-125 in pri-let-7 completely abolished its Drosha processing ( Fig 6C ) . Thus , both the terminal loop and stem base sequence determinants of pri-miR-125 contributed to its inefficient processing by Drosha in vitro , raising the possibility that other post-transcriptional mechanisms may facilitate miR-125’s processing in vivo . To evaluate the Dicer-1 processing of pre-let-7 and pre-miR-125 , we also performed in vitro processing assays with Flag-tagged Dicer-1 that was , like the Drosha-Pasha complexes described above , purified from Kc-167 cell extracts ( S7B Fig ) . In these assays , pre-miR-125 displayed a significantly lower kinetics of processing than pre-let-7 ( Fig 6C ) . Within 10 minutes of incubation with Dicer-1 , 23±3 . 6% of pre-let-7 and 15±0 . 6% of pre-miR-125 were processed to their mature forms . After 60 minutes of incubation , the percentage diced was 71±7 . 2% and 47±0 . 4% for pre-let-7 and pre-miR-125 , respectively ( Fig 6D and 6E ) . Thus , this higher kinetics of processing of let-7 by both Drosha and Dicer likely contributed to its rapid accumulation during metamorphosis . While differential processing was consistent with the more rapid accumulation of let-7 , we hypothesized that the temporal dynamics of miR-125 accumulation ( Fig 6A ) might also reflect an increased stability . In order to monitor the persistence of let-7 and miR-125 in adult nervous system tissue , we measured the expression of these miRNAs after blocking Dicer-1 activity . Total RNA was extracted from heads of 10d-old adult flies that expressed one of two Dicer-1 shRNA constructs , and the levels of let-7 and miR-125 were quantified by Taqman miRNA assays ( Fig 6D and S7C Fig ) . Knockdown of Dicer-1 resulted in a greater reduction of let-7 relative to miR-125 ( Fig 6D ) : expression of Dicer-1shRNA1 or Dicer-1shRNA2 resulted in 0 . 23±0 . 1 or 0 . 31±0 . 37 fold expression of let-7 relative to control but 0 . 62±0 . 18 or 0 . 58±0 . 18 fold expression of miR-125 relative to control , respectively . These data indicated that the decay rate of let-7 was significantly higher than that of miR-125 in the adult nervous system . Finally , to assess whether miR-125 had a longer half-life than let-7 , we measured the decay rates and half-lives of let-7 and miR-125 by analyzing Kc-167 cells transfected with synthetic miRNA duplexes . Cells were washed with fresh medium 5 hours after transfection , and samples were collected at the indicated times for total RNA preparation followed by quantitation of let-7 and miR-125 by Taqman assays . Half-lives were inferred from fitted exponential curves ( S7D Fig ) . As expected , the half-life of miR-125 ( T1/2 , 2 . 7h ) was significantly higher than that of let-7 ( T1/2 , 2h ) . Taken together these experiments suggested a mechanistic basis for the switch in let-7-to-miR-125 activity that occurred during pupal-to-adult transition: while an increased rate of Drosha/Pasha and Dicer processing of let-7 facilitated the attenuation of chinmo in the developing nervous system , the enhanced perdurance of miR-125 ensured that chinmo was silenced in the adult brains ( Fig 7 ) .
let-7 and miR-125 have distinct and non-overlapping functions , despite being co-transcribed and sharing the same target . Loss of either miRNA alone leads to shortened lifespan and premature deterioration of health , as indicated by age-dependent climbing defects and brain degeneration . The aging defects caused specifically by loss of miR-125 are associated with high levels of Chinmo in adult brains , and can be rescued by reducing chinmo levels in the ΔmiR-125 mutant . In contrast , Chinmo is substantially lower in Δlet-7 mutant adult brains and it appears not to contribute to adult Δlet-7 mutant phenotypes: neither adult Δlet-7 mutant climbing defects , brain vacuolization , nor reduced longevity are suppressed by chinmo reduction . Instead , let-7 predominates during development: pupal Chinmo expression is higher and associated defects in neuronal identity are worse in Δlet-7 mutants than ΔmiR-125 mutants . Although deletion of let-7 reduces miR-125 levels , the differences in the Δlet-7 and ΔmiR-125 phenotypes indicate that the Δlet-7 phenotypes are not simply due to loss of miR-125 . In support of this , distinct temporal periods of let-7 and miR-125 activity were also identified using sponges , an independent method for disrupting miRNA activity in which let-7 interference does not affect miR-125 activity . Based on these results , we conclude that a let-7-to-miR-125 switch during the pupal-to-adult transition ensures chinmo repression in adults , maintaining neuronal integrity and promoting life span . Our results illuminate a function of miRNA co-targeting that we term “phasic control , ” which indicates that co-targeting can reflect non-redundant regulation during distinct phases of a cells life , from its birth to its death . Rather than simply reinforcing silencing , such repression at different times may have distinct functions , based not only on the changing status of the cell but also on differences in miRNA::mRNA interactions ( e . g . base-pairing characteristics , trans-acting factors , etc ) . Highlighting such phasic control , we propose a model in which let-7-C miRNAs collectively function as both a rheostat and as a switch but at distinct times ( Fig 7 ) . According to this model , let-7 predominates during nervous system formation , where it shapes the temporal gradient of Chinmo . let-7-dependent attenuation of this dosage-sensitive transcription factor is responsible for the establishment of proper cell fate as neural progenitors divide . Subtle alterations in the rate of let-7 accumulation may adjust the neuronal classes that comprise structures like the mushroom body , whose composition is known to be sensitive to environmental cues [48] . While let-7 adjusts chinmo , miR-125 in contrast switches chinmo off in post-mitotic neurons throughout the adult nervous system . This silencing of a juvenile neuronal marker maintains adult neuronal integrity , since forced Chinmo expression in adults leads to brain deterioration . While our model proposes that miR-125 ensures complete silencing of chinmo , we cannot rule out the possibility that miR-125 repression is alleviated under certain conditions in the adult ( e . g . injury-induced repair ) so that Chinmo can reprogram neurons to a juvenile state that is needed for certain adult functions . Thus , by independently regulating the same target during two different periods , let-7 and miR-125 miRNAs control cell fate establishment and maintenance , respectively . Our model is based in part on results that chinmo repression is achieved predominantly by miR-125 in adult brains , even though let-7 is present . What accounts for the muted let-7 activity that , while present , is not responsible for repression of a verified target ? Perhaps let-7 has many more targets than miR-125 in the adult brain , since miRNAs with a larger repertoire of target genes have a weaker effect on each individual target [49 , 50] . In addition , let-7 targets may be highly expressed in the adult brain , thereby titrating away functional let-7 and leading to its reduced effect on all its targets , including those that are co-targeted by both let-7 and miR-125 . Such a scenario is supported by increasing evidence that the effectiveness of a particular miRNA is influenced by the cellular concentration of available miRNA binding sites [51 , 52] . Alternatively , perhaps let-7 silencing requires cofactors that are only expressed during development . Future studies focused on the identification and characterization of the miR-125-independent targets of let-7 should provide insight into the networks of let-7 targets in adults . While an overarching feature of miRNA regulation is that mRNAs are responsive to multiple miRNAs , our understanding of the biological significance of co-targeting is rudimentary . Supporting the apparently abundant co-targeting identified by miRNA binding site predictions [22–24 , 28 , 53] , there are plenty of examples of multiple miRNAs that can when expressed one-by-one repress the same 3’UTR reporter . Such examples include repression of mtpn 3’UTR by any one of a trio of miRNAs ( miR-375 , miR-125 , and let-7b ) and repression of cdkn1A/p21 3’UTR by any one of a staggering 28 different miRNAs [53 , 54] . Since the effects of combinations of miRNAs are rarely tested , such studies suggest that multiple miRNAs limit the spatial and/or temporal expression of targets and , in cells where they are co-expressed , may function redundantly with one another . In addition to this simple scenario , there are hints of more complex combinatorial scenarios involving either cooperation or competition between co-targeting miRNAs . In the cooperative scenario , miRNAs with overlapping expression patterns lead to enhanced repression of co-targeted mRNAs in cells where they are expressed together . Examples of this include miR-25 and miR-221/222 co-repression of p57 and miR-148a and the miR-206 co-repression of dmpk [55 , 56] , although it is worth noting that the additive effects of these pairs of miRNAs is small though significant . Supporting such cooperative action , additional studies report that multiple binding sites , especially when they are within 15–35 bp of one another , lead to enhanced reporter repression [29 , 57 , 58] . Co-expressed miRNAs can also act competitively , as shown for miR-184 and miR-205 regulation of ship2 [59] . In this scenario , miR-184 does not have repressive activity itself but alleviates the repressive ability that miR-205 exerts via an overlapping binding site , as shown by comparative analysis of miR-205 alone versus in combination with miR-184 as well as analysis of mutated 3’UTR reporters containing intact miR-205 but mutant miR-184 sites . In light of this miR-184 function , systematic assays that have found that many miRNAs when expressed individually have no effect on a 3’UTR reporter do not rule out the possibility that these miRNAs function competitively with others [60 , 61] . Phasic control expands the repertoire of known co-targeting functions , and emphasizes that co-targeting miRNAs may function at different times from one another and for different purposes . Phasic control may be particularly relevant to clustered miRNAs , since clustered miRNAs are enriched for co-targeting relationships [22 , 28] . For example , the vertebrate miR-17~92 cluster , like other miRNA clusters , targets multiple components of related networks and pathways of genes , including the TGF-β pathway [62 , 63] . As with the let-7-C cluster in flies , members of polycistronic clusters , including the miR-17~92 and miR-1/miR-133 clusters , are differentially processed [64 , 65] . The resulting differential accumulation of these co-targeting miRNAs , along with differential base pairing and turnover of co-transcribed miRNAs , may lead to the distinct temporal accumulations of processed miRNAs that are indicative of phasic control . Thus , the staggered accumulation of different miRNAs processed from the same polycistronic transcript over time may be an important feature controlling the progression of temporal features of cell and organismal biology . The biphasic regulation by let-7-C miRNAs may also be relevant to mRNAs co-targeted by let-7-C orthologues in other animals . These include lin-28 and lin-41 , which were originally identified in C . elegans as potential targets of both let-7 and lin-4 , the C . elegans miR-125 orthologue [66 , 67] . These co-targeting relationships are conserved to vertebrates , since mouse lin-28 and lin-41 3’UTRs are responsive to altered levels of both let-7 and miR-125 [68–70] . The neurodevelopmental functions of these co-targetings has been extensively investigated and include , for example , let-7/miR-125-mediated repression of lin-28 to control temporal identity during retinal neurogenesis in zebrafish [71 , 72] . However , the careful dissection of the relative roles of let-7 versus miR-125 as well as their respective post-developmental functions awaits future investigation . While the lin-28/let-7-C relationship does not appear to be conserved to flies [73] , the recent identification of a let-7 and miR-125 target with homology to chinmo suggests that regulation of a chinmo orthologue may be conserved [74] . This target , hypermethylated in cancer 2 ( hic2 ) , encodes a BTB-zinc finger ( BTB-ZF ) transcription factor that contains multiple predicted let-7 and miR-125 sites in its 3’UTR . While reciprocal homology searching predicts equally good amino acid similarity between Chinmo and a number of mammalian BTB-ZFs including Hic2 , the conservation of let-7 and miR-125 sites in the hic2 3’UTR but not other BTB-ZF 3’UTRs suggests that Hic2 is the mammalian orthologue of Chinmo . Thus , our results predict that mammalian let-7 and miR-125 regulate hic2 in a biphasic manner . The persistence and gradual increase of let-7-C miRNAs during adult life may balance the various cellular demands needed for proper tissue and organismal homeostasis over time . Thus , the increasing levels of let-7 that dampen stem cell function in aging tissue , found both in the mouse nervous system and the fly testis [35 , 75] , may be part of a general program that includes the neuronal maintenance function that this study explores . A conserved role for let-7-C miRNAs in such cell maintenance during adult life is supported by the requirement of lin-4 for proper lifespan in C . elegans [2] , since nematodes , like fly brains , exhibit limited cell proliferation during adulthood . In addition , Drosophila let-7 and its target the dp transcription factor promote the maintenance of dopaminergic neurons in the adult brain by regulating the expression of pathogenic Leucine-Rich Repeat Kinase 2 [76] . Interestingly , changes in miR-125b have been linked to Alzheimer’s disease and vertebrate cerebellar neurodegeneration , although the molecular mechanisms underlying these changes have not been addressed [77 , 78] . Taken together , this mounting evidence indicates that let-7 and miR-125 play critical neuroprotective roles in the aging brain so understanding their post-developmental functions in greater detail may be relevant to therapies for human neurodegenerative diseases , including Parkinson’s and Alzheimer’s diseases . In summary , our work has identified a novel in vivo mechanism by which multiple miRNAs repress a common target during distinct stages . Such differential regulation by subsets of co-expressed miRNAs should be considered for designing therapeutic strategies to treat diseases that are frequently caused by de-regulation of highly targeted mRNAs .
All flies were cultured on standard cornmeal medium at 25°C under 12 h light , 12 h dark cycles , except for flies analyzed in the temperature sensitive experiment presented in Fig 3 . These flies were cultured in one of three regimens: at 18°C , at 29°C , and at 18°C until eclosion and then at 29°C thereafter . For steroid mediated UAS-transgene control using the Gene-Switch driver , flies were fed food containing 200μM RU-486 ( Mifepristone , Cayman Chemicals , Ann Arbor MI ) . Staging of pupae and MARCM clone induction was performed as previously described [37 , 38] . Unless otherwise noted , adult male flies of indicated ages were used for experiments . Detailed genotypes of all strains as well as the sources of the genetic mutations and transgenes used in the study are listed in S1 and S2 Tables , respectively . Transgenesis was performed by Rainbow Transgenic Services ( Camarillo , CA ) and BestGene , Inc . ( Chino Hills , CA ) . The let-7-C mutant strains analyzed in this study ( including let-7-Cnull , let-7-Chyp , let-7-Chyp rescue , ΔmiR-100 , Δlet-7 and ΔmiR-125 strains ) were generated by crossing w1118; let-7-CGKI / CyO strains to w1118; let-7-CKO2 , P{neoFRT}40A / CyO strains in which one or both of these strains contained a rescuing transgene inserted on the third chromosome . Since this approach generated trans-heteroyzgous let-7-CGKI / let-7-CKO2 , P{neoFRT}40A animals , it eliminated the effect of any confounding recessive background mutations that might have accumulated on those chromosomes . In addition , because the differing rescuing transgenes were inserted at identical positions on the third chromosome , this approach ensured the pairwise comparison of strains that were otherwise as genetically similar to one another as possible . The detailed genetic scheme for generation of the transgenic samples is described in S1 Fig . Climbing assays were performed as described previously [33] . Lifespan analysis was performed as previously reported [1 , 3 , 79] using let-7-C mutant flies that were generated as described above . Fifteen male flies ( 0–1 day old ) were transferred to each vial . Flies were transferred to fresh food every 3 days at which time dead flies were counted and removed . The survival curves were plotted using Microsoft Excel . Statistical analysis was performed with the Online Application for the Survival Analysis of lifespan assays ( OASIS ) [80] and the p values were calculated using the log-rank ( Mantel-cox ) test . The number of flies used for each experiment are noted in the figure legends , and also included along with the median and maximum lifespans of the tested strains in S3 Table . S4 Table indicates the p values for curves shown in one or more panels . The numbers of flies used for each experiment have been noted in the figure legends . Experiments usually included two independent controls: w1118 as well as a let-7-C mutant strain containing a fully rescuing transgene . The w1118 survival curve was generated with flies that had been back crossed five times . Immunofluorescence was performed as described previously [37 , 38] . Primary antibodies included rat anti-Chinmo [38] ( 1:500 ) , chicken anti-GFP ( Rockland Immunochemicals , 1: 4000 ) , rabbit anti-Woc [81] ( gift from Maurizio Gatti 1:1000 ) , rat anti-Elav ( DSHB , 1:250 ) and mouse anti-Dachshund ( DSHB , 1:100 ) . For quantitating Chinmo levels , pixel intensity of 30 individual cells in single confocal sections of 5 independent dissected brains stained with anti-Chinmo and anti-Woc antibodies were quantified using ImageJ software . The expression of Chinmo was normalized to the pixel intensity of Woc and the average pixel intensity of one Δlet-7-C confocal section showing the highest pixel intensity was designated as 100 Arbitrary Units ( AU ) . Samples whose staining was directly compared were prepared and imaged in parallel and under identical conditions . For c708a neuron counts , mushroom bodies were optically sectioned in 0 . 5 μm increments , and the total number of neurons was determined by manually counting the number of GFP-positive cells section by section , ensuring that cells present on consecutive sections were counted only once . Statistical analysis was performed and histograms generated using GraphPad Prism software . P values were calculated using a two-tailed paired t test . Values are presented as mean ± SEM . All images were collected on a Leica SP5 confocal microscope ( Light Microscopy Imaging Center , Indiana University , Bloomington IN ) . Confocal stacks were merged using Leica LSM software . Total RNA was extracted with Trizol and treated with DNAse I . The reverse transcription was performed as described previously [82] . For analysis of miRNA copy number in Fig 4A , Kc-167 cells were incubated with 20E ( 5×10−6 M ) at 250 C for 24h before being washed with fresh medium . Reverse transcription ( RT ) was carried out on 25ng of total RNA using the Reverse Transcription miRNA Taqman assays ( Applied Biosystem , Foster City , CA ) specific for the miRNA ( dme-let-7 and dme-miR-125 ) . Each cDNA sample was diluted 1:25 and real-time quantitative PCR ( qPCR ) was performed in duplicate using miRNA-specific primers/probe on a StepOnePlus Real Time PCR System ( Applied Biosystem , Foster City , CA ) . For determination of copy number , we generated a standard curve for let-7 and miR-125 using a synthetic let-7 and miR-125 HPLC-purified RNA oligonucleotides synthesized by Integrated DNA Technologies ( Coralville , IA ) corresponding to the 22 nucleotide miR-125-5p ( 5’-rUrCrCrCrUrGrArGrArCrCrCrUrArArCrUrUrGrUrGrA-3’ ) and 21 nucleotide let-7-5p ( 5’- rUrGrArGrGrUrArGrUrArGrGrUrUrGrUrArUrArGrU-3’ ) . For fold change analysis , individual values were normalized to 2S rRNA for Taqman miRNA assays and kinesin levels for Sybr green assays . For qRT-PCR analysis , oligos 2515 , 2516 , 2599 , 2530 , 2728 , 2729 listed in S5 Table were used . Northern blot analysis was performed as described previously [37] . For histochemistry , heads were fixed for 3h in AAF buffer ( 10% Formaldehyde , 5% Acetic Acid , and 85% Ethanol ) . The fixed tissue was serially passaged through 70% ethanol , 95% ethanol , 100% ethanol , and twice in Xylene for 45 minutes each . Following these incubations , the tissues were embedded in paraffin followed by sectioning . The 7 μm tissue sections were mounted on superfrost-plus slides ( VWR International , Radnor PA ) and processed for hematoxylin-eosin staining . For scanning electron microscopy , adult flies were serially passaged through 25% ethanol ( 10h ) , 50% ethanol ( 2h ) , 75% ethanol ( 2h ) , 100% ethanol ( 2h ) , 50% ethanol: 50% hexamethyldisilazane ( HMDS ) ( 3minutes ) and 100% HMDS ( 3 minutes ) , coated with gold-palladium and viewed with a JEOL 5800LV SEM microscope . Drosophila Kc-167 cells were cultured in CCM3 at 23°C . Cells were transfected in 48-well plates with 25 ng of tub-Gal4 plasmid DNA , 25 ng UAS-miRNA plasmid DNA , and 25 ng of 3‘UTR-containing sensor plasmid DNA using Effectene ( Qiagen ) . Luciferase assays were performed using the Dual-Luciferase reporter system ( Promega Life Science , Madison WI ) . Transfections were performed in triplicates and resulting luciferase levels were averaged . Fold repression was calculated by dividing the ratio of Renilla luciferase and firefly luciferase in cells transfected with an empty pUAST attB plasmid with the ratio of Renilla luciferase and firefly luciferase in cells transfected with pUAST attB plasmid containing let-7-C cDNAs . Si-miRNA duplexes were synthesized as single-stranded RNAs by Integrated DNA Technologies ( Coralville , IA ) with HPLC purification , and resuspended in duplex buffer ( 100mM potassium acetate , 30mM HEPES , pH 7 . 5 ) to a concentration of 100μM . Annealing was performed by incubating 50 μM complementary single-stranded RNAs at 92°C for 2 min and leaving them for 30 min at room temperature [86] . si miR-125 sense; 5’UCCCUGAGACCCUAACUUGUGAUU si miR-125 antisense; 5’UCACAAGUUAGGGUCUCAGGGACU si let-7 sense; 5’UGAGGUAGUAGGUUGUAUAGUCU si let-7 antisense; 5’ACUAUACAACCUrArCrUrArCrCrUrCrArUrU The miRNA duplexes were transfected using Dharmafect duo according to manufacturer’s instructions ( GE Life Sciences , Lafayette CO ) . Briefly , 2 . 25 μl of mi-siRNA molecules ( diluted to 4 μM in duplex buffer ) was added such that the final concentration of each siRNA was 5 nM per well ( the volumes indicated are for biological triplicate ) in a 24 well plate . The cells were incubated at 250 C for 5 h before being washed with fresh medium . Quantitative real time PCR was performed to measure the relative levels of the miRNAs in total RNA extracted from transfected cells at different time points . The half lives of let-7 and miR-125 were determined by exponential regression curve fitting using GraphPad Prism version 6 software . Pri-let-7 , and pri-miR-125 were generated by annealing oligos cloned into pLitmus 28i . DNA templates for transcription were generated by PCR with the T7 and 2162 oligo and were transcribed and labelled with 32UTP ( Perkin Elmer , Waltham MA ) using the T7 Megashortscript Kit ( ThermoFisher , Cambridge MA ) . The transcript was purified by running the DNAse I treated reaction on a 4% denaturing PAGE gel and the gel piece corresponding to the labeled transcript was excised from the gel and eluted in a Eppendorf Thermomixer set at 400rpm and 37°C in a buffer containing 0 . 3M Sodium acetate , 0 . 2% Sodium dodecyl sulphate , and 1mM EDTA . The supernatant was precipitated in Ethanol . The precipitated RNA was refolded by heating at 95°C for 2 minutes followed by 37°C for 1 hour . A typical 25μL reaction contained 15μL of the Flag-Drosha-Pasha beads immunoprecipitate , 6 . 4mM MgCl2 , 1 U/μL of Ribonuclease Inhibitor ( ThermoFisher , Cambridge MA ) , and the refolded labeled transcripts ( 0 . 5 × 105 cpm ) . The reaction mixture was incubated at 26°C for 30 to 90 min , and RNA was extracted by phenol followed by ethanol precipitation and analyzed on a 10% denaturing polyacrylamide gel . In vitro dicing assays were typically carried out in 25μl lysis buffer , containing 5% ( v/v ) glycerol , 1 mM DTT , 0 . 1unit μl−1 RNasin Plus RNase Inhibitor ( ThermoFisher , Cambridge MA ) , 1 nM 5′-radiolabeled substrate RNAs ( GE Life Sciences , Lafayette CO; sequences listed below ) and 25 nM Flag-tagged Dicer proteins . The reaction products were resolved by electrophoresis on 10% denaturing Page gel , detected by Typhoon phosphorimager and quantified by ImageQuant software ( GE Life Sciences , Lafayette CO ) .
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Deregulation of mRNAs that are targeted by multiple miRNAs is a common feature of a number of diseased states including neurodegenerative disorders . The currently accepted model is that the combined action of all binding miRNAs ensures target repression . Here , we show that two co-expressed miRNAs exert distinct outcomes on a common target . While miR-125 extends lifespan by repressing its target , chinmo , in adult brains , let-7 downregulates Chinmo in developing animals . Our results indicate that differential processing and turnover rates of let-7 and miR-125 contribute to this switch in miRNA activity . This study has identified the physiological relevance of the targeting of a single mRNA by multiple miRNAs in a scenario where each miRNA exerts a distinct and non-overlapping outcome .
|
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"Methods"
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2016
|
A let-7-to-miR-125 MicroRNA Switch Regulates Neuronal Integrity and Lifespan in Drosophila
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Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained . Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation . We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells ( AEC ) . This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury . Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure . Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation , such as the WNT and transforming growth factor beta ( TGFB ) pathways , and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha ( HNF4A ) , and the retinoid X receptor ( RXR ) signaling pathways . Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation . Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process , demonstrating the power of this approach . Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation .
Over the past two decades the relationship between gene expression and chromatin structure has been increasingly recognized [1]–[4] . Elucidation of the histone code and subsequent insights into the functional implications of post-translational modifications of histone tails have begun to provide a mechanistic understanding of the role that chromatin context plays in gene expression . One of the most widely studied histone marks of active gene transcription is acetylation of lysine residues in the N-terminal tail of histone H3 . Acetylation of Lysines 9 and 14 ( H3K9/14Ac ) , found at promoters and enhancers of actively transcribed genes [5]–[7] , serves as a docking point for chromatin remodeling complexes that open chromatin , facilitating transcriptional activation [8]–[10] . In contrast , trimethylation of lysine 27 of histone H3 ( H3K27me3 ) confers repression through binding of the polycomb repressive complex ( PRC1/2 ) and chromatin compaction [11]–[13] . The H3K9/14Ac and H3K27me3 marks usually occur in distinct cell-type specific genomic regions . Many studies have examined the differentiation of stem cells into a variety of differentiated cell types , in processes that traverse large phenotypic ( and presumably epigenetic ) distances [14] . However , using stem cells to dissect the mechanism ( s ) by which the differentiated epigenotype is reached may be challenging due to the distant relationship between the starting and resulting cell populations . In contrast , examining epigenetic differences between two closely related yet phenotypically distinct cell types might offer more straightforward insights into the relationship between epigenetic changes and the establishment of new expression patterns . Isolated distal lung epithelial cells offer a compelling model system; primary human alveolar epithelial type 2 ( AT2 ) cells can be purified in large numbers from remnant transplant lung , and can be differentiated in vitro in a manner closely mimicking both normal maintenance and regeneration following lung injury [15] , [16] . The distal lung alveolar epithelium consists of two major cell types: cuboidal surfactant-producing AT2 cells and elongated type 1 ( AT1 ) cells that facilitate gas exchange . AT2 cells are the implicated precursors of AT1 cells , and through differentiation restore function of damaged distal lung epithelium [17] , [18] . This phenotypic transition can be recapitulated in vitro when purified primary AT2 cells are plated under defined cell culture conditions [19] , [20] . Previous studies using purified rat AT2 cells showed that AT2 cell-specific markers , such as surfactant protein C and A ( SFTPC , SFTPA1 ) , decrease with time in culture , while AT1 cell markers , such as aquaporin 5 ( AQP5 [21] , [22] ) , caveolin 1 ( CAV1 , [23] , [24] ) and podoplanin ( PDPN , [25] , [26] ) increase over time [27] . These expression changes are accompanied by profound changes in cellular morphology and function [28] . Recent advances have allowed for the isolation of human AT2 cells from remnant human transplant lungs [15] , [29] . Differentiation of purified primary human AT2 cells into AT1-like cells represents a unique kinetic model system to study the process of epithelial cell differentiation without the caveats of cell line immortalization or mixed tissue analysis . Here , we simultaneously profiled the transcriptomic and epigenomic changes of differentiating human alveolar epithelial cells ( AEC ) . Detailed integrated analysis facilitated identification of regulatory networks and participating transcription factors , pointing to roles for known and novel signaling pathways in distal lung epithelial differentiation . To our knowledge this is the first integrated analysis of human primary epithelial cell differentiation .
AT2 cells were isolated from the lungs of three non-smoker donors ( Figure S1A ) and plated on collagen-coated polytetrafluoroethylene membranes . Purity was verified by immunostaining for AT2 cell-specific markers pro-SFTPC and transcription factor NK2 homeobox 1 ( NKX2-1 ) , as well as hematopoietic marker protein tyrosine phosphatase , receptor type , C ( PTPRC , previously CD45 ) and mesenchymal marker vimentin ( VIM ) to check for contaminating cell types . Pro-SFTPC-positive cells averaged 86% purity ( Figure S1A–1B ) . Over the course of 8 days the cells underwent differentiation into AT1-like cells , forming confluent monolayers with tight junctions expressing tight junction protein 1 ( TJP1 , previously ZO-1 ) and showing downregulation of SFTPC , upregulation of AQP5 , and establishment of transepithelial resistance [27] , [30] , [31] ( Figure S1C–1E ) . Raw expression data across all samples had similar distributions ( Figure S2 ) , GEO accession #GSE38571 . Thus , all samples , including a technical duplicate each of day 0 ( D0 ) and day 4 ( D4 ) , were included in normalization ( Figure S3 ) . The relationship between gene expression profiles was examined by unsupervised hierarchical clustering using the top 5% of genes most variant across the dataset . Samples from different lungs clustered together based largely on the timing of differentiation , with the D0 and D2 samples each grouping together ( Figure 1A ) . Importantly , the sample dendrogram indicates the major branch point is between D2 and D4 , which has been observed previously as the point in time when the largest shift from AT2 to AT1 phenotype occurs in gene expression and morphology [32] . Clustering did not change using alternate cutoffs in the number of genes ( top 2% , top 10% ) ( Figure S4 ) . A two-dimensional principal component analysis plot showed that time in culture corresponded to PC1 ( 46% of total variation in the samples ) ( Figure 1B ) , suggesting that the differentiation process contributed most to inter-sample variation . A linear model was fit over the 5 time points ( D0 , D2 , D4 , D6 , and D8 ) excluding the technical duplicates and a moderated t-test was used to determine significance of changes in gene expression from D0 ( AT2 cells ) to D8 ( AT1-like cells ) . 6755 probes ( 5651 genes ) showed statistically significant changes in gene expression; 4196 upregulated probes ( 3500 genes ) and 2559 downregulated probes ( 2171 genes ) ( Figure 1C ) . These changes were distributed throughout the genome ( Figure 1D ) . qRT-PCR of the top 10 up- and down-regulated genes showed a high degree of correlation with the microarray expression results ( Figure 1E , Figure S5 ) . Genes known to become activated during AT2 to AT1 cell differentiation ( PDPN , CAV1 , and AQP5 ) were also assessed using qRT-PCR and values were plotted for comparison alongside the top differentially expressed genes ( Figure 1E , red triangles ) . Ingenuity Pathways Analysis ( IPA ) revealed that the top upregulated pathways were cell assembly and organization , cell movement , cell-cell signaling , tissue development , and lipid metabolism , all of which are consistent with AT2 cells differentiating into larger , flat AT1 cells , while the most downregulated pathways pertained to cell proliferation , cell death , inflammatory response , cell cycle and infectious processes ( Figure 1F ) . Signaling pathways in the upregulated networks included those of v-akt murine thymoma viral oncogene homolog ( AKT ) , protein kinase C ( PRKC ) , and the RAS family RAB genes , implicated in endocytosis , while FBJ murine osteosarcoma viral gene homolog ( FOS ) was the center of interconnectivity in the top downregulated network ( Figure S6 ) . Analysis using another pathway prediction program DAVID yielded results consistent with IPA ( Figure S7 ) . Differentiation of AT2 cells into AT1 cells has been extensively studied in the rat [33] , [19] . To compare differentiation between rat AEC ( rAEC ) and human AEC ( hAEC ) , purified rat AT2 cells were cultured under differentiation-permissive conditions and RNA was subjected to whole genome profiling . Technical variation within raw data was minimal ( Figure S8 ) . Data was preprocessed and clustered similarly to the human expression arrays ( Figure S9 ) . Purified AT2 cells clustered separately from AEC differentiating toward the AT1 cell phenotype ( Figure 2A , Figure S10 ) . We observed 4860 significantly changing probes corresponding to 4799 genes , with 2835 probes ( 2793 genes ) upregulated and 1983 probes ( 1964 genes ) downregulated ( Figure 2B ) . For comparison , human and rat expression arrays were subset to include probes represented on both arrays; 13173 genes were represented in both species . This resulted in 3973 and 3662 statistically significant gene expression alterations in rat and human AEC respectively . Of these statistically significant gene sets derived separately from both species , 1514 genes were significantly differentially expressed during both human and rat AT2 to AT1 differentiation ( Figure 2C–2D , p-value <2 . 2×10−16 ) . Separating significantly up- or downregulated genes yielded similar degrees of overlap ( Figure 2D ) . Therefore , differentiation of AT2 into AT1-like cells involves coordinated changes in thousands of genes , many of which occur in both human and rat . IPA analysis of genes concordantly changing in rat and human AT2 cell differentiation identified many similar altered pathways in both DAVID and IPA results ( Figure 1E , Figure S11A ) . The most significant network from this joint analysis centered on genes involved in lipid metabolism ( Figure S11B ) . The non-overlapping subsets of rAEC and hAEC genes were also of interest because they could indicate potential interspecies variation . While cell cycle control networks featured more prominently in the rat IPA networks , possibly reflecting species-specific differences , IPA of the hAEC and rAEC-specific gene sets revealed that 3 of the top 5 molecular signaling processes were identical ( Figure S12 , Figure S13 ) , suggesting that different genes in a similar pathway were modulated in the two species to achieve similar effects . The top signaling network in hAEC-specific genes , cell growth and proliferation , centered on HNF4A and was the third most significant pathway in rAEC-specific alterations . TGFB featured prominently in rAEC-specific and in overall human IPA analysis . Using subsets of the same batches of human AT2 cells ( D0 ) and fully differentiated AT1-like cells ( D8 ) used for expression analyses , we assessed alterations to the chromatin environment by chromatin immunoprecipitation followed by sequencing ( ChIP-seq ) . Specifically , H3K9/14Ac was used to interrogate active promoter and enhancer elements , and H3K27me3 was used to assess the repressed chromatin state . Peaks were called using both the Spatial clustering approach for the Identification of ChIP Enriched Regions ( SICER ) and the Model-based Analysis of ChIP-Seq ( MACS ) methods . Each analysis revealed thousands of enriched regions , with a significant fraction ( 20–40% ) shared between D0 and D8 and the remainder of them specific to D0 or D8 ( Figure S14 ) . Changes in chromatin were distributed throughout the genome ( Figure 3A ) . The transcription factor binding site ( TFBS ) predictor program Hypergeometric Optimization of Motif EnRichment ( HOMER ) was used to identify conserved sequence motifs enriched within day-specific chromatin marks [34] . For each of the 135 TFBSs in HOMER , the strongest association with either D0 or D8 H3K9/14Ac from SICER-called peaks was determined , and likewise for D0 and D8 H3K27me3 ( Table S1 ) . We then plotted the strongest H3K9/14Ac significance level versus the strongest H3K27me3 significance level for all TFBSs ( with negative significance values representing D0 and positive D8 ) ( Figure 3B ) , revealing a striking correlation – those motifs associated with regions losing H3K9/14Ac from D0 to D8 were consistently associated with regions gaining H3K27me3 from D0 to D8 . Conversely , motifs associated with regions gaining H3K9/14Ac were consistently associated with regions losing H3K27me3 . While this basic trend was expected based on the antagonism between these two marks , the identification of so many transcription factors apparently involved in regulation of both these marks was surprising . Some of the stronger associations with activating chromatin changes included motifs for zinc finger protein 711 ( ZNF711 ) , transcription factor 3 ( TCF3 or E2A ) , TCF4 ( a WNT signaling target ) , and RXR . TFBSs strongly associated with repressive chromatin changes included those for forkhead box ( FOX ) proteins FOXA1 and FOXA2 , TATA-box binding protein TBP , and CCAAT/enhancer-binding protein CEBP . Two example loci illustrate the location of particular motifs within differentially marked chromatin regions – an upregulated gene , frizzled family receptor 2 ( FZD2 ) , which shows spreading acetylation and loss of H3K27me3 , is predicted to have an RXR site within activating chromatin marks ( Figure 3C ) , while a downregulated gene , progastricin ( PGC ) , shows loss of acetylation but no gain of H3K27me3 and is predicted to have numerous FOXA1 sites within silenced regions ( Figure 3D ) . These two examples illustrate that different combinations of marks might be found on activated and repressed genes . To further investigate this , we examined the association between transcriptomic and epigenetic changes by analyzing the relationship between changes in gene expression and all possible combinations of H3K9/14Ac and H3K27me3 from D0 to D8 . Activating chromatin changes were strongly associated with upregulated gene expression , while repressive chromatin changes were loosely associated with downregulated gene expression ( Figure 4A ) . 3011 ( 53% ) of the genes showing altered expression were associated with at least one mark , and the largest single category was that of genes showing only acetylation at D0 ( Figure 4B ) . Since we sampled just two of several dozen known histone tail post-translational modifications , the statistically significantly activated or repressed genes that were not associated with either of these marks may be regulated by epigenetic events and chromatin marks not evaluated in the current study . We performed IPA analysis on sets of genes associated with genomic regions carrying combinations of active or repressive chromatin marks that were associated with positive and negative expression changes respectively ( Figure 4C ) . We simultaneously performed TFBS motif analysis on those genomic regions . For each class , we investigated the most significant TFBS matches , which were then matched to a transcription factor identified in the corresponding IPA gene networks enriched in the same combined chromatin/expression class ( Figure 4C–D ) . Upregulated pathways included TGFB , WNT , HNF4A , RXR , and AKT ( Figure 4E ) , while downregulated pathways included CEBP , RAS-RAF , and tumor necrosis factor ( TNF ) as well as FOXA1 and FOXA2 ( Figure 4F ) . This three-way integration of whole-genome gene expression data , chromatin data , and TFBS identified several pathways already implicated in AEC differentiation , such as the WNT and TGFB signaling pathways [35] , [36] , along with transcription factors previously implicated in lung development such as FOXA1 and FOXA2 [7] , [36] . Data integration allowed us to distinguish those genes ( and regulatory sequences ) likely to be direct transcriptional targets for each pathway , such as PGC , encoding an AT2-specific protease [37] as a potential direct target of FOXA1 ( Figure 3D ) . Our analysis identified several pathways not previously implicated in lung regeneration , such as RXR , HNF4A , and TNF . The chromatin data enabled us to focus on those genes likely to be directly targeted by these novel pathways , such as FZD2 , encoding a WNT receptor and candidate target of RXR ( Figure 3C ) . As noted , one signaling pathway previously reported to be involved in AEC differentiation [35] , [38] , [39] and confirmed through our integrated analysis is the WNT signaling pathway . Specific examination of the microarray data for genes in the WNT signaling pathway showed altered expression of many genes , verified through qRT-PCR ( Figure S15 ) and enrichment of the WNT signaling target transcription factor TCF4 in activated chromatin regions ( Figure 3B , Figure 4E ) . Of the newly implicated pathways , we chose the RXR pathway for validation . Retinoid X receptors can homodimerize or heterodimerize with a large variety of proteins , including retinoic acid receptors ( RARs ) , thyroid hormone receptor ( THR ) , the vitamin D receptor ( VDR ) , farnesoid X receptor ( NR1H4 ) and other nuclear receptors ( NRs ) , as well as the family of peroxisome proliferator-activated receptors ( PPARs ) [40] . While retinoic acid and its receptor has been implicated in lung development and injury [41]–[48] the precise role of RXR remains to be clarified . Because of their many potential binding partners , the function of RXRs can be complex; certain NR heterodimers ( e . g . NR1H4:RXR , PPAR:RXR ) are permissive , responding to an RXR ligand ( “rexinoid” ) or the corresponding NR ligand , while other complexes are non-permissive , responding only to rexinoids in the presence of ligands for the NR partner ( such as RXR:RAR , RXR:VDR , RXR:THR ) [49] . Our integrated data suggested RXR-based activation of certain genes that lose the H3K27me3 mark ( Figure 4E ) . To test a functional role for RXR pathway activation in AEC differentiation , it was important to target these receptors specifically . Thus , freshly isolated rAT2 cells were plated in the presence or absence of UVI-3003 ( 7 . 5 µM ) , a selective RXR inhibitor that blocks transactivation by RXR agonists but has been previously demonstrated to have minimal binding to structurally similar RAR family members [50] . Treatment with UVI-3003 markedly reduced AT1 cell marker induction ( AQP5 ) while delaying downregulation of AT2 cell marker pro-SFTPC ( Figure 5A ) and significantly reducing transepithelial resistance ( p<0 . 0001 ) ( Figure 5B ) . Rescue of UVI-3003 treatment by drug removal on D4 of differentiation restored tight junctions . Analysis of the regulatory region of rat Aqp5 ( a well-established AT1-specific gene ) revealed 34 predicted PPARA:RXR heterodimer binding sites ( Figure 5C ) and an average of 9 . 6 predicted PPARA:RXR sites per kb across 4 kb upstream regions of the rat , mouse , and human AQP5 promoters . A similar phenomenon was seen with other AT1-specific genes ( Pdpn , Cav1 ) ( Figure 5C ) . Luciferase assays using the 4 . 3-kb upstream region of the rat Aqp5 gene transfected into mouse lung epithelial ( MLE-15 ) cells revealed that UVI-3003 inhibited ∼50% of Aqp5 promoter activity ( Figure 5D ) . ChIP assays of cultured rAEC revealed little detectable binding of RXR to the Aqp5 promoter at D0 , but a marked increase in precipitation of a site about 4 kb upstream of the transcription start site was seen at D8 , when the cells had achieved their AT1 cell-like phenotype ( Figure 5E ) . Taken together , these analyses support a function for RXR signaling in AEC differentiation , and illustrate the utility of an integrated transcriptomic/epigenomic approach to identify new pathways involved in differentiation .
AT1-like cells , differentiated in vitro from AT2 cells , exhibit many properties of AT1 cells in vivo , including morphology and expression of known phenotypic markers . Direct isolation of fragile AT1 cells from human lung is very challenging [51] , due in part to the fact that strong cell-specific markers remain to be identified . However , the ability to differentiate AT2 cells in vitro into AT1-like cells offers a tractable model system to study not only the transcriptomic and epigenomic differences between these two cell types , but also the kinetic mechanisms controlling epithelial cell differentiation . Transcriptomic analysis of differentiating primary human and rat AEC identified thousands of genes undergoing significantly altered expression , a large number of which overlap between the two species . ChIP-seq identified D0- and D8-specific acetylation ( H3K9/14Ac ) and methylation events ( H3K27me3 ) and allowed identification of corresponding TFBSs enriched within AT2- or AT1-specific chromatin patterns . Interestingly , almost all of the TFBS enriched within D8-specific H3K9/14Ac regions were also enriched within D0-specific H3K27me3 regions , and conversely for D0-specific H3K9/14Ac and D8-specific H3K27me3 . This near perfect concordance is unexpected since H3K27me3 is thought to represent only one of several silencing mechanisms active in development [3] . Integrated analysis showed that upregulation of gene expression was associated with individual or combined gain of H3K9/14Ac and loss of H3K27me3 , while downregulation was primarily associated with loss of H3K9/14Ac . Approximately half of the genes showing altered expression were not associated with either chromatin mark . This could be because marks were present but distant from the gene , or because other chromatin marks or regulatory mechanisms were involved in their up or downregulation . With application of ChIP-seq to the examination of other chromatin marks , remodeling complexes , and transcription factors , and the addition of information on nucleosome positioning , non-coding RNAs , microRNAs , chromatin conformation capture technologies , and DNA methylation , the current model system will lend itself well to a detailed understanding of the epigenomic basis for the differentiation of adult epithelial cells . This study demonstrates how expression datasets and chromatin mapping are a potent combination to obtain an integrated picture of signaling pathway activity , transcription factors and their genomic targets . Transcriptional profiling can identify altered gene expression and corresponding regulatory pathways , but identifying transcription factors is difficult without knowing which genomic regions are implicated; epigenomic profiling can pinpoint the specific genomic regions where transcription factors and other regulatory proteins are likely to bind . Our approach revealed known regulators , such as TGFB , WNT , FOXA1 and FOXA2 [52]–[55] , [7] as well as new potential regulators of AEC differentiation , including AKT , RXR and HNF4A . As proof-of-principle , we investigated whether RXR signaling was required for the differentiation process of adult alveolar epithelium . Use of an RXR-specific inhibitor delayed differentiation as measured by inhibition of AT1-specific AQP5 expression and delayed TER . A role for RXR in normal AQP5 expression in AT1 cells is further supported by the inhibition of the Aqp5 promoter by UVI-3003 , as well as the specific detection of RXR on the Aqp5 promoter in rAT1-like cells . Retinoic acid receptors ( RARs ) are one of the many potential binding partners with RXRs and numerous reports have implicated retinoic acid and/or RARs in lung development and injury [40]–[49] . Thus , it is possible that RXR effects are mediated through interactions with RAR . However , evaluation of stringently predicted RXR binding sites in the AQP5 promoter in human and rat shows that the presence of adjacent predicted RAR binding sites is rare or absent , while adjacent predicted VDR sites are more common , and predicted estrogen and glucocorticoid receptor sites also abut RXR binding site [56] . Given the multitude of interacting partners of RXRs ( over 19 described [40] ) , dissecting the mechanism of RXR action on the promoter of AQP5 and other genes will require a very detailed examination . While the molecular mechanisms of altered RXR signaling remain to be further defined , inhibition of RXR in vitro as well as ChIP data showing increased promoter occupancy in AT1-like cells on day 8 support its functional role and illustrate the utility of our system and the potential of epigenomic/transcriptomic data integration to reveal novel regulators of biologic processes . In summary , our analysis enabled identification of known and novel signaling pathways , gene regulatory networks and associated TFBS implicated in morphologic and phenotypic changes that occur during AEC differentiation . Full characterization of normal differentiation is critical to determine the precise mechanisms that are perturbed in disease . In the distal lung , this might shed light on the molecular basis of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis . The analysis presented here shows that purified human primary epithelial cells undergoing in vitro differentiation can serve as a powerful tool for the mechanistic investigation of normal and aberrant epithelial cell differentiation .
Remnant human transplant lungs were obtained in compliance with Institutional Review Board-approved protocols for the use of human source material in research ( HS-07-00660 ) and processed within 3 days of death . Rat AT2 cells were isolated in compliance with IACUC protocol #11360 . Human lung tissue was processed as previously described [29] with inclusion of anti-EpCAM conjugated beads to select for epithelial cells . Cells were plated in 50∶50 [DMEM High glucose media ( GIBCO 21063 ) : DMEM-F12 ( Sigma D6421 ) ] . Differentiation into AT1-like cells was verified by measuring SFTPC and AQP5 expression using RNA extracted with the Illustra TriplePrep Kit ( GE LifeSciences , Piscataway , NJ ) , and by measuring transepithelial resistance . Rat AT2 cells were isolated as previously described [57] , [19] . Total RNA , DNA , and protein were simultaneously isolated from AT2 cells ( D0 ) , intermediate cell phenotypes ( D2-6 ) and AT1-like cells ( D8 ) . Chromatin was isolated in tandem at D0 and D8 . Freshly isolated hAT2 cells were fixed with 4% paraformadehyde for 10 min at room temperature ( RT ) , permeabilized with 0 . 3% Triton , and blocked with CAS blocking reagent ( Invitrogen Cat #00-8020 , Camarillo , CA ) for 30 min at RT . Slides were incubated with rabbit anti-pro-SFTPC ( Seven Hills #WRAB-SPC serum ) or anti-PTPRC ( Santa Cruz sc-25590 ) antibodies and diluted in CAS-block at 4°C overnight . Slides were washed in Tris-Buffered Saline & Tween 20 ( TBST ) and incubated with anti-rabbit-FITC fluorescent secondary antibody in CAS-block for 1 hr at RT . For vimentin staining , mouse anti-VIM ( Sigma V2258 ) and biotinylated anti-mouse IgM ( Vector # BA-2020 ) antibodies were used . Sections were viewed with a NIKON Eclipse microscope equipped with a QImaging Retica 200R charge-coupled-device camera ( QImaging , Surrey , BC , Canada ) . Images were processed with the NIS-Elements BR program ( NIKON ) . 1 µg of RNA was converted into cRNA using Illumina TotalPrep RNA amplification kit , ( Life Technologies , USA ) and used for Illumina HT-12v4 or RatRef-12 expression analysis at the Southern California Genotyping Consortium , University of California Los Angeles . BeadStudio was used to convert images to raw signal data . Using R ( version 2 . 11 . 1 ) , Variant Stabilization and Normalization ( VSN ) was performed using LUMI [58] to allow for a large number of differentially expressed genes . Statistical analyses were performed using LIMMA [59] . A linear regression model was fitted over the time-course of differentiation , technical replicates were removed , and t-tests performed between D0 and D8 . False-discovery rate was controlled using the Benjamini-Hochberg ( BH ) correction [60] . R was used for principal component analysis and heatmap generation . Heatmaps were generated by selecting the top 5% of probes most variant across the whole dataset and clustering with Ward's method . Pathways analysis was performed using IPA ( Ingenuity Systems , www . ingenuity . com ) or DAVID [61] , [62] . Correlation of human and rat gene expression was performed using Entrez identifiers and the Mouse Genome Informatics ( MGI ) Web database [63] . RNA was reverse transcribed using random hexamers and M-MLV reverse transcriptase per manufacturer's guidelines ( Invitrogen ) , followed by qRT-PCR using SYBR green ( BioRad , Hercules , CA ) with primers listed in Table S2 . qRT-PCR reactions were performed using a DNA engine Opticon ( MJ Research , Waltham , MA ) and normalized to 18S rRNA . Chromatin immunoprecipitations for H3K9/14Ac ( Millipore , #06-599 ) , H3K27me3 ( Millipore , #07-449 ) , POL2 ( POLR2A , Santa Cruz Biotechnology , sc-899X ) , and RXR ( Santa Cruz Biotechnology , sc-774X ) were performed as previously described [64] . Primer sequences are noted in Table S3 . For rat ChIP at the Gapdh promoter , POL2 was used as a positive control . For human ChIP-seq , positive control loci were the promoter regions of GAPDH and MUC4 for H3K9/14Ac and H3K27me3 respectively; ≥10-fold enrichment was considered successful . ChIP-seq libraries were constructed using the New England Biolabs library prep kit ( NEB Cat#E6200 , Ipswich , MA ) . ChIP products underwent Illumina GAII single-end sequencing; reads were aligned to the hg18 human genome build using the MAQ 0 . 7 . 1 aligner . All datasets are deposited in the public GEO database ( GEO# GSE38571 ) . All datasets are deposited in the public GEO database ( GEO# GSE38571 ) . Integrative Genomics Viewer v1 . 5 ( The Broad Institute ) was used to visually inspect peak quality . SICER [65] peak calling was performed using a window and gap size of 600 bp . Input DNA was used for background normalization . The second peak-calling algorithm , MACS , ( http://cistrome . org/ap/ ) was performed using default parameters and input DNA for background normalization . SICER- and MACS-called peaks had a large degree of overlap , as measured by the correlation coefficient ( Figure S16A ) , calculated using Genome Graphs ( http:/genome . ucsc . edu ) . A lack of correlation was observed between H3K9/14Ac and H3K27me3 ( Figure S16B ) [66]–[70] . ChIP-seq reads from both AT2 and AT1 cells were observed at or near read saturation ( Figure S17 ) . Differential peak occupancy was determined using the UCSC table browser . Manhattan plots were generated using Genome Graphs . Peaks were annotated to the nearest transcription start site ( TSS ) and motif analysis was performed with HOMER [34] . The opposite AEC cell type was used as background . Gene expression data was merged with annotated chromatin peaks using Entrez ID . For the AQP5 TFBS analysis PPAR:RXR predicted binding sites were evaluated using P-match algorithms within ExPlain 3 . 0 across human , mouse and rat species . ExPlain motif V$PPARA_02 with a high probability ( >87% ) were counted and averaged in the region 4 . 3 kb upstream of the promoter for the three species . Similar results were obtained with the Match program in Biobase [56] , which was used to examine the frequency and nature of binding site adjacent to predicted RXR targets . 1×106 rat AT2 ( ≥95% purity ) cells were plated on 1 . 1-cm2 polycarbonate filters ( Corning Costar #3401 ) and treated with either 7 . 5 µM UVI-3003 ( Tocris Biosciences , Ellisville Missouri ) or DMSO vehicle control from the time of plating ( D0 ) through completion of the study . Whole cell lysates were extracted in 2% SDS lysis buffer ( 62 . 5 mM Tris pH 6 . 8 , 2% SDS , 10% glycerol , and protease inhibitor cocktail III ( Calbiochem #539134 ) ) . Western blots were performed as previously described [16] . Primary antibodies ( all rabbit ) were anti-AQP5 ( Alomone Labs AQP-005 ) , anti-CAV1 ( Abcam ab2910 ) , anti-pro-SFTPC ( Millipore AB3786 ) and anti-LAMIN A/C ( sc-20681 , Santa Cruz Biotechnology ) . Blots were analyzed by chemiluminescence and visualized by West Fempto Super Sensitivity Kit ( Thermo Scientific ) with a FluorChem 8900 Imaging System ( Alpha Innotech ) . Mouse lung epithelial cells ( MLE-15 ) were plated at a density of 4×104 cells per well on 24-well plates 1 . 5 days prior to transfection . Duplicate wells were transiently transfected with 500 ng rAqp5-luc containing the 4 . 3 kb rat Aqp5 promoter or pGL2 empty vector using Superfect ( Qiagen ) . Three hours post-transfection , media was changed to contain 7 . 5 µM UVI-3003 or DMSO vehicle control . 48 hours post transfection , luciferase assays were performed as previously described [33] . Raw luciferase values were normalized to protein concentration and then to pGL2 empty vector controls . Significance was measured using the student's t-test .
|
Understanding the role of epigenetic control of gene expression is critical to the full description of biological processes , such as development and regeneration . Herein we utilize the differentiation of cells from the distal lung to gain insight into the correlation between the epigenetic landscape , molecular signaling events , and eventual changes in transcription and phenotype . We found that by integrating epigenetic profiling with whole genome transcriptomic data we were able to determine which molecular signaling events were activated and repressed during adult alveolar epithelial cell differentiation , and we identified epigenetic changes that contributed to these changes . Furthermore , we validated the role of one of these predicted but not previously identified pathways , retinoid X receptor signaling , in this process .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"developmental",
"biology",
"genetics",
"epigenetics",
"biology",
"genomics",
"molecular",
"cell",
"biology"
] |
2013
|
Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation
|
The plant life cycle alternates between two distinct multi-cellular generations , the reduced gametophytes and the dominant sporophyte . Little is known about how generation-specific cell fate , differentiation , and development are controlled by the core regulators of the cell cycle . In Arabidopsis , RETINOBLASTOMA RELATED ( RBR ) , an evolutionarily ancient cell cycle regulator , controls cell proliferation , differentiation , and regulation of a subset of Polycomb Repressive Complex 2 ( PRC2 ) genes and METHYLTRANSFERASE 1 ( MET1 ) in the male and female gametophytes , as well as cell fate establishment in the male gametophyte . Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte , as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR . Maintenance of genome integrity also requires RBR , because diploid plants heterozygous for rbr ( rbr/RBR ) produce an abnormal portion of triploid offspring , likely due to gametic genome duplication . While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR , genetic analysis of tetraploid plants triplex for rbr ( rbr/rbr/rbr/RBR ) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development , trichome differentiation , and regulation of PRC2 subunit genes CURLY LEAF ( CLF ) and VERNALIZATION 2 ( VRN2 ) , and MET1 in leaves . There were , however , no obvious cell cycle and cell proliferation defects in these plant tissues , suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes . Conversely , in leaves of mutants in sporophytic PRC2 subunits , trichome differentiation was also affected and expression of RBR and MET1 was reduced , providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development . Together , dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate , differentiation , and development .
Independent evolution of multicellularity and thus the cell types has implications for adaptation of distinct developmental strategies in plants and animals [1] . Adaptive mechanisms unique to higher plants include alternation between the reduced gametophytic and dominant sporophytic generations , absence of a distinct germ line , and continuous postembryonic development . Unlike animals that develop a germline early in development , the progenitors of gametophytic cell types are derived from sporophytic cells of a mature plant , which acquire competence to undergo meiosis and subsequent mitotic divisions and to establish cell fates of gametic and accessory cell types [2] , [3] . Further , double fertilization of gametes leads to the development of an embryo and endosperm . Upon germination , the mature embryo develops into an adult plant by recurrent morphogenetic patterning . Therefore , plant cells must have a flexible but coordinated molecular machinery that helps to maintain their state of competence for cell fate determination and differentiation of distinct cell types during their developmental ontogeny [4]–[6] . In particular , dynamic control of cell fate and differentiation in plants is achieved by regulators of the cell cycle and chromatin complexes in distinct developmental stages , unlike stable gene repression by the same type of regulators during animal development [7] , [8] . The tumour suppressor Retinoblastoma ( pRB ) and closely related proteins are primarily known as negative regulators of the cell cycle and for their antiproliferative activity in multicellular organisms [9] , [10] . Specifically , pRB forms a repressive complex with E2F transcription factors to control cell cycle progression from G1 into S phase . Less is known how the pRB pathway functions beyond cell cycle , whether in coordinating cell proliferation and differentiation , or to control early cell fate establishment until late developmental processes [9] , [11] . In recent years , pRB homologues have been shown to be necessary in the control of cellular differentiation , stem cell maintenance , and apoptosis in diverse model systems [9] , [12] , [13] including Arabidopsis [14]–[17] . Evolutionary homologues of pRB , either alone or in cooperation with chromatin-associated regulators , can regulate genes involved in cell fate determination and differentiation [9] , [13] , [18] , suggesting a central role of this protein in early cell fate control , as well as subsequent maintenance of the differentiated state and genome integrity [9] , [12] . In Arabidopsis , RETINOBLASTOMA RELATED ( RBR ) is the single homologue of pRB , and the pRB-E2F pathway is largely conserved [7] , [9] . Unlike the mouse embryo-lethal pRB knockouts , Arabidopsis knock-out alleles of RBR are defective in both female and male gametogenesis [14] , [15] , constraining functional dissection of the pre- and post-gametophytic role of RBR in development . Studies that down-regulated RBR in distinct tissues using RBR RNA interference , virus induced gene silencing or by mis-expression of a RBR-binding viral protein to compete with the native RBR , have not elucidated the genetic behaviour of a rbr null mutation during gametophyte or sporophyte development [16] , [17] , [19]–[21] . In addition , it was unclear in these experiments if both RBR mRNA and protein levels were stably reduced throughout development , or aberrantly elevated due to the auto-regulatory function of the pRB-E2F pathway [22] . Nonetheless , these studies have provided an early indication that the RBR pathway functions distinctly in different cell types to prevent cell division , endoreduplication and stem cell maintenance . Recent work demonstrated that RBR genetically interacts with the conserved epigenetic regulators of the Polycomb Repressive Complex 2 ( PRC2 ) to control development of both male and female gametophytes [15] , and that RBR control of cell fate in the male gametophyte is at least partly coupled to its genetic interaction with the cell cycle associated pollen-specific CYCLIN-DEPENDENT KINASE A1 ( CDK A1 ) [17] . Unlike in the sporophytic leaf , RBR is repressed by a maternal and paternal PRC2 complexes during plant reproduction [15] , suggesting that the RBR regulatory network can function differently depending on the developmental context . Together , the developmental role of RBR during sporophytic development remains poorly understood , primarily due to the lack of genetic tools . In this study , we investigated the effects of an Arabidopsis RBR knock-out allele [14] , [15] on the plant life cycle . Detailed analysis of rbr female gametophytes supported the role of RBR in gametophytic cell fate control . Further , we performed a tetraploid genetic analysis that provided direct evidence that at reduced levels of RBR sporophyte development is perturbed . When only one out of four functional RBR alleles was present in tetraploids triplex for rbr ( rbr/rbr/rbr/RBR ) , specific stages of sporophytic differentiation and development were affected . The function of RBR is therefore partially haplo-insufficient during sporophytic plant development , as revealed by RBR dosage analysis in tetraploid plants . Furthermore , we provide genetic evidence that RBR functions in concert with the sporophytic PRC2 subunits to control developmental processes in the sporophyte . In short , our work not only illustrates the coordinated function of the RBR pathway in both gametophytes and the sporophyte , it also demonstrates how tetraploid genetics can be exploited to uncover a novel developmental role of an essential regulator during the entire plant life cycle .
In Arabidopsis , the fully differentiated female gametophyte ( embryo sac ) consists of only four cell types of clonal origin [3]: a haploid egg cell , a homo-diploid central cell derived from the fusion of two haploid polar nuclei , two synergids that facilitate entry of sperm cells into the embryo sac ( Figure 1A ) , and three antipodal cells that undergo early apoptosis . By characterizing one of the RBR knock-out alleles , rbr-3 [14] , we could identify that loss of RBR function did not affect the mitotic divisions and cellularization in the female gametophyte [15] . In the majority of cases , however , all cell types including the central cell with unfused polar nuclei commenced proliferation in this mutant ( Figure 1B and 1C ) . The morphological identity of the proliferating rbr-3 cell types was previously assigned based on their positional information within the embryo sac; however , their molecular identity remained questionable . Therefore , we examined the fate of specific cell types in the absence of RBR using cell type-specific molecular markers that are characteristic for the three cell types of the mature female gametophyte . The marker lines ET1119 , ET2634 , and ET956 express β-glucuronidase ( GUS ) in the egg cell , synergid cells , and the central cell , respectively [23] , [24] ( Figure 1D , 1H , 1L ) . In most proliferating rbr embryo sacs we could not detect GUS expression in the egg , synergid , and central cell ( Figure 1E , 1G , 1I , 1K , 1M , 1N ) . In 3–8% of the cells , cell type-specific markers showed ectopic expression that deviated from their wild-type pattern ( Figure 1F , 1G , 1J , 1K; Figure S1 ) . These findings were further substantiated by loss of gene expression in rbr embryo sacs for central cell-specific FERTILIZATION INDEPENDENT SEED2 ( FIS2 ) [15] , [25] and for two additional unpublished egg cell-specific genes ( A . J . Johnston , H . Bäumlein , T . Dresselhaus , U . Grossniklaus and W . Gruissem , data not shown ) . Therefore , RBR is required for the identity establishment of these gametophytic cell types . In the rare cases where these markers were still present , possibly due to some RBR activity carried over from the rbr/RBR heterozygous megaspore mother cell , they were mis-expressed in the spatial domains of other cell types ( Figure 1F and 1J; Figure S1B , S1C , S1D , S1F ) . For instance , an egg cell marker and a synergid marker were expressed in the central cell and egg cell domains , respectively , in the absence of RBR . Therefore , RBR not only promotes cell differentiation but also seems to coordinate certain positional information in the female gametophyte . In a previous work we have shown that the lack of cell differentiation in rbr gametophytes paralleled deregulation of certain PRC2 genes and MET1 [15] , whose functional orthologues were known for their role in cell specification , differentiation and also cell cycle regulation in diverse animal systems [26]–[29] . Both our present work and a recent report [25] have established that cell fate , cell cycle and ploidy are also impaired in certain RBR-deficient female and male gametophytic cells . There is evidence that RBR directly interacts with MULTICOPYSUPPRESSOR OF IRA1 ( MSI1 ) and FERTILIZATION INDEPENDENT ENDOSPERM ( FIE ) proteins , which are members of distinct PRC2 complexes in plants [25] , [30] , [31] . This is consistent with the findings that central cells in rbr , msi1 and fie mutant female gametophytes aberrantly proliferate and they are defective either in acquiring cellular identity and/or in heterochromatin status [15] , [25] . The phenotypes in rbr mutant gametophytes can be partly attributed to the derepression of MET1 [15] , [25] , which in turn might result in aberrant hypermethylation , heterochromatin maintenance and/or histone turn over . Interestingly , some of these maternal mutant phenotypes including the defective central cell fate in rbr and msi1 could be rescued by suppressing MET1 and associated global methylation , suggesting a complex epigenetic control of development [25] , [32] . Taken together , it is possible that the RBR-PRC2-MET1 network controls cell fate determination either independently , by co-regulating cell cycle activity , and/or by forming a repressive chromatin modifying complex both in male and female gametophytes . Evolutionary homologues of pRB in animal systems have been implicated in the control of ploidy and chromosomal stability [11] . For instance , pRB-deficient tumors are reported to have elevated aberrant ploidy levels , most likely due to the deregulation of mitotic cell cycle [33] , [34] . In Arabidopsis , impairment of the RBR-E2F pathway by ectopic expression of the viral RepA protein [19] increased the endocycles in leaf cells . Similar results were obtained when the RBR pathway was perturbed by over-expressing a D3-type cyclin [35] , [36] or E2F/DP transcription factors [7] . In all these cases , however , it remained unclear if the ploidy changes were the primary effect of loss or reduction of RBR function . Therefore , we investigated if reduced or loss of RBR function in a genetically tractable rbr knock-out allele would change developmentally controlled ploidy . Analysis of cellular ploidy in RBR-deficient female gametophytic cell types is difficult due to the problems in isolating these miniature cells from plants that are heterozygous for rbr . During the morphological analysis of diploid rbr/RBR plants using Nomarski optics [15] , we noticed that in many instances rbr gametophytic nuclei and , in particular , proliferating nuclei in the central cell region were of unusual size . Therefore , we analysed the ploidy of these nuclei by confocal microscopy and subsequent 3D reconstruction of acquired image stacks . We noticed that several rbr supernumerary nuclei derived from the unfused polar nuclei had a diploid rather than haploid chromosome number ( Figure 1B; 14 observations ) . This might be due to endoreduplication events in the absence of RBR activity [19] , as illustrated by an egg-like cell in the inset of Figure 1C as well , where a large excess of metaphase chromosomes was observed . Given that wild-type polar nuclei are haploid [3] and that rbr polar nuclei do not fuse to form a homo-diploid central cell [15] , it is likely that RBR restricts not only ectopic divisions but also polyploidization of haploid polar nuclei . Thus , confocal analysis of the ovules allowed us to demonstrate that absence of RBR leads to events of elevated cellular ploidy in the female gametophyte . Since RBR seems to control ploidy of the female gamete ( s ) , this led us to investigate potential changes in plant genome ploidy in rbr gamete ( s ) -derived progeny . We had previously shown that a selfed diploid rbr/RBR plant ( also referred to as rbr mutant ) produced viable progeny segregating for wild-type RBR/RBR and mutant rbr/RBR genotypes , while the female gametophytically lethal rbr allele was not transmitted to the next generation [14] . Therefore , the observed rare polyploid egg cells ( Figure 1C ) might not produce an offspring . If the rbr mutant produced viable male gametes with altered ploidy , we would expect that the ploidy of a subset of rbr progeny would be different from the parent plant . Indeed , we found that selfed diploid rbr/RBR plants produced 6% triploids among rbr mutant offspring ( n = 56 ) , which produced an array of aneuploid , diploid , and tetraploid plants in the next generation ( Figure S2 ) . This phenomenon is normally not observed in diploid wild-type Arabidopsis . It is most likely that these triploid progeny resulted from the fusion of a haploid RBR egg cell with either a diploid rbr sperm cell or two haploid rbr sperm cells [14] . Unfortunately , we are unable to test these hypotheses in detail because ( i ) the chance occurrence of these events was estimated to be 0 . 1×0 . 06 = 0 . 006 considering the rbr transmission efficiency of 0 . 1 and the presence of 6% triploids among the transmitted mutant progeny , and ( ii ) rbr knock-out male gametophytes rarely formed sperm cells [15] , [17] . Together , our results suggest that RBR controls ploidy maintenance in the gametophytic cells and that it is involved in maintaining genome integrity because in its absence or down-regulation polyploid offspring are produced . Genome-wide polyploidization has played essential role in speciation and thus evolution of plants [37] , however , the factors leading to increased ploidy in plants are not completely understood [38] , [39] . Plant autopolyploidization can be preceded by changes in ploidy either somatically , during meiosis , or during male or female gametogenesis . In case of meiosis , asynaptic mutations and meiotic restitution might lead to formation of unreduced gametes and therefore autopolyploids [40] . Thus far , three Arabidopsis meiotic mutants , dyad [41] , mitosis instead of meiosis ( mime ) [42] and jason [43] , were reported to produce unreduced diploid instead of normal haploid gametophytes . Ploidy alterations in female gametogenesis is partly controlled by the maize INDETERMINATE GAMETOPHYTE 1 ( IG1 ) which encodes for a gene with high similarity to ASYMMETRIC LEAVES2 ( AS2 ) in Arabidopsis [44] . The rbr mutation we report here is the first case in Arabidopsis in which aberrations in gametogenesis could result in triploid offspring due to doubling of haploid gametic genome . A “triploid bridge” leading to production of diploids , aneuploids and tetraploids may act as a transition between diploids and autotetraploids and therefore could play a significant role in polyploidization [39] , [45] . An induction of triploid offspring as observed in rbr knock-out mutants may also occur in the wild-type , should RBR activity be altered by unknown environmental factors . Thus , RBR might have played a crucial role in plant evolution by controlling genome duplication events . The roles we propose for wild-type RBR in female gametophytic cell specification and differentiation as well as in maintaining genome integrity are only valid if the rbr-3 allele is gametophytically recessive and genetic reduction of RBR function had caused the observed effects . Since rbr-3 carries a T-DNA insertion in the middle of the RBR gene [14] , it might generate a truncated protein with a dominant effect . In order to understand the genetic behaviour of rbr in the gametophyte , we subjected the gametophytically lethal rbr mutation to tetraploid genetic analysis . We asked if the rbr-3 mutation behaves recessive or dominant in diploid gametophytes produced by tetraploid plants by analysing seed set phenotypes and segregation of rbr genotypes in the progeny ( Figure S3; see Table 1 and the Materials and Methods section for details ) . An autonomously tetraploidized plant that was heterozygous for the rbr mutation ( Figure S2 ) was subjected to a detailed progeny test ( n = 103 ) , which identified three distinct rbr genotypes . Seed set and progeny segregation phenotypes of rbr simplex ( rbr/RBR/RBR/RBR ) , duplex ( rbr/rbr/RBR/RBR ) and triplex ( rbr/rbr/rbr/RBR ) mutant plants significantly fit the recessive model of rbr-3 inheritance ( χ2 test; p = 0 . 05 ) , implicating abortion of homozygous rbr/rbr gametophytes and rbr/RBR gametophytes giving viable seeds ( Table 1 ) . As an additional step to confirm this genetic model , we examined cytological phenotypes of female gametophytes ( FG ) in these plants . The majority of FGs in both diploid and tetraploid wild-type plants were at stage FG7 upon emasculation , which is typical for wild-type Arabidopsis [15] . We noted that all rbr embryo sacs in a heterozygous rbr/RBR diploid plant showed nuclei proliferation , which significantly fit the expected ratio ( χ2 = 0 . 20 , p = 0 . 65 , n = 194 ) as reported previously [14] , [15] ( Figure S3 ) . In case of the triplex rbr/rbr/rbr/RBR plant , three types of FG genotypes are expected: rbr/rbr , rbr/RBR and rare RBR/RBR . Given that the rbr mutation fits a recessive model of inheritance based on the tetraploid seed set phenotype and progeny test ( Table 1 ) , only those FGs that had completely lost RBR function ( rbr/rbr ) would be expected to show ectopic nuclear divisions , accounting for 54% proliferating FGs ( Figure S2 ) . The observed numbers of embryo sacs with supernumerary nuclei in the triplex plants matched the expectation for proliferation of rbr/rbr embryo sacs ( χ2 = 0 . 076 , p = 0 . 78 , n = 162 ) . These data suggest that the viable rbr/RBR female gametophytes are likely phenotypically and functionally identical to RBR/RBR FGs of wild-type tetraploids , and RBR FGs of wild-type diploids . Together , two independent genetic experiments of seed set and transmission analysis ( Table 1 ) and quantitative analysis of FGs ( Figure S3 ) confirmed that the rbr mutation behaves recessive in the female ( and male ) gametophyte ( s ) . Therefore , we can rule out a dominant ( negative or positive ) effect of a possible truncated version of RBR mRNA or of RBR protein . This situation is perhaps similar to previous reports that premature termination in mouse Rb exons resulted in truncated non-functional proteins [46] , [47] . Hence , we conclude that the rbr-3 allele is a clear loss-of-function mutation of RBR . The male and female gametophytic lethality of the rbr mutation constrains analysis of RBR function during sporophytic development . Tetraploid analysis is therefore an excellent approach to investigate a dosage dependent function of RBR in the sporophyte . We recovered and analysed rbr tetraploid plants with different numbers of rbr-3 alleles by large-scale genotyping and segregation analysis of the tetraploid mutant progeny ( see Figure 2 , Figure S2A and S2B , Table 1 ) . No homozygous tetraploid rbr quadruplex ( rbr/rbr/rbr/rbr ) genotypes could be identified , confirming the genetic data ( see previous section ) that they do not survive post-gametophytically . Specifically , we identified second generation rbr triplex plants ( rbr/rbr/rbr/RBR ) that showed significant quantitative reduction of RBR expression levels in leaves when compared to the wild-type tetraploids ( nulliplex RBR/RBR/RBR/RBR ) ( Figure 3C ) or heterozygous diploids ( data not shown ) . A subsequent independent expression analysis reconfirmed that the third generation rbr triplex plants also maintained significantly lower RBR expression levels in a RBR dosage-dependent manner ( Figure S4 ) . We expected that the rbr triplex plants , which displayed nearly 75% reduction in gene expression compared to the wild type ( Figure S4 ) , could reveal quantitative effects of RBR function more readily than diploid rbr/RBR plants . Wild-type RBR/RBR and heterozygous rbr/RBR plants did not differ in sporophytic phenotypes from germination until maturity , indicating full functionality of a single wild-type RBR copy ( haplo-sufficiency ) at the diploid level . Although rbr triplex plants appeared to grow normally during early sporophyte development ( Figure 2A versus Figure 2B ) , they showed several developmental phenotypes such as stunted growth habit , aberrant leaf size , altered phyllotaxy of siliques , reduced stem thickness , reduced apical dominance , and ectopic floral organs when they were about six weeks old ( Figure 2C–2N ) . In comparison , the growth and development of rbr nulliplex ( Figure 2C–2N ) , simplex and duplex rbr plants ( not shown ) were normal and indistinguishable at similar stages . Thus , the mutant sporophytic phenotypes only occur in rbr triplex plants , indicating that a single RBR copy cannot sustain normal growth and development and , therefore , RBR function is haplo-insufficient in the tetraploid context . While we anticipated that reduction of RBR would alter cell division and/or cell size as was reported from other systems [9] , [20] , organization and size of cells on the abaxial side of cauline leaves in the triplex mutant surprisingly did not deviate from the corresponding wild-type in young and mature leaves ( Figure S5 ) . As rbr gametophytes showed ploidy aberrations ( as discussed before ) , we anticipated that ploidy deregulation could also be observed in the leaf sporophyte with decreased RBR dosage . However , neither diploid ( rbr/RBR versus RBR/RBR ) ( data not shown ) nor tetraploid ( rbr/rbr/rbr/RBR versus RBR/RBR/RBR/RBR ) plants ( Figure S6 ) had significant changes in leaf ploidy when analyzed by flow cytometry . Our genetic results are in contrast to earlier reports that deregulation of RBR had immediate consequences on cell divisions and endocycles during leaf organogenesis [19] , [21] . We reason that retaining one functional RBR copy in diploid and tetraploid systems is sufficient to coordinate cell cycle and specification in the leaf sporophyte and that RBR reduction using a viral RBR-binding protein or virus-induced gene silencing may affect specific functions of RBR that are required for the control of DNA endoreduplication . Next we asked if differentiation of specific cell types was altered in response to RBR dosage change . We examined trichome differentiation patterns in young rosette leaves around 15 days after germination on plates , in diploid and tetraploid plants . As expected , development of trichomes in the 3rd and 4th rosette leaves of diploid rbr/RBR plants did not differ from the corresponding wild-type , confirming haplo-sufficiency of RBR in diploids ( Figure 3A ) . In wild-type tetraploid plants , over 53% of the trichomes had three branches , 34% had four branches and 12% with 5–6 branches ( Figure 3A ) . These data are consistent with the increased DNA content and supernumerary branching in tetraploids as previously reported [5] . Concomitant with a reduction of RBR dosage in rbr triplex plants , however , there was a significant reduction of 4-branched ( 11% ) and 5–6 branched trichomes ( 1% ) along with an increase of less-differentiated 3-branched trichomes ( 84% ) ( Figure 3A ) . In addition , we observed a similar trend in RBR dosage-dependent reduction of 4-branched trichomes in an independent experiment ( Figure S4 ) . Therefore , the single copy of RBR in rbr triplex is sufficient to specify the trichome cells ( Figure 2B versus Figure 2A ) but not sufficient to complete full differentiation of this specialized cell type . It has been proposed that key cell cycle genes that control ploidy restrict trichome branching [5] . Previous studies of down-regulating RBR in diploid leaves provided inconclusive results for ploidy-dependent leaf and trichome differentiation . For example , suppression of RBR in Brassica napus led to elevated ploidy levels in leaves and retarded leaf and trichome development [21] . In contrast , over-expression of a RBR-binding geminivirus RepA protein in diploid Arabidopsis in order to interfere with RBR function revealed only marginal elevation of ploidy levels in mature leaves and supernumerary trichome branching patterns [19] . It is unclear , however , if the RepA protein reduced the endogenous RBR levels in these plants , or if the transcription of RBR was aberrantly elevated due to the autoregulatory function of RBR-E2F pathway [22] . We therefore asked if in single cell trichomes lower RBR levels had caused a concomitant reduction in DNA ploidy , which could explain the fewer branches . By measuring the relative DNA content of individual trichome nuclei by fluorescence microscopy , we found that DNA ploidy in rbr triplex trichome cells was comparable to corresponding tetraploid wild type , and that there was no significant difference within ploidy groups across different genotypes ( Figure S6 ) . Thus , we conclude that cellular differentiation and morphogenesis of trichomes were affected in a RBR dosage-sensitive manner ( Figure 3A ) . Retaining 25% RBR in the triplex ( rbr/rbr/rbr/RBR ) plants does not alter the general leaf , trichome and plant ploidy , cell proliferation and trichome specification , but it appears to be insufficient to complete a full differentiation program . This could be particularly true for trichome differentiation , as is also suggested by a recent report that RBR is a target of the trichome cell specification and differentiation factors GLABRA1 and GLABRA3 [48] . Obtaining homozygous rbr trichomes by inducible methods will be required to analyze how RBR controls early specification and/or differentiation . Taken together , the observed sporophytic developmental anomalies including retarded trichome differentiation are a consequence of partial haplo-insufficiency of RBR in tetraploids , but not due to RBR-mediated cell cycle deregulation . RBR is an essential cell cycle regulatory gene that is expressed in the sporophyte ( embryo , leaves , root and shoot meristems ) and the ovule including the embryo sac [14]–[16] , [49] . To gain better insight into the dynamic expression pattern of RBR throughout the plant life cycle , we analysed RBR RNA and protein accumulation by in situ hybridization and a transgenic RBR protein reporter line RBR::RFP , respectively [49] . We observed RBR::RFP expression throughout sporophyte development in leaves and seedlings ( not shown ) , also during trichome development ( Figure 4P ) . In reproductive tissues , RBR mRNA was detected in developing ovules and anthers ( Figure 4A and 4B ) . In particular , RBR expression was detected in the functional megaspore , the progenitor cell type of the female gametophyte ( Figure 4D ) . In a fully differentiated embryo sac , we observed RBR mRNA expressed in all the embryo sac cell types such as egg cell , central cell and synergid cells , in addition to the sporophytic cells of the ovule ( Figure 4F ) . In contrast , RBR::RFP fusion protein was localized primarily in the central cells [49] suggesting post-translational regulation of RBR in the egg apparatus . A recent study detected RBR::RFP throughout the male gametophyte development [17] . In summary , expression of RBR in all cell types of the gametophytes and the sporophyte , including trichome cells , is consistent with the requirement of RBR for cellular proliferation , cell fate and differentiation of the gametophytic cells , sporophytic development and trichome differentiation . The dynamic expression of RBR throughout plant development , and its cell-cycle inter- and independent functions reported thus far suggests that RBR is also involved in other regulatory networks . Evolutionary homologues of pRB and epigenetic factors such as PRC2 proteins and DNA maintenance methyltransferase ( Dnmt1 ) have essential roles in controlling cell differentiation and development both in plants and animals [8] , [9] , [29] . In animal systems , it has been established that Enhancer of zeste homolog 2 ( Ezh2 ) , a core member of PRC2 , recruits DNA methyltransferase 1 ( Dnmt1 ) , and the resulting maintenance of DNA methylation facilitates formation of more repressive complexes to control distinct developmental processes [50] , [51] . There is evidence that both PRC2 genes and Dnmt1 exert their function in a cell cycle-dependent manner . For instance , several PRC2 members and Dnmt1 homologues seem to be directly repressed by the pRB-E2F complexes in plants and animals [29] , [51] . Furthermore , PRC2 dynamically regulates pRB or RBR via its inherent H3K27me3 activity and possibly through its continuous association throughout the cell cycle [15] , [52] , [53] . In Arabidopsis , there are three distinct orthologues of Ezh2 , namely CURLY LEAF ( CLF ) , which positively regulates cell size and elongation in the leaf sporophyte; MEDEA ( MEA ) , which negatively regulates cell proliferation and cell size during seed development; and SWINGER ( SWN ) , which enhances the function of both CLF and MEA [8] , [54] . Similarly , three orthologues of Supressor of zeste 12 ( Suz12 ) , which are known to be associated with cell cycle and cell differentiation in animal systems , exist in the Arabidopsis genome . FERTILIZATION INDEPENDENT SEED 2 ( FIS2 ) functions similar to MEA during seed development; VERNALIZATION 2 ( VRN2 ) and EMBRYONIC FLOWER 2 ( EMF2 ) are associated with distinct sporophytic pathways [8] . MET1 is the Arabidopsis orthologue of Dnmt1 , which is a key target of RBR and a modifier of several PRC2 genes , and it is critical for coordinated cell division , specification and differentiation of the embryos , and also throughout the sporophytic development [15] , [55]–[57] . The mechanisms by which pRB , PRC2 and Dnmt1 homologues control cellular differentiation and development are not completely understood in plant and animal systems . We recently reported that RBR , several PRC2 genes and MET1 are co-regulated by a negative feedback mechanism during gametophyte differentiation and development [15] . Here we asked if a similar mechanism exists in the leaf sporophyte as well . First , we compared the expression levels of sporophytic PRC2 genes and MET1 in rbr triplex and heterozygous diploid rbr mutant leaf tissues in relation to their corresponding tetraploid or diploid wild-type tissues . Our initial expression analysis in tetraploids suggested that plant to plant variation in expression was quite high . Therefore , we analyzed leaves from individual plants as independent replicates . Prior to gene expression in tetraploids , we examined the expression of MET1 and PRC2 genes in the diploid wild type and rbr/RBR leaf tissues , but we did not detect significant differences in expression levels ( not shown ) . In contrast , we observed that CLF and MET1 were upregulated and VRN2 was downregulated in rbr triplex leaves when compared to the tetraploid wild type ( Figure 3C ) , suggesting the importance of RBR dosage for gene regulation in the tetraploid context . This experiment , however , did not reveal if acute genetic down-regulation of RBR below 25% would be required for the deregulation of the PRC2 genes SWN and EMF2 . Together , RBR regulates MET1 and the PRC2 genes CLF and VRN2 during leaf development in a dosage-dependent manner . Given that RBR-PRC2-MET1 regulatory network functions during gametophyte development [15] , we conclude that RBR control of PRC2 and MET1 is important throughout the plant life cycle . Recent data suggest that RBR can function downstream of chromatin regulators like PRC2 or transcription factors such as SCARECROW , GLABRA1 and GLABRA3 during distinct stages of plant development [15] , [16] , [48] . We therefore asked if PRC2 would reciprocally regulate RBR in the leaf sporophyte . We used two different double mutants that disrupted CLF and SWN , and VRN2 and EMF2 , respectively . Sporophytic PRC2 activity is considerably reduced in these double mutants and , consequently , development of the leaf sporophyte is impaired [58] . Although trichomes were correctly specified in the mutant leaves , we observed that their branching was incomplete ( Figure 3B ) . The majority of the trichomes ( 92–95% ) in diploid wild type leaves differentiated to the mature 3-branched stage . The clf;swn double mutant showed the most severe phenotype in which the majority of trichomes were 2-branched ( 78% ) while only 15% differentiated to the 3-branch stage . The vrn2;emf2 double mutant showed similar phenotypes ( Figure 3B ) , although the percentage of trichomes that fully differentiated was higher than that of clf;swn , likely because the emf2 allele used here was a weak loss-of-function allele of EMF2 [58] . These data collectively suggest that a novel PRC2-dependent epigenetic mechanism operates to control trichome differentiation in addition to leaf development . Intriguingly , expression levels of both RBR and MET1 were significantly reduced in the mutant leaves ( Figure 3B , 3D , 3E ) , suggesting that the sporophytic PRC2 complexes activate both RBR and MET1 in leaves . Previous work demonstrated that MEA is derepressed in leaves of PRC2 mutants and that MEA is a direct target of the sporophytic PRC2 [59] . Given that MEA represses paternal RBR in fertilized female gametophytes [15] , it is probable that indirect repression of RBR by the sporophytic MEA might have led to reduction of RBR levels in PRC2 mutant leaves . Alternatively , reduction of MET1 in PRC2 mutants is consistent with a previous observation in an animal system that depletion of Ezh2 led to downregulation of Dnmt1 concomitant with local reduction of H3K27me3 activity [60] . Therefore , we propose that both RBR and its target gene MET1 are likely independently activated during leaf development and trichome differentiation either by a cell cycle dependent CLF-PRC2 activity , or indirectly via repression by MEA , which in turn is controlled by the CLF-PRC2 ( Figure 5 ) . However , due to the complexity of tetraploid wild type and mutant plants used in this study , diploid plants deregulating RBR in a temporal and spatial manner will be necessary to test our hypothesis . We have provided here direct evidence that RBR has an instructive and dosage-dependent role in cell fate determination , differentiation and development in Arabidopsis . This function is partly mediated by a regulatory loop between RBR and epigenetic regulators such as PRC2 genes and MET1 , which operates distinctly in the gametophyte [15] and the sporophyte generations . When RBR function is abolished , such as in female or male rbr gametophytes [15] , [17] , proper cell fate assignment does not occur . However , quantitative reduction of RBR expression in rbr triplex mutant sporophyte does not prevent cell specification but impairs full differentiation , consistent with an earlier study in which stem cell differentiation was delayed when RBR was reduced [16] . Since we did not observe significant changes in ploidy levels in response to reduced RBR dosage , it is likely that RBR-mediated developmental functions can also be cell cycle-independent , similar to pRB control of cell cycle-unrelated processes in animals [18] , [61] , [62] . In support of the RBR-PRC2-MET1 epigenetic network that we have identified , a recent study revealed that DNA methylation of RBR , CLF , SWN , VRN2 and EMF2 loci is regulated by MET1 in Arabidopsis sporophyte [63] . We propose that the dynamic modulation of this RBR-PRC2-MET1 circuit was adopted to accommodate the regulation of distinct developmental processes in both gametophyte and sporophyte generations .
The rbr-3 allele ( Col background ) [14] , cell-specific marker lines ET956 , ET1119 and ET2634 ( Ler background ) [23] , [24] , RBR::RFP reporter line [64] , and the PRC2 mutant alleles , clf-50 ( Ws ) , swn-3 ( Col ) , emf2-10 ( Ws ) and vrn2-1 ( Ler ) [58] were described previously . Ploidy level of mutant plants was determined using a flow-cytometer ( Partec GmbH , Munster , Germany ) . For trichome quantification , plants were germinated on MS plates without sucrose in growth cabinets , and classes of trichomes were counted on the 3rd and 4th leaves , when the seedlings were at 5–6 leaf stage . Confocal analysis of ovules and spatial analysis of GUS activity in ovules and seed tissues were performed as described earlier [6] , [15] . Scanning electron micrographs of leaves were prepared as published [65] . Cauline leaves of mutant and wild-type tissues were fixed in a formaldehyde/glutaraldehyde fixative . Intact trichome cells were isolated from leaf epidermis by an established protocol based on removal of Ca2+ ions [66] . Nuclear images of guard cells from leaves ( n = 42 and 54 in wild type and mutant , respectively ) , and of the trichome cells of the wild-type ( n = 80 ) and the mutant ( n = 42 ) were recorded for fluorescence measurement upon DAPI staining by confocal microscopy ( LSM 510META , Carl Zeiss , Jena , Germany ) . DAPI was visualized with a 364 nm laser line in combination with a 380–475 nm bandpass filter . Recordings were made with a 20x objective at zoom 4 , with maximum pinhole . Fluorescence intensity was analysed with the LSM software ( release 3 . 2 ) . Data normalization with average fluorescence values of the tetraploid guard cell nuclei ( 4C ) and arbitrary clustering of data points were performed as described elsewhere [67] . Semi-thin paraffin sections of inflorescences , emasculated pistils , and siliques [68] were used for hybridization with the hydrolyzed digoxygenin-UTP-labeled riboprobes ( Roche Diagnostics , Basel , Switzerland ) that were prepared using a RBR cDNA expression clone as a template . In situ hybridization was performed as before [6] . Tests for dominance/recessiveness were performed as previously described [69] . The expected phenotypic ratios for recessive and dominant genetic models were calculated considering ( a ) reduced transmission efficiency of the rbr-3 allele and ( b ) maximal double reduction . Transmission efficiency ( TE ) is an estimate of inheritance of a mutant allele versus the wild-type allele by female or male gametes [14] . It is calculated as a ratio of number of mutant plants to wild-type plants in progenies from reciprocal crosses of a heterozygous mutant . The rbr allele is not transmitted through female gametes ( TE♀ ( rbr ) = 0 ) [14]; the transmission of rbr through pollen was estimated as 0 . 1 based on both TE♀ ( rbr ) in diploid condition , and recovery of triplex plants in triplex progeny ( as a ratio of triplex to duplex plants ) . Double reduction describes the situation in polyploids , in which a heterozygous individual produces homozygous gametes [69] . This can occur if quadrivalents are formed and recombination occurs between the centromere and the locus of interest . Through chromatid segregation both alleles of the sister chromatids can co-exist in the same gamete . Thus , the frequency of double reduction depends on the distance between the locus in question and the centromere . Because the RBR locus is ∼45 cM away from the centromere , these loci can be considered unlinked . Therefore , we used the maximal double reduction frequency of 1/6 for our calculations ( see Text S1 ) . As we did not know how many rbr-3 alleles were present in the tetraploid plants , we compared the observed data to six different models with one , two , or three rbr-3 alleles , them being dominant and recessive , respectively . First we recorded the seed set/sterility phenotypes of a total of 103 progeny plants originating from a selfed autonomously tetraploidized plant , which was heterozygous for the rbr mutation ( Figure S2A , S2B ) . Out of the 103 progeny plants , we took one tetraploid plant group ( consisting of 52 plants ) with similar seed set and subjected the sterility phenotype and progeny segregation data for 6 different models [simplex , recessive ( rbr/RBR/RBR/RBR ) ; simplex , dominant ( rbrD/RBR/RBR/RBR ) ; duplex , recessive ( rbr/rbr/RBR/RBR ) ; duplex , dominant ( rbrD/rbrD/RBR/RBR ) ; triplex , recessive ( rbr/rbr/rbr/RBR ) ; triplex , dominant ( rbrD/rbrD/rbrD/RBR ) ] . These plants were identified as duplex-recessive for rbr ( Table 1 ) . Subsequently , two other distinct tetraploid phenotype groups were fit to simplex-recessive and triplex-recessive models ( 41 and 2 plants , respectively ) ( see Figure S2 and Table 1 for details ) . Progeny analysis of one of these two triplex plants identified in this experiment confirmed stability of the seed set phenotype over subsequent generation ( Figure S4 ) . RNA extraction and reverse transcription were performed as described [15] . Quantitative real-time measurements were performed using SYBR Green Fast Master Mix reagent in an ABI Prism 7500 Sequence Detection System ( Applied Biosystems ) ( Applied Biosystems ) , according to the manufacturer's instructions . For each condition , 2 technical replicates and 3 biological replicates were used . Relative gene expression levels were normalized to the expression levels of a control gene , PP2A ( At1g13320 ) [70] . Primers used in this work are listed in Table S1 .
|
Understanding the convergent developmental mechanisms of core cell cycle genes is highly instructive in biology . When these genes are essential in development , lethality precludes mutation analysis throughout the life cycle of an organism . We subjected a homozygous lethal mutation in RETINOBLASTOMA RELATED ( RBR ) of Arabidopsis for tetraploid genetic analysis to study the function of RBR during the plant life cycle . In diploids , while RBR–deficient female gametophytes with features of aberrant cell fate and differentiation were analogous to what was previously reported for male gametophytes , we provide evidence that RBR controls gametic genome duplication , thus genome integrity in the gametophyte-derived progeny . Quantitative reduction of RBR in tetraploids led to identification of rbr heterozygous plants that displayed novel RBR dosage-dependent phenotypes in differentiation and development of the sporophyte albeit the absence of cell cycle defects . These phenotypes coincided with deregulation of conserved epigenetic factors such as Polycomb Repressive Complex 2 ( PRC2 ) genes and METHYLTRANSFERASE 1 ( MET1 ) in the sporophyte , as shown for the gametophytes as well . However , unlike the repression by the PRC2 in gametophytes , RBR is activated by the sporophytic PRC2 subunits , suggesting that distinct modules of the conserved RBR-PRC2-MET1 loop control gametophyte and sporophyte generations in plants .
|
[
"Abstract",
"Introduction",
"Results/Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"and",
"genomics/epigenetics",
"developmental",
"biology/plant",
"growth",
"and",
"development",
"genetics",
"and",
"genomics/plant",
"genetics",
"and",
"gene",
"expression",
"evolutionary",
"biology/plant",
"genetics",
"and",
"gene",
"expression"
] |
2010
|
Dosage-Sensitive Function of RETINOBLASTOMA RELATED and Convergent Epigenetic Control Are Required during the Arabidopsis Life Cycle
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Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes . This process is controlled by an extensively studied transcriptional program . However , the regulatory events coordinating the progression of such program from development to maturation are largely unknown . Here , we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart . Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12 . 5 . Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production , required for cardiomyocyte maturation , were upregulated . However , mitochondria from mutant cardiomyocytes do not mature properly . In contrast , multiple development regulatory genes near predicted heart enhancers , including genes in the IrxA cluster , were downregulated in Ctcf mutants , suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions . Accordingly , loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing . Furthermore , CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart . Thus , CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development .
The coordinated deployment of genetic programs during lineage commitment and differentiation is a hallmark of developmental processes . Cell specification and maturation are coordinated by controlled activation and repression of specific gene expression programs . In the heart , the first functional organ in the embryo , activation of a core set of cardiogenic transcription factors controls specification of cardiac progenitor cells [1] . Shortly after , high expression of genes encoding sarcomeric components defines the contractile cardiomyocyte as early as embryonic day ( E ) 8 . 5 . Cardiomyocytes then mature by further sarcomere assembly [2] , and increased mitochondrial biogenesis [3 , 4] , and finally exit the cell cycle and become binucleated at early postnatal stages [5] . Even though the genes and regulatory networks controlling morphogenesis and function in the heart are well characterized [6] , the events that coordinate the progression from differentiation to maturation are not understood . Recent studies using both mouse and human pluripotent cells have revealed that epigenomic landscapes and chromatin signatures dynamically change during cardiomyocyte differentiation [7 , 8] , suggesting that chromatin structure might control cardiogenesis . Chromatin conformational changes allow physical interaction of distal regulatory elements in the genome . However , the chromatin interactions controlling expression of cardiac development and maturation are poorly understood . The study of genome function during the last decade [9 , 10] has provided an initial understanding of how functional elements scattered throughout the genome act coordinately to control gene activity . The advent of tools to analyze interactions between distal regions of chromatin [11] has allowed detailed mapping of the three-dimensional genome structure [12 , 13] and its organization in distinct regulatory domains [14] . However , how these domains are established , and their function in gene expression regulation are poorly understood . CTCF ( CCCTC-binding factor ) is one of the best described architectural proteins with a role in chromatin structure organization . Through sequence specific binding to DNA , CTCF acts as a barrier for heterochromatin spreading , establishes boundaries between adjacent topologically associating domains ( TADs ) , defines insulator elements that block enhancer activity on promoters , and contributes to enhancer-promoter interactions [15 , 16] . Loss of function studies using knock-out and knock-down approaches have shown that CTCF is essential in early embryo development [17–19] . Conditional deletion of Ctcf in different developmental contexts leads to defects in cell cycle progression [17] , increased apoptosis [20 , 21] , and its deletion in postmitotic neurons leads to decreases in the expression of clustered protocadherin genes [22] . Yet , we still do not fully understand how CTCF controls chromatin structure to coordinate gene expression . Here , we have studied how CTCF regulates gene expression in the context of the developing mammalian heart . We deleted Ctcf in a population of cardiac progenitor cells , which results in cardiac malformations and embryonic death . Analysis of global transcriptional changes preceding morphological defects caused by loss of CTCF showed downregulation of the cardiac developmental program , and concomitant upregulation of programs involved in cardiomyocyte maturation . This suggests that Ctcf deletion causes a premature arrest of cardiac development and concomitantly promotes cardiomyocyte maturation . Thus , CTCF mediates local chromatin interactions to coordinate transcriptional programs that control developmental transitions in the heart .
To address the role of CTCF during development , we deleted Ctcf in cardiac progenitor cells and their derivatives by using a floxed allele [17] and the Nkx2 . 5-Cre driver , which starts acting as early as E7 . 5-E8 . 0 in cardiomyocyte precursors in the cardiac crescent [23] . E10 . 5 or E11 . 5 mutant ( Ctcffl/fl;Nkx2 . 5-Cre ) embryos appeared normal and showed no gross morphological alterations in the heart ( Fig 1A–1D ) . At E12 . 5 , mutant embryos presented pericardial edema ( Fig 1E ) and the cardiac chambers did not expand properly ( Fig 1F ) . Histological examination showed no defects in E9 . 5 mutant hearts as compared to controls ( Fig 1G and 1H ) . In E10 . 5 mutants , the four chambers and the atrioventricular canal formed properly , although the interventricular septum appeared slightly disorganized ( Fig 1I and 1J ) . This defect was exacerbated by E11 . 5 , when thinning of the myocardial wall was also evident ( Fig 1K and 1L ) . No mutant embryos were recovered beyond E12 . 5 ( S1 Table ) . Control Nkx2 . 5-Cre and compound Ctcffl/+;Nkx2 . 5-Cre heterozygotes showed normal morphology at these stages ( S1 Fig ) . To understand the effect of Ctcf deletion in the developing heart , we determined the time point when CTCF protein was lost in cardiomyocytes . We performed co-immunostaining for CTCF and the cardiomyocyte marker TNNT2 at different stages of heart development ( S2 Fig ) . In E9 . 5 mutant hearts , 46% of cardiomyocytes still had detectable nuclear CTCF , albeit at lower levels than in controls , in which all nuclei were double positive ( S2A and S2B Fig; S2 Table ) . However , by E10 . 5 we were not able to identify any cardiomyocyte expressing CTCF in mutant hearts , although expression was present in endocardium ( S2C and S2D Fig ) . The same pattern was observed at E11 . 5 ( S2E and S2F Fig ) . Persistent CTCF protein in cardiomyocytes at E9 . 5 , despite that Nkx2 . 5-Cre is active from E7 . 5-E8 . 0 [23] , could be explained by protein long half-life [24 , 25] . It has been recently shown that 15% of CTCF is sufficient for proper function [26] . Therefore , the remaining protein we observe can explain why morphological defects are not detected until E10 . 5 . We assessed whether cardiac defects were due to decreased cell proliferation or increased apoptosis . Numbers of cardiomyocytes positive for TUNEL staining ( S3A–S3F , S3M and S3O Fig; S3 Table ) or phosphorylated histone H3 ( S3G–S3L , S3N and S3P Fig; S3 Table ) were not altered in mutant hearts at E10 . 5 and E11 . 5 , indicating that apoptosis and proliferation were unaffected . Accordingly , we did not observe a difference in the number of cardiomyocytes between mutants and controls at these stages ( S3 Table ) . Our results differ from others showing that CTCF loss in other developmental systems cause increased apoptosis [20] , suggesting context-specific effects of CTCF . Our results indicate that Ctcf is required for cardiac morphogenesis . To understand the function of CTCF as a transcriptional regulator in the developing heart , we analyzed the global effects of CTCF loss on gene transcription . We performed RNA-seq analysis on hearts homozygous for cardiac-specific deletion of Ctcf ( Ctcffl/fl;Nkx2 . 5-Cre ) ; heterozygotes , with deletion of only one allele ( Ctcffl/+;Nkx2 . 5-Cre ) ; and control heterozygotes for the floxed allele but not carrying the Nkx2 . 5-Cre driver ( Ctcffl/+ ) . To identify the earliest transcriptional effects of Ctcf deletion this analysis was performed at E10 . 5 , when we first observed a complete loss of CTCF in the mutants . Comparison between homozygous Ctcf-deleted hearts and heterozygotes or controls yielded approximately 2 , 000 differentially expressed genes in each case , of which roughly half were upregulated and half downregulated ( S4 Table ) . Interestingly , comparison between heterozygotes and controls returned only 24 differentially expressed genes , including Ctcf itself and11 pseudogenes ( S4 Table ) . This suggests that , at least in the developing heart [27] , one functional Ctcf allele is sufficient for correct regulation of gene expression . Gene-ontology analysis [28] of all genes differentially expressed upon Ctcf deletion showed enrichment in terms related to developmental processes , including heart development , contractile fibers , translation and mitochondria ( Fig 2A; S5 and S6 Tables ) . When we analyzed upregulated and downregulated genes separately , we found a clear distinction in the functional categories enriched in each case . Numerous upregulated genes were enriched in categories related to translation and mitochondrial function . In contrast , the downregulated genes were over represented in categories related to heart development and the sarcomere ( S5 and S6 Tables ) . Detailed analysis revealed that more than 300 genes related to mitochondrial function were up- and down regulated in Ctcf mutant hearts . Many of such genes encode subunits of mitochondrial respiratory complexes I , III , IV and V/ATP synthase , Sdhd in Complex II , and the large and small mitochondrial ribosome subunits ( S4A Fig; S6 Table ) . This could suggest that CTCF controls expression of core transcriptional regulators of the mitochondrial gene program . However , none of these factors , such as PGC-1α , ERRs , or NRF-1/2 [29] , where dysregulated in mutant hearts ( S4 Table ) . Other upregulated genes are involved in translation and encode most cytoplasmic ribosomal proteins , various initiation and elongation factors , and members of the spliceosomal complex ( S4B Fig; S6 Table ) . CTCF organizes chromatin structure and contributes to the establishment of regulatory domains in the genome [30] . We found that genes misregulated in Ctcf mutant hearts do not cluster in specific genomic regions ( S5A Fig ) , suggesting that CTCF does not control gene expression in large genomic regulatory domains similarly to what has been recently shown in embryonic stem cells [26] . We next examined CTCF chromatin binding near genes whose expression changed upon Ctcf deletion in the heart by mapping the distance between the transcriptional start site ( TSS ) and the nearest CTCF ChIP-seq peak obtained from published datasets on adult 8 week hearts [10] . We found that the up- and downregulated genes are closer to a CTCF binding site than genes whose expression does not change upon Ctcf deletion ( S5B Fig ) . Arbitrarily analyzing 10 or 20 kb windows based on the above distribution , we found that TSS of down- and up-regulated genes are surrounded by CTCF binding sites more frequently than genes whose expression did not change in mutants ( Fig 2B ) . These CTCF binding sites are conserved across multiple tissues , and we did not observe enrichment for heart-specific CTCF peaks near genes deregulated in the mutant . These results suggest that CTCF regulates gene expression mainly by binding nearby genomic regions , possibly by mediating local chromatin interactions . CTCF could define gene regulatory domains by shielding genes from the influence of nearby enhancers or by facilitating enhancer-promoter interactions [15] . In the first scenario , Ctcf loss would lead to upregulation of gene expression and to downregulation in the second . To distinguish between these two possibilities , we determined the distance between dysregulated genes and the nearest heart enhancer [10] . We found that downregulated , but not upregulated , genes are significantly closer to a heart enhancer than expressed genes with no change in Ctcf mutants ( S5B Fig ) . Again , this pattern is preserved when analysis is restricted to a 10 or 20 kb window surrounding the TSS of differentially expressed genes ( Fig 2B ) . As downregulated genes are enriched in developmental regulators , these results suggest that CTCF promotes enhancer-promoter interactions in genes controlling cardiac progenitor establishment and differentiation . The previous results suggest specific genomic features of up- or downregulated genes in Ctcf mutant hearts . To further explore these features , we analyzed in more detail the distribution of CTCF binding peaks on the same dataset used above surrounding the TSS of dysregulated genes . Up- and downregulated genes showed enrichment in CTCF binding immediately upstream of the TSS , and the binding signal was higher than that in genes whose expression did not change in mutants ( Fig 2C ) , which agrees with the previous result ( Fig 2B ) . However , when we analyzed only the dysregulated genes belonging to development , or mitochondria and translation categories ( S6 Table ) we observed a clear difference . Mitochondrial and translation genes showed increased CTCF binding near the TSS , but in developmental genes CTCF binding spread over more distal regions ( Fig 2C ) . We searched promoter-proximal and distal sites for de novo and known sequence motifs to address the possibility that the presence of CTCF sites or other motifs would underlie the differences in distribution between categories . However , we only identified binding motifs for CTCF itself ( S7 Table ) . Our results suggests that Ctcf binds near the TSS to repress genes acting in mitochondria and regulating translation , both crucial for cardiomyocyte maturation [29] . In contrast , CTCF binding to genomic regions more distal to TSS promotes expression of genes located near heart enhancers and controlling cardiac development . Dramatic changes to the nuclear-encoded mitochondrial transcriptome , particularly of proteins involved in oxidative phosphorylation ( OXPHOS ) system , in Ctcf mutant hearts prompted us to analyze the components of this pathway in mutant and control embryonic cardiomyocytes . In agreement with the RNA-seq ( S4 Table ) , Western blot revealed increase in Complex IV subunit I ( Cox I , encoded in mitochondrial DNA ) and Complex IV subunit IV ( Cox IV , encoded by Cox4i1 ) in Ctcf mutant hearts at E10 . 5 and E11 . 5 , as compared to controls ( Fig 3A ) . Similarly increased was Tom20 ( encoded by Tomm20 ) ( Fig 3B ) , which is the mayor receptor of the mitochondrial outer membrane translocase . The abundance of other mitochondrial proteins encoded by genes whose expression did not change in Ctcf mutants ( S4 Table ) , such as Grp75 , a key stress chaperone regulating mitochondrial protein translocation , folding and functions [31]; and Tfam , the transcriptional regulator of mitochondrial DNA [32] , was comparable between control and mutant hearts ( Fig 3B ) . We asked whether upregulation of OXPHOS components favors functional assembly of respiratory complexes and supercomplexes on the mitochondrial inner membrane . Blue-native gel electrophoresis revealed assembled complexes I , IV and V , and supercomplex I+III2 in both control and mutant hearts at E10 . 5 and E11 . 5 ( Fig 3C ) . The mitochondrial voltage-dependent anion channel ( Vdac ) , whose assembly is independent of respiratory complexes and supercomplexes , and whose encoding gene ( Vdac1-3 ) levels did not change in mutants ( S4 Table ) , was used as loading control . In control hearts , Complex I , both in the free form I or in the form of I+III2 , and Complex V substantially increased from E10 . 5 to E11 . 5 ( Fig 3C and 3D ) , consistent with maturation of mitochondrial OXPHOS [4] . In contrast , levels of I+III2 and I did not increase from E10 . 5 to E11 . 5 in Ctcf mutant hearts . Complex V was similarly increased between E10 . 5 and E11 . 5 in control and Ctcf mutant hearts ( Fig 3C and 3D ) . This agrees with maturation/stabilization of the electron transport chain components being regulated independently of their production [33] . Our results suggest that despite increased transcription of subunits of CI and CIII that may lead to more complexes and supercomplexes assembled at the mitochondrial inner membrane , maturation of the respiratory chain is blunted in the Ctcf mutant heart . Transmission electron microscopy ( TEM ) analysis revealed immature , but overall normal mitochondria with healthy cristae packaging in Ctcf mutant cardiomyocytes at E10 . 5 ( Fig 3E ) . Mitochondria in control E11 . 5 cardiomyocytes have a more electro-dense matrix containing more cristae than E10 . 5 , and are embedded in newly assembled sarcomere . This ultrastructure change agrees with blue native gel electrophoresis data and is consistent with cardiomyocyte maturation occurring between E10 . 5 and E11 . 5 [3 , 4] . Mitochondria in Ctcf mutant cardiomyocytes at E11 . 5 are swollen and larger than controls , and are disorganized and scattered through the cytoplasm ( Fig 3E; S6A Fig ) . Immunohistochemistry , targeting the mitochondrial outer membrane component Tom20 , revealed disorganized mitochondria in Ctcf mutant cardiomyocytes at E10 . 5 ( S6B Fig ) . TEM analysis also revealed that E10 . 5 mutant , but not control , cardiomyocytes have long and continuous sarcomeres , which were also visible at E11 . 5 ( Fig 3E ) . Quantification on images of cardiomyocytes from E10 . 5 hearts immunostained for α-actinin revealed comparable numbers of sarcomeric Z-bands between control and Ctcf mutants ( S6C and S6D Fig ) . These results suggest that sarcomere assembly , but not sarcomeric component synthesis , is premature in the Ctcf mutant embryonic heart . The set of genes downregulated in Ctcf mutant hearts that are enriched in heart development functional categories include key transcription factors and members of several signaling pathways controlling cardiac development ( Fig 4A ) . In situ hybridization confirmed downregulation of the transcription factors Nkx2-5 and Hopx in the Ctcf mutant heart at E10 . 5 . ( Fig 4B and 4C ) . This analysis also showed reduced expression of Nppa to a more restricted domain in the left ventricle , and loss of Pitx2 expression in the right ventricle ( Fig 4D and 4E ) . Our RNA-seq showed reciprocal up and downregulation of genes from the Tnnt1/Tnni3 and Tnni2/Tnnt3 troponin clusters ( Fig 4F and 4G ) . Accordingly , in situ hybridization revealed strong downregulation of Tnnt1 and Tnni2 , and upregulation of Tnni3 in atria and ventricles and of Tnnt3 in atria in the Ctcf mutant heart ( Fig 4H–4K ) . These changes in the expression pattern of troponin genes , which are arranged in clusters in the genome , suggest that CTCF coordinates their expression during development . Irx4 , which encodes a transcription factor critical for heart development [34 , 35] was downregulated in Ctcf mutants . We analyzed this gene and its genomic context in more detail as a means to understand the mechanisms through which CTCF regulates transcription in vivo . Irx4 forms part of the 1 . 5 Mb IrxA cluster , which also contains the related Irx1 and Irx2 genes [36] . All three IrxA genes are expressed in the developing heart , in distinct but partially overlapping patterns . Whereas Irx1 and Irx2 express at low levels in the interventricular septum , Irx4 is strongly expressed throughout the ventricles [37] . Previous studies have shown that the IrxA cluster is regulated through long-distance gene-specific interactions [38] . Furthermore , our RNA-seq analysis revealed significant upregulation of the three genes closest to Irx4 outside the IrxA cluster: Ndufs6 , encoding a subunit of mitochondrial Complex I; Mrpl36 , encoding a mitochondrial ribosomal protein; and Lpcat1 , involved in phospholipid metabolism ( Fig 5A; S4 Table ) . To confirm and extend these observations , we examined the expression of the three genes in the IrxA cluster , and of their immediate neighbor Ndufs6 , in control and mutant hearts by in situ hybridization . Expression of Irx4 in mutant hearts was indistinguishable from controls at E9 . 5 ( Fig 5B ) , but expression levels were strongly reduced in mutants at E10 . 5 ( Fig 5C ) and E11 . 5 ( Fig 5D and 5J ) . The RNA-seq analysis showed that Irx1 and Irx2 expression was not significantly different in mutants . However , in situ hybridization showed that their expression domains expanded from the interventricular septum to the adjacent trabecular myocardium in the right and left ventricle of Ctcf mutants ( Fig 5E , 5F , 5H and 5I ) . In control hearts , Ndufs6 was expressed ubiquitously but with higher intensity in the ventricles , similar to Irx4 , but its expression was subtly increased in mutant hearts ( Fig 5G ) . Together , these observations suggest that loss of CTCF leads to overall dysregulation of the IrxA cluster and its neighboring genes , perhaps through modification of its 3D structure . To uncover the function of CTCF in regulating the IrxA chromatin structure we performed chromosome conformation capture followed by deep sequencing ( 4C-seq ) [39] , using as viewpoints the promoters of Irx4 and Ndufs6 in E11 . 5 control and homozygous mutant ( Ctcffl/fl;Nkx2 . 5-Cre ) hearts ( Fig 6A and 6B; S7 Fig ) . In controls , the promoter of Irx4 interacted strongly with the Irx2 promoter and with specific CTCF binding sites located upstream and downstream in the Irx2/Irx4 and Irx4/Ndufs6 intergenic regions , respectively ( asterisks in Fig 6A ) . The CTCF binding sites were previously identified by ChIP-seq in E14 . 5 and 8-week hearts [10] ) . In mutants , the interaction of the Irx4 promoter with the Irx2/Irx4 intergenic CTCF site was lost , and new interactions appeared upstream and 350 kb downstream in the Clptm1l locus ( Fig 6B ) . The Ndufs6 promoter established interactions with the promoters of Irx4 , Lpcat1 and Clptm1l as well as with the two intergenic CTCF sites in control hearts . In CTCF mutants , interactions of the Ndufs6 promoter with both CTCF binding sites and the Irx4 promoter were lost , and only contacts with the promoters of the downstream genes Lpcat1 and Clptm1l were maintained ( Fig 6B ) . Finally , we used a viewpoint for 4C-seq the Irx2/Irx4 intergenic CTCF site , which interacts with the promoters of Irx4 and Ndufs6 ( Fig 6B , S7 Fig ) . In controls , the Irx2/Irx4 intergenic CTCF site interacts upstream and downstream of and extended region spanning Irx4 , Ndufs6 and Mrpl36 , and the downstream Clptm1l locus ( Fig 6B ) . Interaction between the CTCF site and the Irx4 promoter was greatly reduced in Ctcf mutants , reciprocating the pattern observed when using Irx4 as viewpoint . Furthermore , novel interactions appeared that extend to Irx2 ( Fig 6B ) . Overall , these results suggest that CTCF plays a central role in organizing chromatin in the Irx4 regulatory domain by mediating the interaction of several CTCF-bound sites with various promoters in the region . Removal of CTCF from the developing heart re-structures the local 3D organization of the extended IrxA cluster; this results in loss of the interaction between Irx4 and Ndufs6 with flanking CTCF sites . These results also suggest that CTCF limits chromatin interactions domains , as its loss causes an expansion of local contacts . To unambiguously demonstrate that the CTCF binding site located in the Irx2/Irx4 intergenic region is necessary for proper expression of Irx4 , we generated a mouse line in which we deleted such CTCF binding site using the CRISPR/Cas9 system [40] ( S8 Fig ) . Homozygote mice for the deletion are viable and fertile , as expected since Irx4 mutant mice are also viable in homozygosity and only show mild hypertrophy and compromised contractility as adults [34] . We analyzed the expression of Irx4 by in situ hybridization in E10 . 5 embryos from this line . Irx4 was slightly reduced in the ventricles of the mutant line as compared with controls ( Fig 6C and 6D ) . Irx4 was also ectopically expressed in the oral-esophageal region ( Fig 6E and 6F ) , in which Irx1 is normally expressed ( Fig 6G ) . Therefore , deletion of the Irx2/Irx4 intergenic CTCF site leads to expansion of the expression domain of Irx4 , perhaps by allowing Irx1 regulatory elements to contact and activate Irx4 . We have thus shown how CTCF is critical to maintain the correct chromatin structure across the IrxA cluster and neighboring genes , and that specific CTCF binding sites are essential for the proper regulation of gene expression .
Recent years have seen substantial advances in our understanding of the relationship between chromatin structure and gene expression . It is now clear that the spatial organization of the genome sets constraints that determine how different functional elements ( promoters , enhancers , and boundaries ) interact with one another [14] . Nevertheless , we still do not fully understand how this 3D structure is maintained or the role played by chromatin-bound factors in this process . In this study we have explored how one of these factors , CTCF , regulates gene expression and genome structure , by analyzing the effects of its loss during the development of the mammalian heart . Deletion of Ctcf in the developing heart rapidly leads to cardiac defects and embryonic death . However , transcriptomic analysis shows that loss of CTCF does not lead to dysregulation of gene expression across large chromosomal domains; rather , changes appear to be local , as described in other developmental settings [20] . Upregulated and downregulated genes are both more likely to have CTCF binding sites in their vicinity , but only downregulated genes are closer to heart enhancers [10] . Downregulated genes include major regulators of the cardiac developmental program , strongly suggesting that CTCF facilitates enhancer-promoter interactions for these genes in a tissue-specific fashion . In contrast , upregulated genes are highly enriched for genes involved in mitochondrial function and protein translation , which interestingly show higher levels of CTCF binding close to their promoters . Cardiomyocyte maturation involves an increase in the demand for energy and protein production , which is accompanied by increased transcription of mitochondrial and ribosomal genes [8] . Cardiomyocytes lacking CTCF prematurely activate these programs , but fail to maintain functional mitochondria despite the increase in transcription . Therefore , we observe a premature maturation of cardiomyocytes lacking CTCF , accompanied by the shutting down of developmental and patterning processes . Nevertheless , there is a lack of coordination of this precocious differentiation , leading to embryonic lethality at E12 . 5 . To gain insight into the relationship between CTCF , genome structure and the regulation of gene expression , we analyzed the IrxA gene complex . This complex is a paradigm of gene clustering [36] , with genes expressed in overlapping but distinct domains in the developing heart [37] and separated into distinct structural and regulatory domains [38] . We observed changes in the levels and pattern of IrxA gene expression in Ctcf mutants , and interestingly detected similar changes in unrelated genes neighboring Irx4 . This strongly suggests that the changes in the extended IrxA cluster are caused by the loss of CTCF binding in the region , and are not a secondary effect of changes in other genes . Analysis by 4C-seq in wild type and mutant hearts showed that when CTCF is lost , the Irx4 promoter forms fewer contacts with CTCF-flanking sites and gains interactions with regions situated outside of its regulatory domain . Furthermore , the promoter of the mitochondrial Ndufs6 gene also losses interactions with these CTCF sites . These changes in promoter interactions in Ctcf mutants were mirrored by 4C analysis of the Irx2/Irx4 intergenic CTCF site . Consequently , deletion of this CTCF site leads to reduced Irx4 cardiac expression accompanied by gain of expression in novel territories . Therefore , in this context CTCF is acting both as an insulator to separate adjacent regulatory domains , and as a facilitator of promoter-enhancer interactions to ensure proper gene expression within these domains . The structure we have defined for Irx4 is reminiscent of the recently described super-enhancer domains ( SD ) , where regions of a few hundred kilobases located between CTCF sites are organized as insulated neighborhoods within large-scale topological domains [41] . The domain we identify would contain tissue specific enhancers together with their target genes . As is the case with SDs , loss of CTCF leads to downregulation of the gene central to this domain ( Irx4 ) and upregulation of flanking genes ( Irx1 and Irx2 on one side; Ndufs6 , Mrpl36 , and Lpcat1 on the other ) . Overall , our analysis suggests that chromatin structure is relatively stable during development and that the deletion of CTCF does not cause a marked disassembly of this organization , in line with recent reports [42] . However , local intra-TAD domains loops [16 , 43] are affected , leading to dysregulation of genes important in the cardiac developmental program as we observe for the IrxA genes . More puzzling is the observation that only a limited set of genes is altered by Ctcf deletion in the heart . CTCF binds to thousands of sites throughout the genome , most of which are shared between different tissues and cell lines [10 , 12 , 44] . An essential role for CTCF in determining and maintaining chromatin structure and organization was therefore anticipated [30] . Accordingly , constitutive loss of CTCF results in very early embryonic death [17–19] and its selective deletion in different developmental contexts leads to profound defects in the targeted organ or tissue , usually through increased apoptosis [20–22] . However , in all of these cases the same pattern is observed: despite wide distribution of CTCF binding , only a fraction of expressed genes is dysregulated . Furthermore , there is little overlap among genes regulated by CTCF in different systems , indicating a context-specific role of this factor . In the developing heart , we observe the concomitant upregulation and downregulation of maturation and developmental programs , suggesting that the role of CTCF here is to maintain the coordination of expression transitions . In this scenario , only genes subject to dynamic regulation at the time of Ctcf deletion would show changes in expression . The description of different models of neural-specific deletion of Ctcf is compatible with this interpretation . When deleted in precursors during neural development , CTCF loss leads to apoptosis and subsequent death [21 , 22] . However , deletion in postnatal neurons results in long-term survival of Ctcf mutant neurons , but activity-induced changes of gene expression are altered [45] . Together , these observations suggest that CTCF , and possibly local chromatin structure , are not necessary for basal gene activity but essential for dynamic transitions in expression .
Mice were bred in the core animal facility in the Centro Nacional de Investigaciones Cardiovasculares in accordance with national and European legislation . All procedures were approved by the CNIC Committee of Animal Welfare and by the Madrid Autonomous Government Regional Ministry of the Environment and Territorial Organisation ( reference number PROEX 196/14 ) . The Ctcf floxed allele and Nkx2 . 5-Cre line have been previously described [17 , 23] . Primers used for genotyping are detailed in S8 Table . Ctcffl/+ or Ctcffl/fl embryos were used as controls . Mice were bred in the core animal facility in the Centro Nacional de Investigaciones Cardiovasculares in accordance with national and European legislation . Some of the experiments were performed in Toronto , and they were approved by the Toronto Centre for Phenogenomics Animal Care Committee . Whole mount embryos were dissected in cold PBS and imaged using a Nikon SMZ1500 microscope with NIS-Elements BR 4 . 12 . 01 imaging software . For sections , embryos were collected in cold PBS and fixed in 4% PFA overnight at 4°C , dehydrated in an ethanol series , embedded in paraffin , and sectioned at 5 μm for immunostaining amd hematoxylin and eosin , and at 7 μm for in situ hybridization . Sections were observed under an Olympus BX51 microscope and photographed with an Olympus DP71 digital camera . 5 μm paraffin sections were incubated with CTCF 1:1500 ( Bethyl labs A300-543A ) and CT3 1:10 ( Hybridoma Bank ) or processed for histological analysis by hematoxylin and eosin staining . For TUNEL the Terminal Transferase recombinant kit ( Roche 03 333 574 001 ) and biotin-16-dUTP ( Roche 11 093 070 910 ) were used , together with anti-PH3 antibody 1:200 ( Millipore 06–570 ) and CT3 1:10 ( Hybridoma Bank ) ; images were acquired with a Nikon A1R Confocal microscope . Quantification of TUNEL and PH3 staining were performed with ImageJ software . Positive cells for each antibody were counted in 3–5 sections per heart . Three control and three KO hearts were used . Statistical significance was determined using one-tailed Student´s test . α-actinin bands quantification was performed as previously described [3] . Quantification of CTCF staining at E9 . 5 was performed with ImageJ software . Positive cells for CTCF were counted in 5 sections per heart . For cardiomyocyte purification , individual E10 . 5 ventricles were dissected in cold PBS and incubated 5 min at 37°C with 100 μL of 48 mg/mL Collagenase type II and pancreatin 0 . 025gr/mL ( plus NaCl 0 . 0085gr/mL ) diluted in ADS buffer ( 5x: 0 . 034 g/mL NaCl , 0 . 238 g/mL Hepes , 0 . 0006 g/mL NH2PO4 , 0 . 005 g/mL glucose , 0 . 002 KCl , 0 . 001 g/mL MgSO4 ) . After mechanical disruption by pipetting , tubes were left 2 min in the hood , liquid phase was separated , mixed with 100microL of cardiomyocyte media ( 4:1 mixture of of Dulbecco’s Modified Eagle Medium ( DMEM , 1x Gibco , 41965–039 ) and medium M199 ( Sigma , M2154 ) . This is supplemented with 15% inactivated bovine serum; 5mM HEPES , pH7 . 4; 2mM L-glutamine; and 1x penicillin-streptomycin [46] and incubated at 37°C while enzymatic and mechanical disruption was repeated . Both fractions were pooled and centrifuged at 1000 rpm at room temperature . Pellets were resuspended in 300 μL of cardiomyocyte culture media and incubated 45 min in a 24 well plate previously coated with gelatin 1% at 37°C . Cardiomyocytes were counted and incubated in a new 24 well plate without gelatin for 24 hrs . Cardiomyocytes were fixed with PFA diluted in cardiomyocyte media ( 4% for α-actinin sarcomeric staining , 3 . 6% for Tom20 staining ) for 10 min at 4°C , washed 2x with PBS and stored at 4°C until used . Cardiomyocytes were blocked with BSA , and incubated ON 4°C with 1:100 Tom20 or α-actinin . In situ hybridization using digoxigenin-labeled probes ( Roche 11277073910 ) was performed as described [47] and developed with anti-dioxigenin-AP ( Roche 11093274910 ) and BM-Purple ( Roche 11442 074 001 ) . Probes for Irx1 and Irx2 were previously described [48] . Probes for Nkx2 . 5 , Nppa and Pitx2 were kindly provided by Jose Luis de la Pompa ( CNIC ) . All other probes were generated by PCR ( primers used are detailed in S8 Table ) , adding the sequence of the SP6 promoter at the 5’ of forward primers and of the T7 promoter at the 5’ of reverse primers . Sections were observed under an Olympus BX51 microscope and photographed with an Olympus DP71 digital camera . RNA-seq was conducted on three pools of six E10 . 5 hearts each from control ( Ctcffl/+ ) , heterozygotes ( Ctcffl/+;Nkx2 . 5-Cre ) and homozygous mutants ( Ctcffl/fl;Nkx2 . 5-Cre ) . Sequencing was performed by the CNIC Genomics Unit using the GAIIx sequencer , and differential gene expression analysis was performed by the CNIC Bioinformatics Unit using the transcriptome set from Mouse Genome Reference NCBIM37 and Ensembl Gene Build version 65 . GO term enrichment was done using DAVID ( http://david . abcc . ncifcrf . gov ) with cut-offs of minimum 10 genes and an EASE value of 0 . 0001 [28 , 49] . Distances between transcriptional start sites ( TSS ) and between these and CTCF binding sites or predicted heart enhancers were analyzed using R . We used ChIP-seq data for CTCF in adult 8-week hearts . Although data is also available for E14 . 5 embryonic hearts , its quality is much lower ( one replicate with 2 . 8M reads for E14 . 5; two replicates with over 8M reads each for 8w ) . Coordinates for CTCF binding sites , and predicted heart enhancers were obtained from the Ren Lab ( http://chromosome . sdsc . edu/mouse/download . html ) [10] ) . Heat maps were generated using R and the heatmap . 2 package . For density plots of CTCF ChIP signal , values +/- 2kb relative to the TSS were extracted with bwtools [50] from bigWig files available at ENCODE ( ENCSR000CBI ) , and z-scores were calculated transforming each value by substracting the mean over each chromosome and dividing by the standard deviation . After a smoothing step results were plotted using in-house scripts in R . The observed distances between TSS of gene-sets were compared with the expected proportion calculated from 5 , 000 groups randomly sampled without replacement taken from all expressed genes . To identify heart-specific CTCF sites , we first merged all sites identified across multiple tissues from the ENCODE data ( 121 , 147 sites ) and subtracted these from the set of sites bound in heart . This analysis rendered only 1 , 432 heart-specific peaks that were not enriched near different sets of deregulated genes in the Ctcf mutant . For known motif enrichment and de novo motif search using Homer [51] , we defined the promoter-proximal region as the 200 bp immediately upstream from the TSS of differential-expressed genes belonging to each of the “development” , “translation” and “mitochondria” categories , and the distal region as the next 1 . 8 kb upstream and the 2 kb downstream of the TSSs . We then identified all CTCF peaks present in these regions for each category , and used the 200 bp sequences surrounding the summit of CTCF peaks as input . As a background , we used 2 kb upstream of the TSSs of genes expressed but unchanged in our RNAseq data that had no CTCF peaks in that region ( 8 , 445 genes ) . As cut-offs we used p-value < 1e-10 for de novo motif search , and a Benjamini adjusted p-value < 0 , 001 for known motif enrichment . All sequencing data has been deposited at GEO under accession number GSE77644 . Hearts lysates of E10 . 5 and E11 . 5 prepared in RIPA buffer were separated by 12 . 5% SDS polyacrylaimde gel electrophoresis ( SDS PAGE ) and electroblotted onto PVDF transfer membrane ( BioRad ) . For protein detection , the following antibodies were used: CoI ( Complex IV subunit I; Invitrogen ) , Tom20 ( Santa Cruz Biotech ) , αActin ( Sigma Aldrich ) , CoxIV ( Complex IV subunit IV ) , Grp75 and mtFA ( Abcam ) . Quantification of signals arising from near-infrared ( NIR ) fluoropnores conjugated to secondary antibodies was performed by two-channel infrared ( IR ) scanner ( Odyssey v . 3 . 0 ) . Representative gels are shown in figures ( n = 3–4 ) . Mitochondrial isolation from E10 . 5 and E11 . 5 hearts were carried out in duplicate , as described [52] with some modifications . Pools of 4 embryonic hearts were homogenized in a glass homogenizer in buffer A ( Sucrose 0 , 32M; Tris 10mM , EDTA 1mM; pH7 , 4 ) . After centrifugation at 1000g , the nuclei were discarded and mitochondria were collected from the supernatant by a new centrifugation at 12000g . Mitochondrial proteins were solubilized with digitonin ( 4g/g ) ( Sigma D5628 ) and run on a 3%–13% gradient Blue Native gel as described [53] . After electrophoresis , the complexes were electroblotted onto PVDF membrane ( Merckmillipore , IPFL00010 ) and sequentially probed with specific antibodies against Complex I ( anti-Ndufa9 , ab14713 ) , Complex III ( anti-core1 , ab110252 ) , Complex IV ( anti-CoI , Invitrogen ref . 459600 ) , ATPsyntase ( anti-ATPB , ab14730 ) and VDAC1 ( anti-VDA1C , ab14734 ) . Embryonic hearts from E10 . 5 and E11 . 5 were isolated from the specified genotype ( n = 3 ) . Samples were fixed in 2 . 5% glutaraldehyde ( Sigma-Aldrich ) , 4% formaldehyde ( Electron Microscopy Sciences ) in 0 . 1M HEPES buffer for 3–4 h at 4°C , and processed as previously described [54] . Briefly , after buffer washes samples were post-fixed for 1h at room temperature in a 1:1 solution of 2% osmium tetroxide ( Electron Microscopy Sciences ) and 3% aqueous potassium ferrocyanide ( Sigma-Aldrich ) . Samples were rinsed in distilled water . Tissues were dehydrated through a graded acetone series and embedded in Spurr's Low Viscosity embedding mixture ( Electron Microscopy Sciences ) . Ultrathin sections ( 60 nm ) were then mounted on copper grids and stained with lead citrate . Samples were examined on a JEOL 10–10 electron microscope and analyzed by ImageJ v . 1 . 6 . 0 Software . 4C was performed as previously described [55 , 56] . Briefly , hearts were dissected from control and mutant ( Ctcffl/fl;Nkx2 . 5-Cre ) E11 . 5 embryos ( in order to be able to obtain more starting material ) . Each heart was minced and passed throw a 70 micrometers cell strainer , crosslinked with 2% PFA , frozen with liquid nitrogen , and stored at -80° for later processing once genotypes were established . Pools of 45–65 hearts from controls or mutants were lysed and digested first with DpnII ( New England Biolabs , cat . no . R0543M ) followed by Csp6I ( Fermentas , cat . no . ER0211 ) . For all experiments , 100 to 200 ng of the resulting 4C template was used for the subsequent PCR reaction ( primers used are detailed in S8 Table ) . Sequencing was performed by the CNIC Genomics Unit . All sequencing data has been deposited at GEO under accession number GSE77644 . For sample comparison between controls and mutants , 4C-seq data was normalized by total weight in a window of 15 Mb surrounding the IrxA cluster . The mean and standard deviation for each fragment were calculated for each group of replicates , and the difference was determined between the means in control and mutants for each viewpoint . Data were compared using the R package edgeR [57] , which applies statistical tests based on negative binomial distributions . For contact estimation , aligned reads were assigned to their corresponding virtual first cutter digested genome fragment . Each fragment end was considered as a captured site ( ligated to the viewpoint ) if one or more sequences mapped to it starting at the end of the fragment and in the right orientation , facing the center of the fragment . The number of captured sites was summarized per 30 fragment window ( max of 60 captured sites per window ) . The frequency of captured sites per window was used to fit a distance decreasing monotone function and z-scores were calculated from its residuals using a modified version of FourCSeq [58] . Significant contacts were considered in cases where the z-score was >2 in both replicates and deviated significantly ( adjusted p value <0 . 05 ) from its normal cumulative distribution in at least one of the replicates . Two guide RNAs ( g5 . 3-g3; S8 Table ) , together with the Cas9 protein with NLS ( PNA Bio ) , were injected into the pronucleus of E0 . 5 B6/CBA F1 embryos , which were then transferred to CD1 pseudo-pregnant females . Guide RNAs were injected at 25ng-μL , and the Cas9 protein at 30 ng/μL . Embryos from the established line were collected at E10 . 5 and processed for in situ hybridization and genotyping ( primers listed in S8 Table ) .
|
Properly regulated gene expression in time and space during development and differentiation requires not only transcriptional inputs , but also specific structuring of the chromatin . CTCF is a DNA binding factor that is believed to be critical for this process through binding to tens of thousands of sites across the genome . Despite the knowledge gained in recent years on the role of CTCF in genome organization , its functions in vivo are poorly understood . To address this issue , we studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development . Surprisingly only a fraction of genes change their expression when CTCF is removed . Importantly , misregulated genes control opposing genetic programs in charge of development and patterning on one hand , and cardiomyocyte maturation on the other . This imbalance leads to faulty mitochondria and incorrect expression of cardiac patterning genes , and subsequent embryonic lethality . Our results suggest that CTCF is not necessary for maintenance of global genome structure , but coordinates dynamic genetic programs controlling phenotypic transitions in developing cells and tissues .
|
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"and",
"methods"
] |
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"medicine",
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2017
|
CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart
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The clustering of transcription factor binding sites in developmental enhancers and the apparent preferential conservation of clustered sites have been widely interpreted as proof that spatially constrained physical interactions between transcription factors are required for regulatory function . However , we show here that selection on the composition of enhancers alone , and not their internal structure , leads to the accumulation of clustered sites with evolutionary dynamics that suggest they are preferentially conserved . We simulated the evolution of idealized enhancers from Drosophila melanogaster constrained to contain only a minimum number of binding sites for one or more factors . Under this constraint , mutations that destroy an existing binding site are tolerated only if a compensating site has emerged elsewhere in the enhancer . Overlapping sites , such as those frequently observed for the activator Bicoid and repressor Krüppel , had significantly longer evolutionary half-lives than isolated sites for the same factors . This leads to a substantially higher density of overlapping sites than expected by chance and the appearance that such sites are preferentially conserved . Because D . melanogaster ( like many other species ) has a bias for deletions over insertions , sites tended to become closer together over time , leading to an overall clustering of sites in the absence of any selection for clustered sites . Since this effect is strongest for the oldest sites , clustered sites also incorrectly appear to be preferentially conserved . Following speciation , sites tend to be closer together in all descendent species than in their common ancestors , violating the common assumption that shared features of species' genomes reflect their ancestral state . Finally , we show that selection on binding site composition alone recapitulates the observed number of overlapping and closely neighboring sites in real D . melanogaster enhancers . Thus , this study calls into question the common practice of inferring “cis-regulatory grammars” from the organization and evolutionary dynamics of developmental enhancers .
The transcriptional output of developmental enhancers is affected by the spatial organization of the transcription factor binding sites they contain . The relative positioning of sites is known from individual cases to modulate direct competition between factors for the same site [1] , [2] , cooperative and repressive interactions between transcription factors [3] , [4] , and the formation of higher-order regulatory complexes [5]–[7] . However , we have a precise understanding of the relationship between binding site organization and function for few , if any , developmental enhancers . In the absence of efficient experimental protocols for dissecting enhancer function , recent efforts have attempted to infer functional constraints on binding site organization from the distribution and evolution of binding sites in enhancers of interest . We recently examined developmental enhancers in species distantly related to D . melanogaster and found a strong preferential conservation of overlapping and proximal sites [8] , a result which was confirmed by a recent survey of enhancer evolution across the twelve sequenced Drosophila genomes [9] . Others have focused on the density of overlapping and proximal sites , finding that both are significantly enriched [10] , [11] . All of these studies , including ours , reached a similar conclusion: the evolutionary dynamics of binding sites in developmental enhancers suggest that clustered and/or overlapping sites are common functional necessities for enhancer activity . This shared conclusion was premised on the idea that the observed non-random arrangement of sites must be a result of selection on the relative positioning of sites within enhancers . However , alternative explanations for these phenomena , especially the possibility that such arrangements might arise as a byproduct of other mutational and selective pressures [12] , have not been explored . Here we simulate the evolution of real and synthetic D . melanogaster enhancers constrained only to maintain their binding site composition and investigate the spatial organization of binding sites within enhancers evolving with no direct selection on the arrangement of sites within them . We show that this simple global constraint on enhancer composition is sufficient to produce many of the organizational and evolutionary features observed in real enhancers , including enrichment and apparent conservation of overlapping and clustered sites .
We used simulations to explore the properties of enhancers evolving under selection on binding site composition . We subjected synthetic enhancers , in which a predefined number of binding sites for one or more transcription factors were randomly positioned in randomly generated sequence with the same composition as D . melanogaster non-coding DNA , to mutations sampled from the distribution of substitutions , insertions and deletions observed in D . melanogaster [13] . We applied a strong selective constraint to these mutated sequences . If the number of sites in the enhancer fell below a specified threshold , we rejected the new sequence . Otherwise , it was carried through to the next mutational step ( Figure 1 ) . Because such a strict cutoff might not be realistic , we compared the results of these simulations to those involving a large population of enhancers in which suboptimal sequences were assigned a fitness penalty rather than immediately removed . None of the measures of binding site distribution and evolution discussed below differed appreciably between these models ( see Text S1 ) . Since these population simulations required significantly greater computational resources , we present only the results of the simpler model below . The most basic property of our model of enhancer evolution is that most mutations that destroy a binding site will be rejected , as they bring the number of sites present in the enhancer below the specified fitness threshold ( Figure 1B ) . However , the small size of most binding sites means that they are generated de novo by random mutation at an appreciable rate . And , once new sites are generated , mutations that destroy existing sites will be tolerated ( Figure 1C ) , leading to non-homologous site conservation , or “binding site turnover” [14]–[17] . The rate at which mutations destroy existing sites for a given factor and create new ones depends on the size and degeneracy of the site recognized by the factor . To examine how specificity affects these rates , we simulated the evolution of enhancers constrained only to have a single site matching real , or randomly generated , transcription factor specificities . The rate of turnover varied considerably , depending on the size of the recognition site , its base composition and degeneracy ( Figure 2 ) , with the variance primarily explained by the rate at which new binding sites are generated from random DNA . Longer , less degenerate and GC-rich sites are generated from random sequence at a lower rate and thus turn over more slowly . The expected half-life ( measured in mutational distance ) of binding sites for the typical D . melanogaster transcription factor was between one and two substitutions per site , or around 50 to 100 million years . This is consistent with previous studies of turnover rates for functional sites in real enhancers that have estimated that there have been around one to two turnover events per site per hundred million years [17] , [18] . Some transcription factors , such as the D . melanogaster proteins Bicoid ( BCD ) and Krüppel ( KR ) , overlap in their binding specificities , so that the same bases can be part of binding sites for multiple factors [19]–[21] . In specific cases competition between BCD and KR for overlapping sites plays an important role in producing specific expression patterns [22] , [23] . The high frequency of overlapping BCD and KR sites in other embryonic enhancers has been used as evidence for the generality of this mechanism [10] . However , when we simulated the evolution of synthetic 1 , 000 bp enhancers constrained to contain five BCD and five KR sites , we find an almost twofold elevation in the frequency of overlapping BCD and KR sites compared to the random expectation ( Figure 3A ) . Thus selection acting to preserve enhancer composition alone indirectly leads to “higher order” structure in enhancers . This phenomenon is not specific to BCD and KR: rather it is a general property of factors with overlapping binding specificities ( data not shown ) . The increase in the density of overlapping sites is almost entirely due to their increased half-life relative to isolated sites . In the BCD/KR simulations described above , which had no explicit selection to maintain overlapping sites , overlapping sites persisted 1 . 5 to 2 . 0 times longer ( depending on the specific choice of matrix ) than isolated sites ( Figure 3B , Figure S1 , and Figure S2 ) . This difference in half-life between overlapping and isolated sites not only increases the density of overlapping sites; it also significantly alters how they are classified in comparative genomic analyses . Their longer half-life means that overlapping sites are more likely to be found at orthologous positions in related species . In particular , at evolutionary distances in the range typically used for comparative analyses ( around one substitution per site ) the likelihood of finding an orthologous overlapping pair of BCD and KR sites is two times larger than the likelihood of finding an orthologous singleton site ( Figure 3B , Figure S1 , and Figure S2 ) . Thus , our simulations show that selection to maintain enhancer composition not only leads to an increase in the density of overlapping sites , it also makes it appear that selection is acting to specifically preserve them . Binding sites in real enhancers are clustered , with an excess of short inter-binding-site distances at the expense of long ones [10] , [11] . This clustering has been interpreted as evidence that long-range interactions between transcription factors or between transcription factors and nucleosomes are required for proper gene regulation [10] , [11] . However , in our simulations , we also observed an increase in the proportion of small spacers ( Figure 4A ) . This induced binding site clustering occurred whenever the mutation model included a bias for deletions over insertions , a known property of Drosophila species [24] . When simulations were run with only point mutations , or with balanced insertions and deletions , no increase in short spacers was observed . Unlike point mutations , deletions can disrupt multiple non-overlapping binding sites . In our simulations , deletions affecting two or more sites were less than half as likely to be accepted as were deletions affecting single sites ( 10 . 5% compared to 23 . 2% of the time ) . Thus it is possible that the induced binding site clustering arises from the protective effect proximal sites have against each other's deletion ( Figure 4B ) . Indeed , in simulations that exclusively involved deletions , tightly spaced but non-overlapping sites showed a substantial increase in half-life ( Figure S3 ) . However , in simulations with a realistic balance of mutations and indels this effect was minimal ( Figure S4 ) , as the frequency of multi-site deletions was low relative to single site deletions and point mutations . Instead , the induced binding site clustering appears to be driven simply by the deletion of spacer DNA between sites . Since , in our simulations , deletions between sites occur more frequently than sites are lost , sites get closer together over time , distorting the distribution of inter-site distances . A corollary of this phenomenon is that sites that are observed to be close together tend to be older , and therefore more likely to be labeled as conserved , than isolated sites ( Figure 4C ) . Thus , both binding site clustering and an apparent preferential conservation of clustered sites are expected to occur even in the absence of any selection on enhancer organization . Sequence features present in multiple related species are generally considered to reflect those found in the shared ancestor , whether through selection or common descent . However , the deletion bias-induced tendency for sites to get closer together over time distorts this relationship . To illustrate this , we placed two sites at a fixed distance and monitored the distance between them over time in a large number of independent simulations . With indels , but no bias towards deletions either in frequency or in average length , the intersite spacing quickly diverges between simulations ( Figure 5A ) . However , with the observed Drosophila deletion bias , the spacing between sites in the different simulations is strongly correlated ( Figure 5B ) . Thus , with a deletion bias , the spacing between sites after speciation will appear conserved and yet reflect neither selection nor the ancestral state . To examine how this relationship between inter-site spacing and age might affect evolutionary inference , we simulated the divergence after speciation of regulatory sequences containing pairs of binding sites separated by varying distances . We then compared , at different times after divergence , the inter-site spacing in orthologous evolved sequences . Roughly following practice in the field , we considered the spacing to be “conserved” if the sites were within 30 bp in both species . Even where the starting spacing was 30 bp , the probability that it remained within 30 bp in both species in the absence of a deletion bias is small at evolutionary distances beyond one substitution per site ( Figure 5C ) . But with a deletion bias , the probability is substantially higher , and is appreciable even for starting spacings of 50 or 100 bp ( Figure 5C–5E ) . Thus , comparison of binding site spacing in multiple species with deletion biases will often lead to the incorrect inference that selection has acted to preserve close spacing of binding sites . To assess whether the above-described effects could replicate the degree of binding site overlap and clustering that is observed in extant enhancers , we simulated the evolution of the well-characterized eve stripe 2 enhancer [23] , with compositional constraints derived from the extensive biochemical and genetic literature on this enhancer . In particular we required five Krüppel , ten Bicoid , three Hunchback , five Giant [25] , and a single Zelda [26] binding site ( see Table 1 ) . We also required that a certain number of sites for each factor be predicted high-affinity sites ( based on the number of high-affinity sites in the D . melanogaster enhancer ) . We simulated 1 , 000 replicates of this enhancer to twenty substitutions per site , and found that both the number of overlapping BCD and KR sites , and the number of sites in close proximity to others , in the real enhancer were well within the range typically generated by this architecture-free evolutionary model ( Figure 6A and 6B ) .
In retrospect , the properties we observed are straightforward consequences of coupling selection on binding site composition with a deletion-biased mutational process . One does not need simulations to see why overlapping sites will clearly turn over less frequently than isolated sites , that a deletion bias will drive sites closer together over time , and how both phenomena distort comparative analyses . But as self-evident as these results may appear , they have never been noted before , despite more than a decade of intense comparative genomic analysis of enhancer structure and function in Drosophila . Indeed , prior to performing these simulations we did not consider that the clustering of binding sites in Drosophila enhancers might arise from a deletion bias . We simply attempted to have our simulations accurately reflect the underlying mutational process in our simulations , with the consequences evident only in the results . This highlights the value of simulations of simple evolutionary processes in uncovering unappreciated consequences of our models and assumptions . Furthermore , although the general effects of selection on binding site composition and of a deletion bias can be intuited , specific quantitative aspects of the model are difficult to work out analytically . For example , while we have developed a mathematical model for the effect on half-life of overlapping sites in enhancers ( see Text S2 ) , it is difficult to extend this model to enhancers with multiple sites . Simulations can answer these questions simply and effectively . The simulations we performed here used non-coding DNA , transcription factor binding sites , and mutation patterns from D . melanogaster . Interspecies differences in the composition of non-coding DNA , specificity of transcription factors , and base substitution patterns will have minimal effect on our conclusions . However , differences in the indel rate and the balance of insertions and deletions could significantly alter the existence or magnitude of the induced binding site clustering . Although the deletion biased mutation process we used in our model is often thought of as a Drosophila-specific phenomena , there is increasing evidence that short indels are deletion biased in all species [30]–[35] . Thus , we expect this effect to be general , although the magnitude will differ depending on the indel rate and bias ( see Text S3 ) . Lynch has eloquently argued that biologists are often too quick to assume that organismal and genomic complexity must arise from selection for complex structures and too slow to adopt non-adaptive hypotheses [12] . Our results lend additional support to this view , and extend it to show that indirect and non-adaptive forces can not only produce structure , but also create an illusion that this structure is being conserved . We do not doubt that many aspects of transcriptional regulation constrain the location of transcription factor binding sites within enhancers . Indeed a large body of experimental evidence supports this notion , and we remain committed to identifying and characterizing these constraints . But if this process is to be fueled by comparative sequence analysis , as we believe it must be , it is essential that we give careful consideration to the neutral and indirect forces that we now know can produce evolutionary mirages of structure and function .
Starting sequences 1 , 000 basepairs in length were generated randomly to match the base composition of D . melanogaster non-coding DNA , and binding sites were added to bring the starting density of sites to the specified thresholds . Mutations were sampled randomly from point mutations , insertions , and deletions . 80% of mutations were point mutations generated from an HKY85 [36] model with GC content 40% and kappa two; 12% were deletions and 8% insertions with size distributions drawn from [13] . The deletion bias ( 60% ) , and proportion of all mutations that were indels ( 20% ) , were also according to [13] . Except where noted , simulations took place for 100 , 000 mutation/selection rounds . To compensate for the change in the size of the enhancer when insertions and deletions occurred , bases were removed or added from the nearest edge of the sequence . New base pairs added with a 40% GC content . The simulation software was written in Python and utilizes the Motility [37] binding site identification package . Simulations using BCD and KR used matrices from in vitro footprinting [38] , one-hybrid assays [39] , and SELEX [40] , with cutoff scores chosen to match expected numbers of their sites in the even-skipped stripe two enhancer: 5 . 5 , 4 . 9 , and 4 . 1 for BCD and 5 . 6 , 4 . 1 , and 0 . 0 for KR for the three sources of matrices . Unless noted otherwise , simulations used matrices from the footprinting data set . In the simulations in presented in Figure 5 , we sought only to examine the evolution of site spacing over time and not the conservation and/or turnover of individual binding sites . Thus , we preconditioned in each case that neither could binding sites be generated from random sequence nor could existing binding sites be disrupted . To this end , in these simulations , all mutations affecting positions contained within existing binding sites were considered precluded by selection and discarded , and , similarly , the sequence was not scored for new binding sites created by mutations . We generated Figure 5A and 5B by simulating 980 , 000 300 base pair sequences to 30 substitutions per site , and Figure 5C–5E by simulating 480 , 000 300 base pair sequences to ten substitutions per site . In the even indels case , the distribution of insertion lengths was set equal to the distribution of deletion lengths . Properties of the simulations were computed following a lengthy ( ∼30 subs/site ) burn-in period that allowed the randomly generated starting model to reach equilibrium . We tested several sets of neutral mutation and selective parameters to make sure this burn-in period was sufficient ( Figure S5 , Figure S6 , and Figure S7 ) . We chose binding site lengths randomly between five and twelve . At each position , we chose a consensus nucleotide and assigned its frequency by sampling a Gaussian with mean 0 . 8 and standard deviation 0 . 2 . Subsequent nucleotide frequencies were chosen similarly , each being given a frequency chosen from a Gaussian with a mean and standard deviation of 80% and 20% of the remaining probability mass , respectively . Weight matrices were constructed against a 40% GC bias and threshold scores were sampled from a uniform distribution spanning zero to the maximum scores of the sites . Information content was calculated by weighting all N-mers above the score threshold with the GC bias and subtracting the information in an N-mer of random sequence of equal length and GC bias . To find the expected probability KR and BCD sites would overlap in random DNA , we sampled random ten-mers from a 40% GC background distribution . If this sequence contained a KR site , then we added flanking sequence of length N-1 , where N is the length of a BCD site . If this sequence also contained a BCD site , then we considered it as an overlap . The probability of a BCD site generating a KR site was found in an analogous manner . The post-selection conditional probability was directly calculated by simulating an enhancer with five sites for each transcription factor as described above and counting observations of singleton and overlapped binding sites . We determined the half-lives of sets of binding sites by randomly sampling individual sites in our simulations and observing their degradation as the simulations progressed . Our data consisted of simulations of 1 , 000 enhancers , each run for 30 , 000 iterations . For each enhancer , after a burn-in period of 10 , 000 iterations , we took a ‘snapshot’ of the binding sites present every 3 , 000 iterations . In each subsequent iteration of the simulation , the presence or absence of each binding site in the snapshot was assessed: if it had been destroyed by a point mutation or indel in that iteration , then a site ‘death’ was recorded . This process was repeated for 2 , 000 post-snapshot iterations of the simulation . We used one-hybrid binding sequences for Hunchback , Giant , Bicoid , and Krüppel from [39] and created weighted matrices as described . We used the same methods to generate a Zelda-consensus matrix from the sequences listed in [41] . Our enhancer sequence and matrices are available in Dataset S1 . In order to determine the required number of sites for each matrix , we assessed the number of hits it had to the eve stripe 2 sequence at several score cutoffs . If the number of hits at a given score cutoff exceeded the number expected by chance , then this number/score cutoff pair was accepted as a requirement , provided that it did not substantially increase the total required number of sites for that factor beyond that described in [23] . The constraint on the enhancer is described in Table 1 .
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Because mutation is a random process , most biologists assume that apparently non-random features of genome sequences must be the result of natural selection acting to create and preserve them . Where this is true , genome sequences provide a powerful means to infer aspects of molecular , cellular , and organismal biology from the signatures of selection they have left behind . However , recent analyses have shown that many aspects of genome structure and organization that have traditionally been attributed to selection can often arise from random processes . Several groups—including ours—studying the sequences that specify when and where genes should be produced have identified common , seemingly conserved , architectural features , based on which we have proposed new models for the activity of the complex molecular machines that regulate gene expression . However , in the work described here we simulate the evolution of these regulatory sequences and show that many of the features that we and others have identified can arise as a byproduct of random mutational processes and selection for other properties . This calls into question many conclusions of comparative genome analysis , and more generally highlights what Michael Lynch has called the “frailty of adaptive hypotheses” for the origins of complex genomic structures .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"genetics",
"and",
"genomics/gene",
"expression",
"genetics",
"and",
"genomics/comparative",
"genomics",
"evolutionary",
"biology/genomics"
] |
2010
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Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers
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Transmissible spongiform encephalopathies ( TSE ) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein ( PrP ) to a misfolded , protease-resistant form ( PrPres ) . Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases . The N171S PrP polymorphism is found mainly in humans of African descent , but its low incidence has precluded study of its possible influence on prion disease . Similar to previous experiments of others , for laboratory studies we created a transgenic model expressing the mouse PrP homolog , PrP-170S , of human PrP-171S . Since PrP polymorphisms can vary in their effects on different TSE diseases , we tested these mice with four different strains of mouse-adapted scrapie . Whereas 22L and ME7 scrapie strains induced typical clinical disease , neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation , strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times . When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie , dominant-negative inhibition of disease did not occur , possibly because interaction of strain RML with PrP-170S was minimal . Surprisingly , in vitro PrP conversion using protein misfolding cyclic amplification ( PMCA ) , did not reproduce the in vivo findings , suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro . These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient . PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms .
TSE or prion diseases are transmissible neurodegenerative diseases occurring in a variety of mammalian species including domestic and wild animals as well as humans [1] . Examples include scrapie in sheep , chronic wasting disease ( CWD ) in cervids and bovine spongiform encephalopathy ( BSE ) in cattle . Human diseases include sporadic and variant Creutzfeldt-Jakob disease ( sCJD and vCJD ) and familial diseases such as Gerstmann–Sträussler–Scheinker syndrome ( GSS ) , Fatal Familial Insomnia ( FFI ) and familial CJD . A hallmark of prion diseases is the conversion of the normal protease-sensitive host prion protein ( PrPsen ) into a misfolded partially protease-resistant form ( PrPres ) which may be in part responsible for the generation of the disease [2] . PrP expression is required for prion disease , and PrP knockout mice are resistant to both infection and disease [3] . PrP gene polymorphisms occur naturally in many species . In both sheep and in mice such polymorphisms have been found to influence susceptibility to scrapie infection and disease . For example , sheep expressing Alanine , Arginine , Arginine at positions 136 , 154 and 171 ( A136R154R171 ) respectively , are highly resistant to most strains of sheep scrapie [4] , [5] , [6] . However , recent observations indicate that sheep with the A136R154R171 genotype can be susceptible to atypical scrapie ( Nor98 ) suggesting that resistance might be strain specific [7] , [8] . Similarly in inbred mice there are two known PrP alleles ( Prnpa and Prnpb ) which are characterized by amino acid differences at PrP residues 108 and 189 [9] , [10] , [11] . Mice with Prnpa have short incubation times with one set of scrapie strains and prolonged incubation times with another set , while mice with Prnpb show an opposite pattern of incubation times with these same scrapie strains [12] , [13] . Thus there is a different strain-specific pattern of susceptibility associated with each of these Prnp genotypes . In humans , the methionine vs . valine polymorphism at PrP residue 129 appears to influence the clinical phenotype of familial prion disease associated with the D178N PrP mutation [14] , [15] , as well as susceptibility to sCJD , vCJD and kuru [16] , [17] , [18] , [19] , [20] . In addition , the human polymorphism E219K may be associated with resistance to sCJD [21] , but does not appear to correlate with resistance to vCJD in humans or mouse models [22] , [23] . The human PrP polymorphism , G127V , was recently shown to be associated with resistance to kuru [24] . Other naturally occurring human polymorphisms , such as G142S and N171S , have not been tested for prion disease susceptibility [25] . N171S is a PrP polymorphism found in Sub-Saharan Africans , Jamaicans and Sardinians [26] . The rarity of this polymorphism and the fact that it is present mainly in geographical regions with limited CJD surveillance make it difficult to detect possible associations with prion disease . Therefore , in order to initiate laboratory studies of the possible effects of this polymorphism on TSE diseases and possibly also other CNS disorders , we generated transgenic mice expressing mouse PrP-170S , the mouse homolog of human PrP-171S ( Table 1 ) . This approach of using transgenic mice expressing mouse or hamster PrP with human PrP mutations and/or polymorphic residues at homologous sites has been taken in numerous previous studies of prion diseases . These include important studies of models of Gerstmann-Sträussler-Scheinker syndrome ( GSS ) [27] , [28] , [29] , [30] , [31] , [32] , FFI [33] and familial CJD [34] , [35] , as well as a non-infectious neurodegenerative disease associated with expression of prion protein with a nine octapeptide insertion [36] . In addition , transgenic mice expressing mouse PrP with cervid PrP residues associated with a “rigid loop” in PrP have also revealed interesting in vivo pathogenic effects [37] , [38] . Because PrP variations in animals and humans can have different effects on different TSE strains , we tested our mice with four different scrapie strains which have been maintained in continuous mouse passage for many years . In these studies PrP-170S expressing mice were highly susceptible to scrapie strains 22L and ME7 , but were markedly resistant to scrapie strains RML and 79A . In contrast , control mice were susceptible to all four strains . This in vivo strain-specific influence on scrapie susceptibility was not reproduced in cell-free in vitro PrP conversion studies , suggesting that in vivo conditions not replicated in our in vitro system were required . PrP-170S transgenic mice appear to be an interesting new model to study the interactions between TSE agent strains and PrP .
All mice were housed at the Rocky Mountain Laboratories ( RML ) in an AAALAC-accredited facility and experimentation followed NIH RML Animal Care and Use Committee approved protocols ( NIH/RML Protocol #2007-31 ) . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . To study the effect of the human N171S PrP polymorphism ( Ref SNP#rs16990018 ) we constructed transgenic mice expressing serine instead of asparagine at mouse PrP residue 170 ( homologous to human PrP residue 171 ) ( Table 1 ) . The mutation was made at residue 170 in mouse PrP using a cDNA clone of mouse PrP , p1-5 ( E48-16 ) [39] using oligonucleotides 2077U ( 5′- CCA GTG GAT CAG TAC AGC AGC CAG AAC AAC TTC GTG C -3′ ) and 2078L ( 5′- GCA CGA AGT TGT TCT GGC TGC TGT ACT GAT CCA CTG G -3′ ) with a site directed mutagenesis kit ( Stratagene/Agilent , Santa Clara , CA ) . The plasmid with the mutation was recloned , and the mutation was confirmed by sequencing . A DNA fragment from PshAI to SfoI with PrP sequences containing the mutation was excised , purified and religated into a subclone , p44-3 ( E58-6 ) [39] , [40] , derived from the pHGPrP half-genomic clone [41] by digesting with AgeI and SfoI to remove a portion of the PrP ORF and replacing this segment with an oligonucleotide polylinker containing these and other sites . Upstream sequences previously excised between two BamHI sites to remove an unwanted SfoI site were replaced by digestion with adjacent sites NotI and BspEI and the original 6 . 2kb fragment from PHGPrP was reinserted . The resulting plasmid , p188-6 , was digested with NotI and SbfI to separate mouse sequences from bacterial plasmid sequences , and the fragment containing the mouse sequences was used to inoculate C57BL/6 mouse eggs to generate transgenic mice expressing PrP-170S [39] . Of the five transgenic founding lines produced , three lines ( Tg330 , Tg340 and Tg290 ) were selected for experimentation . These transgenic lines were hemizygous for the transgene and homozygous for Prnp , the gene encoding normal mouse PrP ( MoPrP ) . The Prnp gene encoding PrP-170N in C57BL/6 mice was removed by serial backcrossing to C57BL/10Sn mice with a knocked-out Prnp gene ( B10 PrP-/- ) derived from the original Edinburgh Prnp knockout mouse as previously described [39] , [42] . Transgenic lines were maintained as transgene heterozygotes by crossing to C57BL/10 PrP-/- mice and selection of transgene positive mice by PCR analysis of tail DNA . Genotyping was conducted using standard PCR reactions as previously described [39] . Briefly , detection of the modified knock-out version of Prnp and the neo cassette in the PrP-/- mice was accomplished using primers RK1 and Mut217 as previously described [43] . These primers yielded a C57BL/10 MoPrP product of approximately 700 bp and a PrP-/- neomycin cassette gene product of approximately 1700bp . Detection of the half-genomic PrP transgene , expressing the N170S PrP construct , was accomplished using primers pE2+ and Mut217 as described previously [43] . PCR products were visualized via electrophoresis in a 2% agarose gel . All mice were housed at the Rocky Mountain Laboratories ( RML ) in an AAALAC-accredited facility and experimentation followed NIH RML Animal Care and Use Committee approved protocols ( NIH/RML Protocol #2007-31 ) . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . Mice were bred and genotyped at RML . For use as controls , weanling C57BL/10Hsd mice were obtained from Harlan Sprague Dawley , Madison , WI . Transgenic tga20 mice were obtained from EMMA ( Munich , Germany ) [41] . Mice were injected intracerebrally ( i . c . ) with 50μl of a 1% ( wt/vol ) dilution of brain homogenate pools from C57BL mice terminally ill from 22L , RML , 79A or ME7 scrapie . Two stocks of RML scrapie ( RML-06 , RML-81 ) were used . Titers of scrapie stocks were determined in previous i . c . endpoint titration experiments and were as follows; 22L = 2 . 5×109 , ME7 = 4 . 0×108 , RML-06 = 3 . 2×108 , RML-81 = 4 . 8×108 , 79A = 1 . 6×108 ( units = 50% infectious dose ( ID50 ) /gm of brain ) . Brain homogenates were diluted for inoculation in phosphate buffered balanced saline ( PBBS ) pH 7 . 2 , supplemented with 2% fetal bovine serum ( Hyclone , Logan , UT ) . Observations were made daily to assess clinical signs of scrapie disease , which included ataxia , altered gait , wasting , kyphosis , hind limb weakness , aimless wandering , somnolence , immobility and leg clasping reflex . Mice with clinical signs were euthanized and brain tissue was analyzed for PrPres by immunoblotting . Mice with both clinical signs and PrPres by immunoblot were defined as diseased , and the day of euthanasia was recorded as the incubation period in the data presented . Uninoculated control transgenic mice were followed up to 700 days of age and showed no clinical signs of scrapie differing from normal signs of senescence . Several uninoculated mice were analyzed by histopathology after euthanasia at 465 days of age , and brain tissue had no detectable grey matter vacuolation or abnormal PrPres deposition . Statistical analysis of data in the coexpression experiment was done by a one way ANOVA with Dunnett's multiple comparison test using GraphPad Prism software . Tissue samples from mice were analyzed for PrPres and PrPsen by immunoblot . Brain homogenates ( 20%w/v ) were prepared in 10 mM Tris-HCl [pH 7 . 4] using a mini-beadbeater ( Biospec products , Bartlesville , OK ) . All homogenates were sonicated for 1 min using a Vibracell cup-horn sonicator ( Sonics , Newtown , NJ ) as previously described [40] . To test for PrPres , samples were proteinase K treated as follows; 20 µl of a 20% ( w/v ) tissue homogenate was adjusted to 100 mM Tris HCl ( pH 8 . 3 ) , 1% Triton X-100 , 1% sodium deoxycholate and 50 µg/ml proteinase K ( PK ) , in a total volume of 31 µl . Tubes were mixed and incubated for 45 minutes at 37°C . The reaction was stopped by adding 2 µl of 100 mM Pefabloc ( Roche Diagnostics , Indianapolis , Indiana ) and placed on ice for 5 min . Samples tested for PrPsen were treated with proteinase inhibitors: 10 µM leupeptin , 1 µM pepstatin A , and 7 µg/ml aprotinin . PrPsen samples were not treated with PK . An equal volume of 2X Laemmli sample buffer ( Biorad , Hercules , CA ) was added to both PrPsen and PrPres samples , and then tubes were boiled 5 minutes . Samples were frozen at −20°C until the day of analysis , when samples were thawed , reboiled for 5 minutes and then electrophoresed on a 16% Tris-Glycine SDS-PAGE gel ( Invitrogen , Carlsbad , CA ) and blotted to PVDF membranes ( Biorad ) using a 7 minute transfer , program 3 ( P3 ) on an iBlot ( Invitrogen ) device . Immunoblots were blocked in a solution of 2 . 5% milk ( Biorad ) in 0 . 1 M Tris , 1 . 5M NaCl and 0 . 05% Tween20 , for 1 hour . Then blots were probed with D13 anti-PrP monoclonal antibody [44] , ( InPro , San Francisco , CA ) at a final concentration of 0 . 2 µg/ml , followed by a peroxidase-conjugated anti-human IgG secondary antibody ( Sigma , St . Louis , MO ) at a final dilution of 1∶5000 in the blocking buffer described above . Bands were detected using enhanced chemiluminescence substrate ( ECL ) as directed by manufacturer ( GE Healthcare , Pittsburgh , PA ) . For histopathological analysis mice were deeply anesthetized and euthanized by cervical dislocation . Tissues were removed and placed in 3 . 7% phosphate-buffered formalin for 3 to 5 days before dehydration and embedding in paraffin . Serial 5 µm sections were cut using a standard Leica microtome , placed on positively charged glass slides and dried overnight at 43°C . Slides were then deparaffinized using standard procedures . Slides were stained with hematoxylin and eosin and analyzed for pathological changes . Immunohistochemical detection of PrPres using DAB chromogen ( DAB Map kit; Ventana Medical Systems , Tucson , AZ . ) was done as follows: antigen retrieval and staining were performed using the Ventana automated Discovery XT stainer . PrP antigens were exposed by incubation in CC1 buffer ( Ventana ) containing Tris-Borate-EDTA , pH 8 . 0 for 20 minutes at 95°C . Staining for PrP was done using human anti-mouse PrP monoclonal antibody D13 at a dilution of 1∶500 at 37°C for 2 hours , followed by a biotinylated anti-human IgG at 1∶500 ( Jackson ImmunoResearch , West Grove , PA . ) , and avidin-horseradish peroxidase with DAB as chromogen . Slides were examined and photomicrographs were taken observed using an Olympus BX51 microscope and Microsuite FIVE software . For preparation of normal brain homogenates containing PrPsen to be used as the substrate for PMCA reactions healthy transgenic mice expressing either PrP-170S ( Tg330 ) or PrP-170N ( Tga20 ) [41] were deeply anesthetized and then perfused with PBS containing 5 mM EDTA . Brains were removed and homogenized in a beadbeater at a concentration of 20% ( w/v ) in PMCA conversion buffer ( PBS containing 4 mM EDTA , 1% Triton X-100 and complete mini-protease inhibitor cocktail ( Roche ) , sterilized by filtration with a 0 . 2 µm filter ) then cooled on ice for 10–15 min . Homogenates were diluted with PMCA conversion buffer to 10% ( w/v ) then clarified by a brief 1500 X g spin [45] . Supernatants were stored at −80° C in 1 ml aliquots until used in PMCA reactions . Brain homogenates used as seeds for the PMCA reactions came from clinically sick 22L or RML-infected C57BL/6 mice . Brains were homogenized at a 20% ( w/v ) concentration using 0 . 1 M Tris pH 7 . 4 , then diluted in the same buffer to a final concentration of 10% ( w/v ) prior to storage at −80°C . Scrapie-positive brain homogenates were used to “seed” normal brain homogenate in the following manner . A 10% scrapie-positive brain homogenate ( seed ) was added to normal brain homogenate ( substrate ) at desired dilutions and then these master mixes were aliquoted into multiple 0 . 2ml reaction tubes ( GeneMate , ISCBioexpress , Kaysville , Utah ) . One of these tubes was frozen as an unsonicated control and the remaining tubes were repeatedly sonicated and incubated as described below . Tubes were positioned in a plastic tube rack PMCA adapter ( Misonix , Farmingdale , NY ) and placed on the rim of a microplate horn of a Misonix Model 3000 microsonicator so that the 50 µl samples were immersed in the sonicator bath . The microplate horn was covered with a plastic lid to minimize evaporation from the water bath . The sonicator was located inside an incubator set to 37°C and was programmed to perform cycles , each consisting of a 40 second pulse of sonication set at 60% maximum followed by a 30 min incubation . Forty-eight cycles ( i . e . 24 h ) constituted one round of PMCA . PMCA reaction tubes were removed from the sonicator and vortexed and 10 µl of the 50 µl volume was sampled and mixed with 10 µl PK at 100 µg/ml for a final PK concentration of 50 µg/ml . Samples were maintained at 37°C in a water bath for one hour . The PK digestions were halted with 2 µl of 100 mM Pefabloc ( Roche Diagnostics ) and placed on ice for 5 min . An equal volume of 2X Laemmli sample buffer ( Biorad , Hercules , CA ) was added to the PMCA samples , and then tubes were boiled 5 minutes . Samples were frozen at −20°C until the day of analysis , when samples were thawed , and reboiled for 5 minutes . PMCA products were visualized by SDS page gel electrophoresis and immunoblotting as described above for brain tissues , with the following exceptions . Gels were blotted to HyBond ECL nitrocellulose membranes ( GE Healthcare Life Sciences ) instead of PVDF membrane . Immunoblots were blocked in Near-Infrared Fluorescence Western Blotting Blocking Buffer ( Rockland Immunochemicals Inc . , Gilbertsville , PA ) and PBS mixed in equal parts . Primary antibody was D13 1∶100 diluted supernatant derived from CHO cells expressing the D13 antibody construct [44] . These cells were obtained from R . Anthony Williamson , The Scripps Research Institute , La Jolla , CA . The secondary antibody was IRDye800CW-conjugated goat-anti-mouse IgG ( LiCor , Lincoln , NE ) diluted at 1∶10 , 000 . Both antibodies were diluted in the blocking buffer described above with the addition of 0 . 2% Tween 20 . Finally , bands were detected using an Odyssey near-infrared fluorescence scanner ( LiCor ) . Groups were compared using a Mann-Whitney test with GraphPad Prism software .
To study the effect of the human PrP polymorphism , N171S , on susceptibility to prion disease we generated transgenic mice expressing mouse PrP-170S , the mouse homolog of the human PrP-171S ( Table 1 ) . Five founder lines were produced on the C57BL/6 background , and these were crossed serially to C57BL/10 mice homozygous for the Edinburgh version of the PrP null gene ( Prnp-/- ) [42] . Three lines of the transgenic mice ( Tg290 , Tg330 , and Tg340 ) with the Prnp-/- gene , and hemizygous for the PrP-170S transgene , were obtained and used for further study . Because PrPsen expression is known to influence scrapie incubation period [41] , [46] , we determined PrPsen levels in brain by immunoblot . Both Tg330 and Tg340 mice expressed PrPsen at levels 2 to 3-fold higher than were seen in non-transgenic control C57BL/10 mice ( Prnp+/+ ) which express PrP-170N ( Figure 1 ) . Tg290 expressed approximately 10-fold lower PrP levels than did control mice ( data not shown ) . All three strains of PrP-170S mice were next tested for susceptibility to scrapie infection . Four strains of scrapie were used to test the influence of the N170S polymorphism on susceptibility to TSE disease . In Tg330 and Tg340 mice , the 22L and ME7 scrapie strains produced 100% incidence of typical fatal prion disease with clinical signs similar to control C57BL/10 mice ( Table 2 ) . Shorter incubation periods were observed in the Tg330 and Tg340 mice than in C57BL/10 mice , probably due to higher PrPsen expression levels in the transgenic mice . The diagnosis of scrapie was confirmed by the detection of PrPres in brain by immunoblotting . Brain PrPres levels in transgenic mice were slightly less than those in control mice , possibly due to shorter incubation periods ( Figure 2A ) . In Tg290 mice inoculation with scrapie strain 22L produced disease in 6 of 8 mice at an average incubation period of 615 dpi ( data not shown ) . This lower incidence of disease and slower tempo appeared to be related to the low PrPsen expression in Tg290 mice , and this line was not studied further . Unexpected results were observed upon inoculation of RML and 79A scrapie strains . Whereas control C57BL/10 mice were uniformly susceptible to these two strains , transgenic Tg 330 and Tg340 mice were quite resistant . Only two mice inoculated with RML and two mice inoculated with 79A had clinical disease and PrPres detectable by immunoblot ( Figure 2 ) , and all four occurred at late times ranging from 439-545dpi ( Table 2 ) . Brains were also examined microscopically for pathology and presence of PrPres . In Tg mice infected with strains ME7 or 22L , at the time of clinical disease vacuolation and PrPres deposition were widespread in many brain regions ( Figure 3A , B , C ) similar to non-transgenic control mice ( not shown ) . In contrast , after infection with strains 79A or RML , Tg mice with clinical signs or PrPres detectable by immunoblot showed localized vacuolation and PrPres deposition ( Figures 3E and 3H ) mostly limited to the thalamus , hippocampus and pons . In addition , ten RML or 79A-infected Tg mice , who were negative for PrPres by immunoblot , had subclinical infection as shown by brain PrPres deposits mainly localized to the vestibular nuclei in the pons or to the anterodorsal region of the thalamus detected at 545-603dpi ( Figure 3I , J , K , L ) . In these same areas gray matter vacuolation was either absent or minimal ( Figure 3F ) . Detection of these subclinical mice provided evidence of a higher incidence of infection than was shown by standard clinical observation and analysis of brain PrPres by immunoblotting . Perhaps these mice should be considered preclinical as they might have developed clinical disease if allowed to survive for a longer time . These results suggested that infection of Tg PrP-170S mice by RML or 79A scrapie occurred at slow and reduced levels , and this appeared to account for the rare presence of clinical signs in this experiment . Our in vivo experiments showed a dramatic difference in the tempo and levels of PrPres generation in brains of Tg330 and Tg340 mice after infection with scrapie strains RML and 79A compared to strains 22L and ME7 . Possibly a delay in PrP conversion and subsequent accumulation of PrPres might account for our results . Therefore to test PrP conversion under in vitro conditions we used the PMCA cell-free system [47] . As a source of PrPsen , brain homogenates derived from either Tga20 mice expressing PrP-170N or Tg330 mice expressing PrP-170S were used , and these reactions were initiated by seeding with PrPres from PrP-170N mice infected with scrapie strains 22L or RML . Surprisingly , RML PrPres seed generated PrPres conversion with both PrP-170N and PrP-170S brain homogenates . Quantification of PrPres produced in the PMCA revealed slightly greater amounts of product in the PrP-170N reactions , but these differences were not statistically significant ( Figure 4A , 4B and 4C ) . However , the positive conversion of PrP-170S by RML PrPres indicated that there was not a basic inability of RML scrapie PrPres to convert PrP-170S . This was in contrast to the slow conversion in vivo requiring over 400 days when Tg330 and Tg340 mice were inoculated with RML scrapie ( Figures 2 and 3 ) . In similar PMCA reactions 22L PrPres seed also gave higher PrPres generation using PrP-170N compared to PrP-170S substrate . These differences were statistically different ( Figure 4D , 4E , 4F ) , and might in part be due to the higher PrP expression in substrates from tga20 versus Tg330 mice . In summary , comparison of PrP-170N and PrP-170S mice by seeding with PrPres from RML or 22L gave no evidence that in vitro conversion of PrP-170S by RML PrPres was abnormal as detected by PMCA . In numerous previous studies expression of two different PrP alleles from the same or different species has been shown to reduce the level of prion infection as well as the incidence and tempo of disease [22] , [48] , [49] . The resistance of the Tg330 and Tg340 mice to RML and 79A scrapie prompted us to test whether the presence of both PrP-170S and PrP-170N in the same mouse would result in lower disease incidence or increased incubation times . To investigate this question , 22L or RML scrapie was inoculated into mice expressing two PrP alleles , one allele of the PrP-170S transgene and one allele of the mouse Prnp gene expressing PrP-170N . Mice expressing only one allele of PrP-170N ( Prnp+/− ) were inoculated as controls . With the 22L strain , mice expressing both 170S and 170N PrP had significantly faster incubation times ( 106–115dpi ) than mice expressing only 170N ( 259 dpi ) ( Table 3 ) . Therefore interference between these PrP variants did not seem to occur . The decrease in incubation period seen when both PrP alleles were expressed could be explained if the PrP-170S variant could contribute to a more rapid incubation time , as would be predicted by the susceptibility of the original Tg330 and Tg340 mice to 22L infection . In the case of strain RML , the expression of both alleles was associated with a decrease in incubation time compared to expression of PrP-170N alone , but these differences were not statistically significant ( Table 3 ) . Therefore , the more highly expressed PrP-170S protein appeared to contribute very little towards accelerating the disease tempo in these mice , and also there was no evidence for interference . This outcome might be predicted from the very low susceptibility of the PrP-170S transgenic mice to RML scrapie infection ( Table 2 ) . In summary , the PrP-170S variant appeared to be “neutral” during the infection by RML scrapie , showing no interference with the PrP-170N in the co-expression experiment .
In the present experiments the influence of a normal human prion protein gene allelic variation , N171S , on prion disease susceptibility was studied in a mouse system using transgenic mice expressing the mouse homolog , N170S . Susceptibility to prion disease induced by mouse scrapie strains , 22L and ME7 , was identical in control mice expressing PrP-170N versus transgenic mice expressing PrP-170S . In contrast , after inoculation with scrapie strains , RML or 79A , mice expressing PrP-170S were markedly resistant compared to control mice expressing PrP-170N . Of the 43 transgenic mice inoculated with these two scrapie strains , disease occurred only after 439 days and this was seen in only 4 mice . An additional 10 mice had subclinical infection after 545–603 days , as shown by detection of PrPres by IHC , however the remaining 29 mice had no evidence of clinical signs or PrPres accumulation in brain after observation up to 603 days . These results demonstrated that mice expressing PrP-170S were highly resistant to infection by strains RML and 79A , but this resistance was strain-specific since there was no resistance to two other strains ( ME7 and 22L ) . One explanation for the scrapie strain-specific differences seen in our experiments may lie in the origins of the scrapie strains used ( Figure 5 ) . Strain ME7 was the result of a passage of natural scrapie in Suffolk sheep directly to mice and therefore was not related to other scrapie strains[50] . On the other hand , 22L , RML and 79A share a common origin in that they were all derived from the Moredun Institute's sheep scrapie brain pool 1 ( SSBP/1 ) [51] . However , there were major differences in the passage history of each of these 3 strains , and strains 79A and RML were more closely related to each other than to strain 22L [51] , [52] ( Figure 5 ) . This may explain the similar resistance pattern of strains 79A and RML in PrP-170S mice . Although the biochemical explanation for scrapie strains in general remains a mystery at this time , it has been hypothesized that PrPres from each strain adopts a slightly different conformation which is conferred on successive PrPsen molecules as they are converted to PrPres [53] . Accordingly , our current data might be explained by the conformational selection model of prion strains and species barriers [54] , [55] which was first developed to explain prion strain differences involving the Sup35 protein in yeast [56] , [57] , [58] . This model suggests that only a subset of all possible PrPres conformations is compatible with any individual PrP primary structure . Thus incompatibility between the infecting prion strain and the host PrP would result in a transmission barrier [54] . For example , mouse PrP-170S might easily assume the conformations required by strains ME7 or 22L , but might be less adept at assuming the conformations associated with strains RML or 79A . In practice most mutations studied lead to altered incubation periods [30] , [31] , but when strong species barriers exist , either no transmission or low level subclinical cross-species transmission has been observed [59] , [60] , [61] . Another possible explanation for our results is that in vivo conversion of PrP-170S by RML or 79A strains might generate PrPres with a lower than usual stability . This might be due to either high dissociation of PrPres into smaller oligomers or increased susceptibility of PrPres to catabolic degradation . However , we were not able to detect any evidence for the presence of PrPres with increased susceptibility to proteinase K in PrP-170S transgenic mice infected by either RML or 79A scrapie strains ( data not shown ) . Effects of PrP amino acid variations on prion disease species and strain transmission barriers in some cases appear to correlate with differences in PrP conversion . For example , sheep homozygous for PrP with V136R154Q 171 or A136R154Q 171 show opposite patterns of susceptibility to scrapie strains SSBP1 and CH1641 [62] , [63] , and in vitro generation of PrPres in PMCA reactions agreed with the in vivo resistance observed [64] . However , when we tested PrP conversion in vitro using PMCA , both 22L and RML PrPres were able to seed the generation of PrPres derived from PrP-170S ( Figure 4 ) . This conversion of PrP-170S by RML scrapie was in contrast to the ineffective production of disease and slow and low PrPres generation in PrP-170S mice infected with RML scrapie . Biological and biochemical differences between conditions in brain tissue of live mice and PMCA test tube reactions might account for the discrepancies between our in vivo and in vitro results . Similar discrepancies between PrPres generation by PMCA in vitro and clinical susceptibility have been noted previously , and in some cases strong in vivo transmission barriers between species have been easily overcome by using PMCA with minor alterations in conditions [65] . Thus PrP conversion by PMCA would appear to be less selective than in vivo infection by TSE agents . Interestingly , the N171S polymorphism in humans ( homologous to the N170S change in our Tg330 and Tg340 mice ) occurs near other PrP residues implicated in influencing PrP structure and folding as well as susceptibility to prion diseases in animals . For example , alterations in PrP folding in vitro have been noted after mutations homologous to human residues 168 [66] and 170 [67] . At the structural level , PrP mutation at human residue 170 ( S170N ) appears to create a stabilized loop structure located near residues 165-175 [68] , and this change may influence susceptibility to CWD and other prion agents [37] , [38] . However , others have reported no effect of S170N on species-specific PrP conversion in vitro in a mouse-hamster system [69] , [70] , [71] . In contrast , resistance of rabbits to prion disease appears to be associated with a serine at human PrP residue 174 [72] . Similarly , the sheep polymorphism at residue 171 ( human residue 168 ) is important in the resistance of sheep to classical scrapie strains [6] , [73] , [74] . Possibly the N171S polymorphism examined in the present study might be able to modulate prion disease because of its location near the PrP loop structure and other nearby influential residues . In humans , familial prion diseases have been associated with PrP mutations in the near vicinity of the PrP loop structure , i . e . Q160X [75] , Y163X [76] , D178N [77] and V180I [78] . The N171S polymorphism is not by itself associated with familial prion disease [26] . However , a previous study identified a family with an unusual psychiatric disorder associated with PrP N171S [79] . More recently , in an African-American family with unusual psychiatric signs and sleep abnormalities preceding onset of familial CJD , disease was linked to expression of a PrP molecule containing both PrP N171S and D178N mutations [80] . Interestingly this is the first family with African ancestry where the D178N mutation has been detected , as the 12 previously reported families were of European or Japanese descent [78] , [80] . This family might be an example where the N171S polymorphism altered the clinical disease signs when expressed in combination with a known pathogenic PrP mutation . It remains unclear whether this effect is mediated by a direct influence of these mutations on PrP misfolding or whether indirect effects involving other non-PrP molecules might also play a role . Nevertheless , the fact that the N171S polymorphism is present in healthy populations of humans [81] , suggests that N171S is likely non-pathogenic by itself and that there may even be a selective advantage for maintaining its presence in human genomes .
|
Transmissible spongiform encephalopathies ( TSE ) or prion diseases are infectious fatal neurological diseases that affect many mammals , including humans . In these diseases a misfolded form of host prion protein ( PrP ) leads to brain degeneration and death . The genetic code of PrP in individual animals or humans has minor variations , which in some cases are associated with altered susceptibility to disease . In humans a variation at residue 171 ( N171S ) has been found in people mainly of African descent . However , due to the low incidence of the variation and difficult accessibility of these individuals , studies of prion diseases in these populations have not been carried out . Therefore , to create a laboratory animal model to study the effect of this variation on prion diseases , we generated transgenic mice expressing the mouse version of the human PrP variation at residue 171 . We then studied the susceptibility of these mice to 4 strains of mouse-adapted scrapie . In our experiments these transgenic mice were uniquely resistant to two scrapie strains , but showed high sensitivity to two others . This resistance appeared to be related to a slow or inefficient generation of the aggregated disease-associated form of PrP in these mice , and was not duplicated using in vitro assays . In summary , transgenic mice expressing this variant PrP provide an interesting model to study differences among prion strains and their interactions with PrP in vivo .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"neurobiology",
"of",
"disease",
"and",
"regeneration",
"neurodegenerative",
"diseases",
"neuroscience",
"animal",
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"neurological",
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] |
2011
|
Strain Specific Resistance to Murine Scrapie Associated with a Naturally Occurring Human Prion Protein Polymorphism at Residue 171
|
Metastatic dissemination employs both the blood and lymphatic vascular systems . Solid tumors dynamically remodel and generate both vessel types during cancer progression . Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes ( LNs ) are prognostic markers for breast cancer metastasis and patient outcome , and tumor-induced lymphangiogenesis likely influences metastasis . Deregulated tumor tissue fluid homeostasis and immune trafficking associated with tumor lymphangiogenesis may contribute to metastatic spreading; however , the precise functional characterization of lymphatic endothelial cells ( LECs ) in tumors is challenged by the lack of specific reagents to decipher their rate-limiting role in metastasis . Therefore , we generated novel transgenic mice ( PDPN promoter-driven Cre recombinase transgene [PDPN-Cre] and PDPN promoter-driven thymidine kinase transgene [PDPN-tk] ) that allow for the identification and genetically controlled depletion of proliferating podoplanin ( Pdpn ) -expressing LECs . We demonstrate that suppression of lymphangiogenesis is successfully achieved in lymphangioma lesions induced in the PDPN-tk mice . In multiple metastatic breast cancer mouse models , we identified distinct roles for LECs in primary and metastatic tumors . Our findings support the functional contribution of primary tumor lymphangiogenesis in controlling metastasis to axillary LNs and lung parenchyma . Reduced lymphatic vessel density enhanced primary tumor lymphedema and increased the frequency of intratumoral macrophages but was not associated with a significant impact on primary tumor growth despite a marked reduction in metastatic dissemination . Our findings identify the rate-limiting contribution of the breast tumor lymphatic vessels for lung metastasis .
Metastasis is responsible for 90% of deaths of breast cancer patients [1 , 2] . The contribution of both cancer cells and stromal cells ( such as fibroblasts , endothelial cells , pericytes , and immune cells ) is important for cancer development and metastasis , including in breast cancer [2–5] . The lymphatic system , consisting of lymphatic vessels and lymphoid organs , is an essential regulator of tissue fluid homeostasis , immune cell trafficking , and immunological surveillance [6–8] . Lymphatic vessels can be divided into several subtypes: initial lymphatic with incomplete basement membrane and no pericyte/smooth-muscle-cell coverage; transitional precollecting lymphatics; and larger collecting lymphatics with a complete basement membrane and smooth muscle investment . In the context of cancers , these different types of lymphatic vessels can be actively regulated by tumor-derived growth factors [8 , 9] . Lymphangiogenesis , the formation of new lymphatic vessels , has been associated with metastasis of solid tumors to lymph nodes ( LNs ) and distant organs [8–13] . Recent studies demonstrate that lymphatic vessels undergo dynamic remodeling , including lymphangiogenesis and lymphatic enlargement , which facilitates tumor metastasis [14–16] . Furthermore , two recent studies further confirmed the dissemination of cancer cells from LN to distant organs through LN blood vessels in tumor-bearing mice [17 , 18] . Previous studies using various transgenic mouse models employing vascular endothelial growth factor C/D ( VEGF-C/D ) overexpression or VEGF-C/D trap suggested a potential role for lymphangiogenesis in cancer progression [19–24] . Given that VEGF-C/D can also target nonlymphatic processes , we aim at establishing new mouse models that can specifically target lymphangiogenesis via genetic depletion of proliferating lymphatic endothelial cells ( LECs ) . Lymphatic vessel markers include Prox1 [25] , the lymphatic vessel endothelial hyaluronan receptor-1 ( LYVE1 ) [26] , podoplanin ( PDPN ) , and VEGF receptor 3 ( VEGFR3 ) [27] . PDPN , a 43-kDa membrane protein , is present in podocytes [28] and is one of the most widely employed markers of LECs [29 , 30] . To functionally evaluate the specific role of lymphatic vessels in cancer progression and metastasis , we generated novel transgenic mice that express the herpes simplex virus ( HSV ) thymidine kinase ( tk ) under the control of the PDPN gene promoter ( PDPN-tk mice ) . Upon ganciclovir ( GCV ) administration to PDPN-tk mice , PDPN-positive cells that also express tk will convert GCV into a nucleoside analog that irreversibly arrests DNA replication , resulting over time in the depletion of proliferating PDPN-expressing LECs . Here , we demonstrate that the depletion of proliferating PDPN-expressing LECs significantly inhibits lymphangiogenesis in mammary tumors , resulting in decreased distant metastasis without an impact on primary tumor growth .
The PDPN-tk mouse model ( BALB/c background ) was generated using a 4-kb PDPN promoter sequence cloned and ligated to HSV viral tk sequence using the topoisomerase I-activated pCR2 . 1-TOPO ( pCR2 . 1-TOPO ) vector . The PDPN-Cre mouse model was generated using the same PDPN promoter sequence cloned and ligated to Cre recombinase sequence ( Fig 1A ) . The final constructs were confirmed by DNA sequencing . To examine the specificity of the PDPN promoter , we generated the PDPN-Cre; LoxP-Stop-LoxP ( LSL ) -yellow fluorescent protein ( YFP ) transgenic mice ( BALB/c background ) to lineage trace the PDPN+ cells . The YFP expression colocalized with LYVE1- or PDPN-positive lymphatic endothelium in normal organs ( S1 Fig ) . Additionally , the eyes of PDPN-Cre; LSL-YFP mice exhibited YFP/green fluorescent protein ( GFP ) expression ( Fig 1B ) , consistent with previous observation using Prox1-GFP transgenic mice [31] . Primary LECs were isolated as previously documented [32] from incomplete Freund’s adjuvant ( IFA ) -induced benign mouse lymphangioma . Briefly , LECs were isolated from hyperplastic lymphatic vessels , cultured , and expanded ( S2A Fig ) . These cells exhibited typical LEC morphology , intrinsic YFP expression , and positive immunostaining for PDPN ( Fig 1B ) and LYVE1 ( S2B Fig ) . Robust expression of intrinsic YFP was observed in LECs from IFA-induced lymphangioma in PDPN-Cre; LSL-YFP mice , showing the YFP-expressing LECs as the dominant cell population ( 80% of all nucleated cells ) within the lymphangioma tissue ( S2C Fig ) . These results confirmed the recombination efficacy of PDPN-Cre in LECs . Our previous study identified that blood-vascular endothelial-cell–specific deletion of β1 integrin ( Tie2-Cre; β1 integrin ( Int ) loxP/loxP mice ) resulted in embryonic lethality due to severe vascular defects [33] , while others demonstrated that blood-vascular endothelial-cell–specific deletion of transforming growth factor ( TGF ) β type II receptor ( cadherin 5 promoter-driven tamoxifen-inducible Cre recombinase transgene ( Cdh5-CreERT2 ) ; TGFBRIIloxP/loxP mice ) also resulted in embryonic lethality [34] . In contrast , PDPN-Cre; β1 IntloxP/loxP and PDPN-Cre; TGFBRIIloxP/loxP mice were born in the expected Mendelian ratio without any noticeable abnormality/defect ( S3A and S3B Fig ) , supporting the specificity of the PDPN-Cre transgenic in targeting gene deletion in lymphatic vessels and not blood vessels . To evaluate the efficacy of the PDPN-tk transgene , PDPN+ LECs separated from lymphangioma tissues of PDPN-tk or wild-type ( WT ) mice were cultured ( S4A Fig ) and treated with increasing concentrations of GCV ( Fig 1C ) . A dose-dependent depletion of LECs ( derived from PDPN-tk mice but not WT mice ) was observed , reaching 56% of LEC depletion at an exposure of 50 μM GCV . In addition , in vivo administration of GCV to PDPN-tk mice ( daily 50 mg/kg body weight ) inhibited the formation of IFA-induced lymphangioma when compared to control ( WT ) mice ( Fig 1D ) . As previously documented [32] , IFA-induced benign mouse lymphangioma formed white solid masses on the abdominal surface of the diaphragm and on the surface ( under the Glisson’s capsule ) of the liver ( Fig 1D ) . The hyperplastic LECs forming these masses present with enlarged lumens that are distinct from adipose tissue ( S4B Fig ) . LEC lumen formation within lymphangioma tissue was specifically impaired in the PDPN-tk mice when compared to control mice ( Fig 1E ) , indicative of the depletion of hyperplastic LECs . A significant and specific decrease in proliferating LECs is recorded in lymphangioma of PDPN-tk mice compared to control mice ( Fig 1F and 1G ) . Notably , lymphangioma formation was not altered in control mice , including WT mice ( with or without GCV treatment ) and non-GCV-treated PDPN-tk mice . The depletion of proliferating LECs resulted in a decrease in the size of lumen structures of lymphangioma , and this was accompanied with a modest increase in α-smooth muscle actin ( αSMA ) -expressing myofibroblasts in these benign lesions ( S4C Fig ) . However , these myofibroblasts did not appear to play a role in IFA-induced lymphangioma because the formation of these lesions was not impaired in αSMA-tk transgenic mice ( depletion of proliferating myofibroblasts that exhibit αSMA expression [35] , S4D Fig ) . These results underscore the specificity of PDPN-tk mice and support that LEC proliferation , but not myofibroblast proliferation , is essential for the formation of IFA-induced lymphangioma . Matrigel plug assay was conducted to determine the functional role of PDPN+ LECs in lymphangiogenesis . Growth-factor–reduced matrigel supplemented with VEGF-C induced robust lymphatic vessel formation as well as blood vessel formation after subcutaneous implantation ( 400 μL matrigel per plug; one plug per mouse ) . In contrast with WT + GCV mice , PDPN-tk + GCV mice exhibited reduced lymphangiogenesis ( Fig 2A ) and LEC proliferation ( S5A Fig ) in the matrigel plugs , while the angiogenesis response , measured by cluster of differentiation ( CD ) 31 immunolabeling , was unaffected ( Fig 2A ) . Decreased lymphatic vessel density in matrigel plugs of PDPN-tk mice was also confirmed by immunohistochemical assessment of lymphatic markers , PDPN ( Fig 2B ) and LYVE1 ( S5B Fig ) . The PDPN-expressing cells within the matrigel plugs were predominantly co-immunolabeled with the LEC marker LYVE1 but did not express the cancer-associated fibroblast marker αSMA ( S5C Fig ) . These results support that the cell population targeted by the PDPN-tk transgene in the aforementioned matrigel plug assays comprises of LECs and not fibroblasts . Additionally , we examined the LN , intestine , and kidney of WT + GCV mice and PDPN-tk + GCV mice bearing the VEGF-C enriched matrigel plug . Although specific depletion of PDPN-expressing LECs was observed in the plug with active lymphangiogenesis ( Fig 2A ) , no changes were noted for PDPN immunolabeling in these normal , unaffected tissues ( S6A Fig ) , supporting that our genetic strategy only targets proliferating PDPN-expressing cells . Tumor lymphangiogenesis was examined in orthotopic 4T1 mammary tumors established in either PDPN-tk or WT female mice ( all treated with GCV ) . Tumor tissues were scanned for lymphatic vessels and blood vessels . GCV-treated PDPN-tk mice revealed significantly suppressed lymphangiogenesis in both tumor center and tumor margin/periphery , defined as 100 μm from the tumor edge [36 , 37] , while angiogenesis was not significantly altered ( Fig 2C , S6B Fig , and S7A Fig ) . Despite a significant suppression of lymphangiogenesis , the growth of orthotopic 4T1 mammary tumors in GCV-treated PDPN-tk mice was unchanged when compared with GCV-treated WT control mice ( Fig 3A ) . Interestingly , despite the unchanged primary tumor growth , PDPN-tk-GCV mice with tumors exhibited significantly fewer surface metastatic lung nodules ( Fig 3B ) and histologically identified lung metastases ( Fig 3C ) when compared to WT mice . No tumor-infiltrated axillary or inguinal LN was observed in PDPN-tk-GCV mice ( 0 out of 9 mice ) , whereas WT mice in the control group occasionally presented with axillary and/or inguinal LN metastases ( 2 out of 8 mice ) ( Fig 3A ) . We also employed the mouse mammary tumor virus–polyoma middle tumor antigen ( MMTV-PyMT ) model , in which spontaneous mammary carcinomas and lung metastasis develop , to examine the impact of lymphatic/LEC depletion on cancer progression . MMTV-PyMT mice were bred with PDPN-tk mice to generate the MMTV-PyMT; PDPN-tk mice , as well as the MMTV-PyMT; WT littermate control mice . Female mice were monitored for tumor growth . The growth of MMTV-PyMT tumors was not significantly altered in GCV-treated MMTV-PyMT; PDPN-tk mice when compared to MMTV-PyMT; WT mice ( Fig 4A ) . Decreased lymphatic vessel density in MMTV-PyMT; PDPN-tk tumors was confirmed by immunohistochemical staining for LYVE1 ( S7B Fig ) . The MMTV-PyMT; PDPN-tk mice exhibited increased incidence of cystic tumors , possibly resulting from enhanced lymphedema , when compared to control mice ( Fig 4A and 4B ) . Previous studies have established that impaired lymphatic function can result in the accumulation of macromolecular proteins ( such as albumin ) because of compromised lymphatic drainage [38–40] . The increased level of lymphedema in primary tumor tissues of MMTV-PyMT; PDPN-tk mice was confirmed by albumin immunohistochemistry ( Fig 4B ) . The total number of surface metastatic lung nodules , histologically identified lung metastatic lesions , and axillary LN metastasis was significantly reduced in MMTV-PyMT; PDPN-tk mice when compared to MMTV-PyMT; WT mice ( Fig 4C and 4D ) . Taken together , these results support that suppression of lymphangiogenesis in primary mammary tumors did not impact their growth but limited their metastatic dissemination . Previous studies indicated that PDPN may also be expressed by cancer-associated fibroblasts [41–43] or macrophages [44] . Our analyses revealed that PDPN-expressing cells did not coexpress the breast-tumor–associated fibroblast marker αSMA but predominantly coexpressed the LEC-associated marker VEGFR3 ( S7C Fig ) and weakly coexpressed or failed to coexpress the vascular marker CD31 ( S7D Fig ) , consistent with previous observations [19] . We also noted that PDPN+ cells did not show colocalization with the macrophage marker CD68 in 4T1 tumors ( WT mice ) , although close contact between CD68+ macrophages and PDPN+/LYVE1+ lymphatic vessels could be occasionally observed ( S7E Fig ) . Further , The Cancer Genome Atlas ( TCGA ) data set of 844 patients with invasive breast carcinoma ( RNA sequencing version 2 analysis [RNA Seq V2] normalized gene expression with RSEM output [RSEM] ) revealed a correlation between PDPN mRNA level and LN metastasis , showing higher levels of PDPN mRNA ( PDPN mRNA expression normalized to Gapdh ) associated with more LN metastasis ( Fig 5A ) . These results were consistent with previous reports regarding the correlation between PDPN level ( as examined by immunohistochemistry ) and LN metastasis in breast cancer patients [45 , 46] . We also found marginally decreased occurrence of metastasis in distant organs ( such as bone and lung ) in PDPN-low patients compared to PDPN-high patients ( S8A Fig ) . However , the number of cases with known distant organ metastasis was too low to offer conclusive evidence regarding the correlation between PDPN level and occurrence of distant metastases . Given that lymphatic vessels support immune cell trafficking , we next examined the immune infiltration in lymphatic-depleted tumors compared to control tumors . We employed an established flow-cytometry–based analysis ( S8B Fig ) , as previously detailed [35 , 47] . Upon depletion of PDPN-expressing LECs , the frequencies of most immune cell subpopulations ( CD45+ , CD3+ , CD4+ , CD8+ , CD19+ , and natural killer [NK] 1 . 1+ cells ) remained unaltered ( Fig 5B and S8C Fig ) . In contrast , the percentage of CD11b+ and CD11b+Gr1− macrophage population significantly increased in lymphatic-depleted tumors compared to control tumors ( Fig 5C ) . This result is consistent with the reports that suggest that elevated macrophage accumulation is associated with lymphedema [48 , 49] . The percentage of CD11b+Gr1+-myeloid–derived suppressor cells ( either CD11b+Ly6G+ or CD11b+Ly6C+ ) or CD11c+ dendritic cells remained unchanged ( Fig 5C ) . The ratio of CD4+FoxP3+ effector T cells ( Teff ) to CD4+FoxP3+ regulatory T cells ( Treg ) was not affected ( Fig 5D ) .
Lymphangiogenesis , the formation of new lymphatic vessels , is associated with the progression of solid tumors [8–12] . It is known that lymphatics in the tumors are related to distant metastasis and contribute to immune surveillance and tissue fluid homeostasis . In this study , we performed experiments to determine the functional contribution of lymphatic vessels in lung metastasis associated with breast cancer . To achieve this goal , we generated two new transgenic mouse strains that allowed for the selective depletion of proliferating PDPN-positive LECs ( PDPN-tk mice ) and for the fate mapping/lineage tracing of PDPN-positive LECs ( PDPN-Cre mice ) . The inhibition of lymphangiogenesis employing PDPN-tk mice supports that breast-cancer–associated LN and lung metastasis is in part relying upon dissemination of cancer cells via lymphatic vessels . Interestingly , two recent studies highlighted that the dissemination route of cancer cells from LN to distant organs employs LN blood vessels in tumor-bearing mice [17 , 18] . In our studies , the vascular density in the mammary tumors was unchanged upon depletion of PDPN+ cells . Lineage tracing experiments employing the PDPN-Cre mice showed that PDPN+ cells are associated with lymphatic vessels but not the blood vessels . Interestingly , the growth of primary mammary tumors was not markedly altered when lymphangiogenesis was inhibited . These observations are also in alignment with a previous study showing that lymphangiogenesis induced by VEGF-C overexpression facilitates tumor metastasis without contributing to any growth advantage of primary tumor cells [19] . Various cancer types have distinct preferences in metastatic routes ( such as a hematogenous route or a lymphatic route ) , yet the underlying mechanisms of such phenomena are still poorly understood . A recent study demonstrated the hematogenous route for ovarian cancer metastasis [50] in contrast to a peritoneal circulation-facilitated spread as previously proposed . Notably , depletion of lymphatic vessels did not alter the vascular density or lead to suppression of tumor growth but resulted in intratumor lymphedema due to potential imbalance in tissue fluid homeostasis . The new mouse models described herein may prove helpful for future studies related to breast-cancer–associated lymphedema , a substantial clinical problem observed in breast cancer patients . Although our results support that the newly generated PDPN-tk transgenic mice enable the specific targeting of LECs in various models of lymphangiogenesis , including tumor lymphangiogenesis , it remains possible that immunolabeling for PDPN could be observed in other stromal cells in the tumor microenvironment , including cancer-associated fibroblasts , as noted in human breast cancer tissues [41–43] . The prognostic value of PDPN-expressing mesenchymal cells in the tumor microenvironment remains to be further studied . The lymphatic system can regulate immune cell trafficking and tissue fluid homeostasis , yet our results indicated that suppression of tumor lymphangiogenesis did not significantly alter tumor immune infiltration . This may reflect a cancer-type–specific observation since it was reported in melanomas of mice lacking dermal lymphatic vessels that lymphatics were critical in establishing tumor-associated inflammation and immunity [24] . The percentage of intratumoral CD11b+Gr1− macrophages , however , was significantly elevated with PDPN+ LEC depletion . This may reflect a host response to compensate decreased lymphangiogenesis , in particular since macrophages play a role in regulating lymphangiogenesis and releasing lymphangiogenic factors [51–54] . Increased numbers of CD11b+Gr1− macrophages in mammary tumors with PDPN+ LEC depletion is also consistent with previous reports on increased macrophage infiltration as a hallmark of lymphedema [48 , 49] and could support a potential role of these cells in metastasis , albeit further study is still needed . Intriguingly , our results suggest that the decreased metastatic burden associated with suppressed lymphangiogenesis may be independent of a lymphocytic polarization in the primary tumor microenvironment .
Mice were euthanized using CO2 inhalation . All mice were maintained under standard housing conditions at the MD Anderson Cancer Center ( MDACC ) animal facility and the Beth Israel Deaconess Medical Center ( BIDMC ) animal facility , and all animal procedures were approved by the MDACC Institutional Animal Care and Use Committee and the BIDMC Institutional Animal Care and Use Committee ( IACUC number: 1033 ) . The PDPN-tk mouse strain was generated by cloning and ligating the 4-kb PDPN promoter sequence to HSV viral tk sequence using the pCR2 . 1-TOPO vector ( Invitrogen , Carlsbad , CA , USA ) . A similar approach was used to generate the PDPN-Cre mouse strain . Both transgenic mice were generated by the Transgenic Mouse Core Facility at Harvard Medical School . The mice were backcrossed ( over 20 generations ) and maintained on the BALB/c genetic background . Primers for PDPN-tk genotyping PCR are PDPN-forward 5′-ACCGGAGACATAAATGCCGA-3′ and TK-reverse 5′-AGCACCCGCCAGTAAGTC-3′ . Primers for PDPN-Cre genotyping PCR are PDPN-forward 5′-ACCGGAGACATAAATGCCGA-3′ and Cre-reverse 5′-CGCCGCATAACCAGTGAAAC-3′ . αSMA-tk mice were generated and characterized in our previous study [55] . TGFBRII flox mice were kindly provided by H . Moses , Vanderbilt University [56] . β1 integrin flox mice were purchased from the Jackson Laboratory ( Bar Harbor , ME , USA ) . Investigators were not blinded to group allocation but were blinded for the histological assessment of phenotypic outcome . No randomization method was used , and no animal was excluded from the analysis . The experimental endpoint is defined as when tumor burden reaches 1 , 500 mm3 or 1 . 5 cm in diameter ( whichever comes first ) . For the evaluation of surface lung nodules in mouse mammary tumor models , all surfaces of all of the lobes were ascertained for the presence of surface lung nodules . For the microscopic evaluation of lung metastases in mouse mammary tumor models , we counted the number of nodules observed on a single H&E-stained cross section of the lungs . Matrigel plug assay was conducted to determine the functional role of PDPN+ LECs in lymphangiogenesis . Growth-factor–reduced matrigel ( Corning , Corning , NY , USA ) supplemented with VEGF-C ( 1 μg /400 μL matrigel ) was subcutaneously implanted in WT and PDPN-tk mice ( 400 μL matrigel per plug; one plug per mouse; n = 5 mice per group ) . Primary antibodies are as follows: albumin ( A90-134A , Bethyl , 1:100 ) , αSMA ( M0851 , Dako , 1:100 ) , CD31 ( ab28364 , Abcam , 1:300 ) , CD68 ( M0814 , Dako , 1:200 ) , Ki67 ( RM-9106 , Thermo Scientific , 1:400 ) , LYVE1 ( ab14917 , Abcam , 1:200 ) , PDPN ( ab11936 , Abcam , 1:400 ) , VEGFR3 ( RM0003-5F63 , Novus Biologicals , 1:100 ) , and YFP/GFP ( ab13970 , Abcam , 1:200 ) . For all immunohistochemical stainings , sections were incubated with biotinylated secondary antibody and then streptavidin-HRP ( Vector Labs , Burlingame , CA , USA ) . Counterstaining with hematoxylin was conducted , and DAB positivity was examined in randomly selected visual fields . For all immunofluorescence stainings , sections were incubated with fluorescent-labeled secondary antibodies according to the primary antibody usage . For the YFP staining of tissue samples from PDPN-Cre; LSL-YFP mice , optimized protocols for tissue collection and immunofluorescence staining were used in order to minimize the autofluorescence in the skin and intestine sections . These optimized protocols include: conducting PBS perfusion before collecting the organs from mice; using Sudan Black B ( Sigma-Aldrich , St . Louis , MO , USA ) incubation on the sections before the staining [57]; blocking with 4% cold water fish gel before primary antibody incubation; and decreased secondary antibody concentration . Staining for αSMA was performed with Mouse-on-Mouse ( MOM ) kit ( Vector Laboratories ) following the manufacturer's instructions . The images of at least 3 random visual fields for each sample section were quantified for positive area using NIH ImageJ analysis software ( albumin , CD31 , Ki67 , LYVE1 , or PDPN ) . Quantified values for multiple visual fields were averaged to produce a single value for each animal , which was then averaged again to represent the mean bar for the group in each graph . Either WT or PDPN-tk mice ( 3 months old , female ) were intraperitoneally injected twice ( day 1 and day 14 ) with IFA ( 200 μL , 1:1 mixed with PBS ) to induce the formation of mouse hyperplastic lymphatic tissue ( lymphangioma ) , as previously described [32] . The lymphangioma confluence was quantified as the percentage coverage by lymphangioma area among the total area of the diaphragm , as quantified by ImageJ software . The average diameter of lumen structures within lymphangioma tissues was calculated by measuring 10–20 randomly selected lumens within microscopic ( 40× ) images of H&E-stained tissue slides using ImageJ software . Mouse lymphangioma tissue , formed by hyperplastic lymphatic vessels on the diaphragm and liver of mice in response to IFA treatment , was collected on day 21 and digested with 1 mg/mL collagenase solution ( collagenase I:collagenase II = 1:1 ) at 37°C for 30 min . Cell suspension was filtered and purified for LECs using anti-PDPN antibody ( Abcam , ab11936 ) and Magnetic Dynabeads ( Thermo Fisher Scientific , Waltham , MA , USA ) . Isolated primary mouse LECs were cultured in endothelial cell growth medium ( Lonza , Basel , Switzerland ) . For the detection of YFP-positive LECs in IFA-induced lymphangioma by flow cytometry , mouse lymphangioma tissue was collected , prepared as a single-cell suspension according to the same protocol above , and examined for YFP fluorescence signal by flow cytometry . For in vitro treatment of GCV , LECs from WT or PDPN-tk mice were isolated using the same method listed above , cultured , treated with 0 , 5 , or 50 μM GCV for 48 h , and then examined for cell viability ( measured as the absorbance at 450 nm by a microplate reader ) using the Cell Counting Kit-8 ( Dojindo Molecular Technologies , Kumamoto , Japan ) . Results of cell viability were expressed as percentage of viable cell counts using the control vehicle-treated group as the reference . Either WT or PDPN-tk female mice , around 3 months old , were used for orthotopic implantation of 4T1 mammary epithelial cancer cells . 4T1 Cells were from American Type Culture Collection ( ATCC ) and cultured in DMEM with 10% FBS and 100 U/mL penicillin–streptomycin . Cells were examined monthly to ensure a negative result for mycoplasma test . Mice were anesthetized with ketamine/xylazine , the skin near the mammary gland was incised , and 4T1 cancer cells were injected into the mammary glands ( in total , 1 × 106 cells per mouse; 5 × 105 cells at each side , for both left and right sides ) , as previously described [4] . PDPN-tk and WT control mice were treated with daily intraperitoneal injections of 50 mg/kg body weight of GCV ( InvivoGen , San Diego , CA , USA ) , when the sum of the tumor volumes per mouse reached approximately 300 mm3 ( approximately 8–9 days post cancer cell inoculation ) . Tumor volumes were measured every other day using digital calipers and calculated using the equation length × width2 × 0 . 52 . Mice were sacrificed when the sum of the tumor volumes reached approximately 1 , 500 mm3 ( approximately 22–25 days post-cancer cell inoculation ) . MMTV-PyMT transgenic mice from the BALB/c genetic background were provided by Dr . Jack Lawler ( BIDMC and Harvard Medical School , Boston , MA , USA ) . MMTV-PyMT mice were bred with PDPN-tk mice to generate the MMTV-PyMT; PDPN-tk mice . Female MMTV-PyMT; PDPN-tk mice and female MMTV-PyMT; WT littermate control mice were used for mammary tumor studies . GCV treatment was conducted as daily intraperitoneal injections of 50 mg/kg body weight of GCV ( InvivoGen , San Diego , CA ) , starting when the sum tumor volumes per mouse reached approximately 300 mm3 . Tumor volumes were measured twice per week using digital calipers and calculated using the equation length × width2 × 0 . 52 . Mice were sacrificed when tumor volume reached approximately 1 , 500 mm3 or 1 . 5 cm in diameter ( whichever came first ) . A tumor was counted as a cystic tumor when it formed prominent fluid-filled cyst with a volume greater than 100 mm3 . Tumors and other organs , including the lungs , were collected as previously described [58] . For the characterization of immune infiltration , tumors ( from 3-month-old WT or PDPN-tk mice harboring 4T1 orthotopic mammary tumors and treated with GCV ) were examined by flow-cytometry–based immunotyping methodology ( BD LSRFortessa X-20 Cytometer; BD Biosciences , San Jose , CA , USA ) . Tumors were weighed , minced with gentleMACS Dissociator ( Miltenyi Biotec , Bergisch Gladbach , Germany ) , and digested in 2 mL solution containing 1 mg/mL Liberase TL ( Roche , Indianapolis , IN , USA ) and 0 . 2 mg/mL DNase I in RPMI media at 37°C for 30 min . The tissue lysates were filtered through a 100-μm mesh before immunostaining [35 , 47] . The subsequent single-cell suspension was stained with fixable viability dye eFluor 780 , anti-CD45 . 2 Pacific Blue , anti-CD3 PE-Cy7 , anti-CD3 Alexa Fluor 700 , anti-FoxP3 Alexa Fluor 700 , anti-CD11c eFluor 615 , and anti-NK1 . 1 PE ( eBioscience , San Diego , CA , USA ) ; anti-CD4 Qdot 605 ( Life Technologies , Gaithersburg , MD , USA ) ; anti-CD8 Brilliant Violet 650 , anti-CD11b Brilliant Violet 570 , anti-CD19 Qdot655 , and anti-F4/80 FITC ( BioLegend , San Diego , CA , USA ) ; and anti-Ly6C APC and anti-Ly6G PE-Cy7 ( BD Biosciences ) . The percentage positive cells were analyzed by FlowJo 10 . 1 . Unstained , live/dead stain only , and single-stained beads ( eBioscience ) served as compensation controls . Singlets were gated using forward-scatter ( FSC ) height ( FSC-H ) and FSC area ( FSC-A ) event characteristics . Data were derived from multiple experiments with 9 mice per group . The mRNA data ( RNA Seq V2 RSEM ) and clinical data of 844 patients with invasive breast carcinoma from the TCGA data set were obtained using the cBioPortal for Cancer Genomics ( http://www . cbioportal . org/ ) [59] . χ2 analyses , using SPSS statistical software , were performed comparing LN metastatic frequency between PDPN-High and PDPN-Low groups of patients . The metastasis occurrence at distant organs of these patients was also analyzed based on the detailed clinical information from TCGA data set . Statistical analyses of flow cytometry and immunostaining quantifications were performed with unpaired , two-tailed t test , one-way ANOVA with Tukey’s multiple comparison test , or Fisher’s exact test with GraphPad Prism ( GraphPad Software , San Diego , CA , USA ) . A p value < 0 . 05 was considered statistically significant . Error bars represent SEM when multiple visual fields were averaged to produce a single value for each animal , which was then averaged again to represent the mean bar for the group in each graph .
|
Cancer progression and metastasis of solid tumors can occur in association with the generation of new lymphatic vessels ( lymphangiogenesis ) . Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes are used as prognostic markers for breast cancer metastasis and patient outcome . However , the specific role of newly formed lymphatic vessels in breast cancer metastasis to the lung remains unknown . In this study , we have analyzed this process by generating novel transgenic mice that enabled the identification of podoplanin ( Pdpn ) -expressing lymphatic endothelial cells , as well as the controlled depletion of these cells during lymphangiogenesis in breast cancer progression . We show that in multiple metastatic breast cancer mouse models , the specific suppression of lymphangiogenesis , without impacting blood vessel formation ( angiogenesis ) , does not limit primary tumor growth but reduces cancer cell dissemination to the lung and metastatic disease . We conclude that inhibition of breast tumor lymphangiogenesis decreases lung metastasis without affecting primary tumor growth .
|
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2018
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Podoplanin+ tumor lymphatics are rate limiting for breast cancer metastasis
|
This paper shows that the various computations underlying spatial cognition can be implemented using statistical inference in a single probabilistic model . Inference is implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time . For example , to estimate where you are in a known environment , forward inference is used to optimally combine location estimates from path integration with those from sensory input . To decide which way to turn to reach a goal , forward inference is used to compute the likelihood of reaching that goal under each option . To work out which environment you are in , forward inference is used to compute the likelihood of sensory observations under the different hypotheses . For reaching sensory goals that require a chaining together of decisions , forward inference can be used to compute a state trajectory that will lead to that goal , and backward inference to refine the route and estimate control signals that produce the required trajectory . We propose that these computations are reflected in recent findings of pattern replay in the mammalian brain . Specifically , that theta sequences reflect decision making , theta flickering reflects model selection , and remote replay reflects route and motor planning . We also propose a mapping of the above computational processes onto lateral and medial entorhinal cortex and hippocampus .
This paper describes a dynamic Bayesian model of spatial cognition . Here we define spatial cognition as including the tasks of localisation ( estimating where you are in a known environment ) , sensory imagery ( constructing a virtual scene ) , decision making ( deciding which way to turn to reach a goal ) , model selection ( working out which environment you are in ) and motor planning ( computing a sequence of motor commands that will lead to a sensory goal ) . We show that all of these tasks can be implemented using statistical inference in a single probabilistic model . We note that the above formulation is slightly different to previous definitions by OKeefe and Nadel [1] , Gallistel [2] , and Redish [3] which stress the capacity of determining and performing a path from a current position towards a desired location . The model has hidden states comprising speed , direction and allocentric location , control variables comprising change in direction and speed , and sensory states representing olfactory , somatosensory and visual information . The model describes the dynamical evolution of hidden states , and provides a mapping from hidden to sensory states . Inference in the model is then implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time . We propose that these computations are reflected in recent empirical findings of pattern replay in the mammalian brain [4] , [5] . Specifically , we propose that theta sequences reflect decision making , theta flickering reflects model selection , and remote replay reflects route and motor planning . Our use of the terms ‘forward’ and ‘backward’ here relate to time and should not be confused with the direction of message passing in a cortical hierarchy [6] . Our approach falls into the general category of ‘map-based’ or ‘model-based’ planning [1] , [7]–[10] , or ‘model-based decision making’ [11] . The term ‘model-based’ refers to making and updating a representation of the world ( such as a cognitive map ) . This is to be contrasted , for example , with ‘model-free’ approaches in which agents merely react to stimuli , after having previously learnt stimulus-response mappings through extensive exposure to an environment [12] . More generally , agents will use a variety of navigation strategies depending on their cognitive capabilities and familiarity with an environment . Spatial decisions can , for example , be classified [13] as being cue-guided ( eg . move towards the red house ) , stimulus triggered ( eg . turn left at the red house ) , route based ( turn left at the red house then right at the blue house ) . There is a good deal of evidence showing that the brain has multiple decision making or control systems , each with its own strengths and weaknesses [14]–[16] . The usefulness of model-based planning is most apparent after an agent has sufficient experience to learn a model of an environment and when , subsequently , local changes to that environment are made which affect the optimal route to a goal [15] . In statistical terms , these would be referred to as nonstationarities . For spatial models this could be , for example , a hole appearing in a wall enabling an agent to take a shortcut , or a new object appearing preventing an agent taking a habitual route . Another strength of model-based control is that it can reduce learning time . Tse et al . [17] , for example , studied decision making in rats and found that learning required fewer trials when it occurred against a background of prior knowledge . This allows new information to be assimilated into an existing schema or model . The model-based versus model-free distinction has become important for the study of decision making in general as the underlying neuroanatomical differences are being delineated [11] , [15] . Khamassi and Humphries [18] argue that , due to the shared underlying neuroanatomy , spatial navigation strategies that were previously described as being either place-driven or cue-driven are better thought of as being model-based versus model-free . Daw et al . [15] propose that arbitration between model-based and model-free controllers is based on the relative uncertainty of the decisions and more recently , Pezzulo et al . [19] have embedded both types of decision making systems into a single ‘mixed instrumental controller’ . This paper describes the computations underlying spatial cognition , initially , at a rather abstract level of manipulations of probability densities and then employs vector and matrix representations of variables and connectivities . Although we later on go on to describe how our model relates to underlying neuronal implementations , the model itself is not specified at a neuronal level . This style of modelling has many precedents in the literature . For example , Bousquet et al . [20] have conceived of the hippocampus as a Kalman filter . This requires that the hippocampus has an ‘observation model’ relating hidden states ( places specified in allocentric coordinates ) to sensory cues , and a dynamic model relating previous to current state via path integration . Kalman filtering then refers to the forward inference algorithm that combines path integral estimates of state with current sensory cues to provide optimal updates of the agent's location . The main function of Kalman filtering in this context is therefore one of localisation . One of the key points of this paper is that if an agent has taken the trouble to construct a ‘dynamic model’ and an ‘observation model’ then they can be used for more than just localisation; the same models , when combined with additional inference steps , can also be used for model selection , decision making and motor planning and to construct sensory imagery . Other statistical treatments of hippocampal function address the issue of context learning [21] . Here , a context is defined in statistical terms as a stationary distribution of experiences . The problem of context learning is then reduced to one of clustering together an agent's experiences into a finite number of contexts . This is addressed through the use of Hidden Markov Models ( HMMs ) and it is shown how this perspective explains experimental findings in rat navigation concerning sequence and reversal learning and place-cell remapping . Johnson et al . [22] provide a normative statistical model of exploratory behaviour called Information Foraging ( IF ) . ‘Passive IF’ describes the temporal distribution of an agent's sampling process ( eg . spending longer investigating novel versus familiar objects ) whereas ‘Directed IF’ describes its spatial distribution ( eg . where it should move to next ) . Additionally , IF is conceived to apply both to the environment and the agent's memory of the environment . Directed IF proposes a common hippocampal substrate for constructive memory ( eg . scene construction ) , vicarious trial and error behaviour , model-based facilitation of memory performance , and memory consolidation . The IF framework samples spatial locations , or episodic memories using an information theoretic criterion . To compute this criterion it is necessary for the agent to possess an observation model of the sort described in our article below . A further statistical treatment of hippocampal function comprises a two-stage processing model of memory formation in the entorhinal-hippocampal loop [23] . The first stage , which is proposed to take place during theta activity , allows hippocampus to temporally decorrelate and sparsify its input , and develop representations based on an Independent Component Analysis . The second stage , which is proposed to take place during Sharp Wave Ripples [24] , allows hippocampus to replay these new representations to neocortex where long term memories are held to be instantiated . This paper is concerned with computational processes underlying spatial cognition and we describe how the underlying computations may be instantiated in hippocampus and associated brain regions . The hippocampal formation is , however , implicated in a much broader array of functions [25] , such as episodic memory , that our model does not address . Indeed one of the key differences between our approach and some other models of spatial cognition [10] , [16] is that the approach we describe has no episodic component . Specifically , the sequences that are generated in our model are the result of online computation rather than memory recall . However , as we highlight in the discussion , the interactions between episodic memory and the computations we describe would be especially interesting to examine in future work . The paper is structured as follows . The computer simulations in this paper describe an agent acting in a simple two-dimensional environment . This environment produces visual , somatosensory and olfactory cues as described in the methods section on the ‘Environmental Model’ . The agent then develops its own model of the environment as described in the ‘Probabilistic Model’ section . This describes the two elements of the model ( i ) a dynamical model describing the evolution of hidden states and ( ii ) a mapping from hidden states to sensory states . The section on ‘Spatial Cognition as Statistical Inference’ then describes how the various tasks of localisation , decision making ( and sensory imagery ) , model selection and motor planning can be described in probabilistic terms . The section on ‘Forward and Backward Inference’ describes the common set of forward and backward recursions for estimating the required probability densities . The section on ‘Results’ describes an implementation of the above algorithms and provides some numerical results . The discussion section on ‘Neuronal Implementation’ then describes our proposal for how these algorithms are implemented in the brain and how functional connectivity among a candidate set of brain regions changes as a function of task . We conclude with a discussion of how the above computations might relate to pattern replay and what are the specific predictions of our model .
Computer simulations are implemented in Matlab ( R2012a , The MathWorks , Inc . ) and are based on an agent navigating in a simple 2D environment depicted in Figure 1 . The location of the agent is specified using orthogonal allocentric coordinates and its direction of heading ( clockwise from positive ) is . The environment contains two inner walls and four boundary walls . The agent is equipped with a touch sensor that detects the minimum Euclidian distance to a wall , . It is also equipped with a nose that detects olfactory input , . In this paper we consider a single olfactory source located at allocentric coordinates . We assume this source diffuses isotropically with scale parameter so that olfactory input at location is given by an exponential function ( 1 ) All of the simulations use a single olfactory source with , and . More realistic environments with multiple olfactory sources and turbulence [26] are beyond the scope of this paper . The agent is also equipped with a retina that is aligned with the direction of heading . The retina provides one-dimensional visual input , , from −45 to +45 degrees of visual angle around and comprises pixels . The retina provides information about the ‘colour’ of the walls within its field of view . In our simulations ‘colour’ is a scalar variable which we have displayed using colormaps for ease of visualisation . The scalar values corresponding to the various walls are 0 . 14 ( north border ) , 0 . 29 ( east border ) , 0 . 43 ( south border ) , 0 . 57 ( west border ) , 0 . 71 ( west wall ) , 0 . 86 ( east wall ) . These map onto the colours shown in Figure 1 using Matlab's default colour map . Although classical laboratory navigation tasks do not involve walls with different colours , they employ extra-maze cues which enable experimental subjects to localize themselves . For the sake of simplicity , here we provide such visual information to the simulated agent by variation of wall colour . The environmental model of retinal input takes the values of and and produces using calculations based on the two-dimensional geometrical relation of the agent with the environment . This uses a simple ray-tracing algorithm . The agent then has its own predictive model of retinal input , described in the ‘vision’ section below , which predicts from and using a basis set expansion . The agent has similar models of olfactory and somatosensory input ( see ‘Olfaction’ and ‘Touch’ below ) . Overall , the environmental model produces the signals , and which form the sensory inputs to the agent's spatial cognition model ( see next section ) . We write this as to denote sensory signals from the environment . For a sequence of signals we write . These sensory inputs are surrogates for the compact codes produced by predictive coding in sensory cortices [27] . We emphasise that the agent has its own model of sensory input ( an ‘observation model’ ) which is distinct from the environmental input itself . The agent's observation model is learnt from exposure to the environment . We investigate agents having a model comprising two parts ( i ) a dynamical model and ( ii ) an observation model . The dynamical model describes how the agent's internal state , is updated from the previous time step and motor efference copy . The observation model is a mapping from hidden states to sensory states . Our probabilistic model falls into the general class of discrete-time nonlinear state-space models ( 2 ) where is a control input , is state noise and is sensory noise . The noise components are Gaussian distributed with and . This is a Nonlinear Dynamical System ( NDS ) with inputs and hidden variables . We consider a series of time points and denote sequences of sensory states , hidden states , and controls using , , and . These are also referred to as trajectories . The above equations implicitly specify the state transition probability density and the observation probability density . This latter probability depends on the agent's model of its environment , . Together these densities comprise the agent's generative model , as depicted in Figure 2 ( top left ) . This section describes , initially at the level of manipulations of probability densities , how the various computations underlying spatial cognition can be implemented . It then describes a practical algorithm based on local linearisation . If an agent has a probabilistic model of its environment , , then the various tasks that together comprise spatial cognition are optimally implemented using statistical inference in that model . These inferences will be optimal in the sense of maximising likelihood . The various tasks - localisation , imagery , decision making , model selection and planning - all rely on the same statistical model . They are differentiated by what variables are known and what the agent wishes to compute . This is depicted in the panels in Figure 2 where shaded circles denote known quantities . Additionally , for each task , the information entering the system may be of a different nature . For example , for imagery , the inputs , , are virtual motor commands and for localisation they are motor efference copies . Similarly , during localisation and model selection the agent receives inputs from sensory cortices . For the simulations in this paper these come from the environmental model , . However , during decision making and motor planning these inputs do not derive from the agent's environment but are generated internally and correspond to the agent's goals . Text S2 describes how the required probability densities can be computed at the very general level of manipulations of probability densities . However , these operations cannot be implemented exactly . They can only be implemented approximately and there are basically two types of approximate inference methods . These are based either on sampling [39] or Local Linearization ( LL ) [40] . In this paper we adopt an LL approach although this is not without disadvantages . We return to this important issue in the discussion . The following subsections describe the forward and backward inference algorithms under LL assumptions . Readers unfamiliar with statistical inference for dynamical systems models may benefit from textbook material [38] .
This section describes a preliminary learning phase in which an agent is exposed to an environment to learn the sensory mapping from states to observations . Here the agent is provided with the observations and also exact knowledge of the hidden states . More realistic simulations would also require the agent to infer the hidden states whilst learning . This is in principle straightforward but is beyond the scope of the current paper , as our focus is on temporal dynamics . We return to this point in the discussion . The olfactory and sensorimotor models use a 10-by-10 grid of basis cells giving 100 cells in all . We assume that the parameters governing the location and width of these cells have been set in a previous learning phase . The weight vectors and ( see equations 11 and 12 ) were optimised using least squares regression and 225 training exemplars with uniform spatial sampling . The retinal model used the same number and location of basis cells . It additionally used 32 head direction cells each having a directional precision parameter . The conjunctive representation comprised 3200 basis cells . The weight vector ( see equation 14 ) was optimised using least squares and a training set comprising 10 , 575 exemplars . These were generated from spatial positions taken uniformly throughout the maze . Visual input from the environmental model for multiple directions at each spatial location was used to create the training examples . At the end of this learning phase the agent is exquisitely familiar with the environment . A trained model can then be used to generate visual imagery . This is implemented by specifying a synthetic control sequence , running path integration and generating predictions from the model . For example , Figure 3A shows a control sequence that is used to generate the ‘north-east’ trajectory shown in Figure 3C . We also generated ‘north-west’ , ‘south-west’ and ‘south-east’ trajectories by changing the sign of direction change , , and/or the initial direction , . To quantitatively assess the accuracy of these imagery sequences , , we compared them to the sequence of visual inputs that would have been received from the environmental model , . Figure 3D plots the proportion of variance explained by the agent's model as a function of retinal angle . These plots were computed separately for each trajectory , and show that only activity in the central retina is accurately predicted . This is due to the increased optic flow in peripheral regions of the agent's retina . The asymmetry in Figure 3D is due to the particular spatial arrangement and numerical values of the visual cues . These results suggest that it would be better to have a retina with lower spatial resolution in the periphery . This simulation shows how an agent can localise itself in an environment . The agent was located centrally and moved according to the south-east trajectory . Its exact path was computed using noiseless path integration and the appropriate environmental inputs were provided to the agent . In the discussion section below we propose a mapping of the forward and backward inference equations onto the hippocampal-entorhinal complex . We now report the results of two simulations . The first used the standard forward inference updates in equations 23 and 24 . This corresponds to the algorithm that an agent with an intact hippocampus would use . The second , however , had a ‘lesioned hippocampus’ in that only the path integral updates in equation 23 were used ( we set ) . This in effect removed the top down input from hippocampus to MEC ( see ‘Localisation’ subsection in the discussion ) so that path integral errors are not corrected by sensory input . In both cases the agent's path updates , , were subject to a small amount of noise ( with standard deviation 0 . 01 ) at each time step . Figure 4 shows the results for single and multiple trials . Here , localisation with an intact hippocampus results in better tracking of the agent's location . Localisation accuracy was assessed over multiple trials ( ) and found to be significantly more accurate with , rather than without , a hippocampus ( ) . The mean localisation error was 60 per cent smaller with a hippocampus . For the above simulations we disabled somatosensory input by setting . This was found to be necessary as this input is not a reliable predictor of location ( the distance from a boundary is the same at very many locations in an environment ) . This simulation shows how an agent can make a decision about which direction to turn by computing likelihood ratios . To demonstrate this principle , we selected the ‘north-west’ and ‘north-east’ trajectories as two possible control sequences . The sensory goal was set equal to the sensory input that would be received at the end of the ‘north-east’ trajectory . This goal was set to be identical at all time points . The agent's starting location was and with initial speed set to zero . The log of the likelihood ratio ( see equation 28 ) , , for model 1 versus model 2 was then computed at each time step . Figure 5 shows the accumulated as a function of the to time points along the trajectory . A of 3 corresponds to a probability of 95% [42] . This indicates that a confident decision can be made early on in the hypothesized trajectories . The degree to which each sensory modality is used in the above computations is determined by the relative values of observation noise covariances ( see Text S4 ) . These were initially fixed to the values described at the beginning of the simulations section . Whilst a confident decision could soon be reached using the above default values , decomposition of the LR into modality specific terms showed a strong contribution from both olfactory and visual modalities , but a somatosensory contribution that was initially rather noisy . This is due to small idiosyncrasies in the predictions of somatosensory values . We therefore experimented with the level of somatosensory noise covariance . Figure 5 was produced using a value of which means LR effectively ignores this contribution ( although we also have , there are 20 visual inputs ) . This simulation shows how likelihood ratios can also be used to estimate what environment an agent is located in . We first trained an agent on the maze as described in the imagery section . We refer to this as environment one and the model , described by the set of estimated weights , as model one . We then trained the agent on a second environment and allowed it to develop a separate model . These are referred to as environment two and model two . The second environment was exactly the same as the first except that the east and west boundary walls had their colours swapped . We then placed the agent in the first maze and used the ‘north-east’ control trajectory , , and allowed the agent to compute the likelihood of observed data under its two models , and , as described earlier . The log of the likelihood ratio , for model 1 versus model 2 was then computed at each time step . Figure 6 shows the as a function of the number of time points along the trajectory . The degree to which each sensory modality is used in the above computations is determined by the relative values of observation noise covariances . These were fixed to the values described at the beginning of the simulations section . However , because the only difference between the two models is in their predictions of retinal input ( due to the swapping of wall colours ) , the above computation is driven solely by vision . For the decision making example , described above , the likelihood of reaching the goal given the two trajectories is also differentiated by the olfactory inputs at the goal location ( as the olfactory source is located in the south west corner and diffuses isotropically , there will be weaker input in the north east than north west corner ) . This explains the scaling differences in the likelihood ratios - decision making is easier , in this example , as it is guided by olfaction as well as vision . This is not generally the case , however , and only occurred here due to the specifics of the environments and goals ( same olfactory sources at same locations in both mazes , different olfactory inputs at the two goals ) . This simulation gives an example of how route and motor planning can be implemented . The agent is placed in maze 1 at starting location , with initial speed and direction . This initial state , , is known with high precision ( see equation 27 ) . The initial distribution over motor controls has mean and precision ( see equation 32 ) . The covariance of the noise on the motor controls is set to ( see equation 31 ) . This specifies that the control signals for changes in acceleration ( first element ) are expected to be larger than those for direction ( second element ) . For this simulation we augmented the sensory vector with observations of the agent's speed . The sensory goal is multimodal with components for olfaction , touch , vision and speed . For olfaction , touch and speed we set , and . The goal is therefore to navigate to the point in space with olfactory code most similar to . The environmental location with this value is , . The observation noise covariance for speed was set to . A second aim is that the distance to the nearest boundary should be close to . A third aim is that the speed should be as near to as possible . That is , the agent should be stationary at the target . The visual component is set to correspond to an image of the left wall with all ‘yellow’ values . The desired goal trajectory , , is set to be equal to the goal at all time points . The degree to which each sensory modality is used in motor planning is determined by the relative values of observation noise covariance . We used the values described at the beginning of the simulations section . This means that motor planning is guided most by olfaction and touch , and least by vision . The estimated hidden states and inputs were then computed as shown in the earlier section on ‘Inference over Inputs’ . Figure 7 shows the planned route traced out by forward and backward inference . For forward inference we are plotting the and elements of ( see equation 24 ) , and for backward inference the and elements of ( see equation 30 ) . The paths for backward inference are smoother and more direct . Figure 7 also shows the estimated motor control sequence . These sequences correspond to the mean from backward inference , , as described in the section on ‘Inference over Inputs’ ( see equation 33 ) . Simple decisions such as ‘turn left’ or ‘turn right’ can be implemented using the ‘decision making’ procedure described in the above section . This is a rudimentary form of planning . The route and motor planning described here is a more powerful approach that we envisage is engaged when the optimal route to a goal involves the chaining together of multiple decisions ( eg . ‘turn left’ , ‘straight on’ , ‘turn right’ ) .
This section discusses how and where in the brain the above computational processes might be implemented . Our starting point here is Figure 8 which describes a candidate set of brain regions . Entorhinal cortex is partitioned into Lateral ( LEC ) and Medial ( MEC ) components , with the latter representing spatial and the former non-spatial information [43] . The LEC receives substantial input from perirhinal cortex which in turn receives major projections from temporal cortices , whereas the MEC receives substantial input from parahippocampal cortex which in turn receives projections from parietal cortices . The anatomical connectivity supporting this architecture is described in Figure 3 of [44] . We assume that temporal , parietal , parahippocampal and perirhinal cortices and the machinery that feeds into them , together produce a compact coding of spatial and non-spatial aspects of the agent's environment . These processes are not explicitly modelled in this paper . Our simple and tentative mapping onto hippocampal neuroanatomy currently does not distinguish between CA3 and CA1 , instead we consider a single hippocampal node encompassing the activity of CA3-CA1 place cells . Our model then comprises two hippocampal-entorhinal loops , one spatial and one non-spatial , as shown in Figure 8 ( top left ) . The spatial loop proceeds from superficial MEC layers to CA3-CA1 , and returns to deep layers of MEC . This partitioning into deep and superficial layers is consistent with known anatomy and previous functional models [45] . Anatomically , entorhinal-hippocampal connectivity is more complex with , for example , direct connections from EC layer three to CA1 [46] , and return connections via proximal CA1 ( CA1p ) and distal Subiculum ( SUBd ) [47] , but our model does not have this level of detail . The non-spatial loop proceeds from superficial LEC layers to CA3-CA1 , and returns to deep layers of LEC . The sensory states of our spatial model , , are compact codes representing non-spatial information in the superficial layers of LEC . Predictions of these sensory states from the agent's model , , are made via the CA3-CA1 to LEC pathway . In our model , the function of CA3-CA1 is to integrate spatial input from MEC with non-spatial input from LEC . This is consistent with a recent schematic model [48] , where it is argued that this functionality is preserved across mammals . The mapping from CA3-CA1 to LEC generates the agent's predictions of sensory states , whereas the mapping from LEC to CA3-CA1 implements the ( approximate ) inverse of this mapping . Together , these recurrent connections constitute the agent's model of its environment , , and different models will be instantiated in different subsets of these connections . That populations of cells in LEC encode sensory prediction errors , , is supported by recent recordings in rats [49] . This study identified cells that fired at locations where objects had been located on previous trials ( high prediction error ) , but did not respond when the object was actually present ( no prediction error ) . As with other proposals that the brain may implement some form of approximate Bayesian inference [71] , to formally test this idea it is necessary to have a proposal for how neural populations represent uncertainty . Ma et al . [72] , for example , have shown how populations of cells can represent probability distributions using probabilistic population codes in which simple linear combinations of firing rates can implement Bayesian inference . Beck at al . [73] have shown how such a scheme can implement Kalman filtering . As we have locally linearised the dynamic and observation nonlinearities , the forward inference step in this paper closely corresponds to Kalman filtering . It therefore seems plausible that forward inference using EKF can be implemented using similar principles . Thus , although equations 23 to 26 perhaps seem rather removed from neurobiology there may well be a plausible neural implementation . It has yet to be demonstrated how the gamma recursions underlying backward inference could be implemented using probabilistic population codes . However , given that the gamma recursions comprise an implementation of Bayes rule followed by a marginalisation ( see Text S2 ) whereas Kalman filtering is a marginalisation followed by Bayes rule ( see Text S2 ) we imagine a similar instantiation is possible . The Beck at al . [73] approach assumes that trial-to-trial variability in population firing rates is in a class of distributions from the linear-exponential family . This includes distributions where cells have independent Poisson rates . There is good evidence to suggest that MTL cell firing is not independent and Poisson [74] , but it is not known if their activity falls into the more general linear-exponential family . Other proposals as to how the brain might implement Bayesian inference are specific to the hippocampus . One proposal [75] suggests that higher certainty is encoded by spike patterns containing more spikes and where the spikes are closer together . If this is true then our perspective makes a number of simple predictions . For example , because backward inference produces higher certainty estimates than forward inference , backward replays should produce burstier spike trains . This should be simple to test using existing data [76] . An important part of our proposal is that the multiple tasks that together comprise spatial cognition can all be implemented using probabilistic inference in a single model . A caveat here is that our approach is restricted to goal-direction navigation . Whilst the forward inference in nonlinear dynamical systems that gives rise to the EKF algorithm , has a long history in estimates of localisation , there have been no proposals , to our knowledge , that also consider planning . However , in the machine learning literature , similar approaches for solving planning or control problems have been developed under the generic term ‘Planning as Inference’ . For example , Attias [77] has proposed that planning problems can be solved using Bayesian inference . The central idea is to infer the control signals , , conditioned on known initial state , and desired goal states . Similarly , Toussaint [78] describes the estimation of control signals using a Bayesian message passing algorithm which defaults to the classic Linear Quadratic Regulator ( LQR ) for linear Gaussian dynamics . Proposals have been made regarding how this Planning as Inference framework maps onto neural architectures in the brain [79] , [80] . A key difference to our proposal is that Toussaint solves a closed-loop ( feedback ) control problem . This finds a mapping from state-space to the optimal action , also known as the ‘policy’ . In terms of the underlying generative model in Figure 2 , this requires extra links from to . In this paper we solve an open-loop control problem . Our estimated control trajectory is a set of ballistic commands that cannot be updated in light of future information regarding the state of the system . Nevertheless , these commands can be rapidly computed at arbitrary time scales ‘on the fly’ , and this type of control strategy may be sufficient for a compliant motor system . In our simulations the agent learnt to predict sensory input using a pre-developed set of place cells with fixed centres and widths . This allowed us to use a simple regression approach for learning the basis function weights , which is similar to the standard two-stage optimisation process in machine learning . In the first stage basis functions are estimated in an initial unsupervised learning phase ( eg . based purely on MEC input ) , and basis function weights are learnt in a second , supervised learning phase [81] . Our simulations also assumed the agent had exact knowledge of its hidden state during learning , whereas more realistic simulations would also require the agent to infer these states . In principle this requires a straightforward implementation of the Expectation-Maximisation ( EM ) algorithm [38] , [82] for learning in dynamical systems . A more powerful alternative which integrates out the dependence on model parameters in the forward and backward passes is Variational Bayes ( VB ) [83] , [84] . Implementation of these VB schemes would mean that the maximum likelihood approach described in this paper would be replaced by a maximum evidence approach . Agents would implement decision making , model selection and motor planning by maximising the model evidence . Given that VB approximates the model evidence using free energy , the resulting scheme would then be broadly consistent with the Free Energy Principle [85] . A further detail here is that in previous applications of VB [83] , [84] , backward inference was implemented using the beta not the gamma recursions . In this paper we propose that it is the gamma recursions that are implemented in the brain , as they do not require storage of sensory observation sequences . The forward and backward algorithms are general purpose computations which may be implemented in a number of ways and this paper has focussed on an implementation based on local linearisation . The benefit of this is that the state probability distributions are Gaussian and so may be described with a small number of parameters; means and covariances . Additionally , there are analytic formulae for updating the parameters . A drawback of the LL approach is that the true probability distributions may be non-Gaussian . One possibility is that the distribution over the agent's location may be multimodal . This will be the case when an agent is placed in a familiar environment at an unknown location where there are multiple locations consistent with sensory data . For this scenario inferential methods based on sampling , such as particle filtering , would be more appropriate [37] . A second concern is that a single iteration of forward and backward inference may not be sufficient to find the controls that maximise the planning likelihood . It may be possible to improve the estimated controls by running multiple forward and backward replays such that the linearisation takes place around a different and improved trajectory each time . This iterated local linearisation would be analogous to the iterative Local Quadratic Gaussian ( iLQG ) approach from control theory [86] . This second concern may also be addressed by treating space as discrete rather than continuous . In this perspective the agent is currently located in one of a finite number of ‘bins’ each of which may correspond to the support of a place cell . The optimal trajectory through these bins can then be computed by solving a discrete Bellman equation . Todorov has shown that this corresponds to backward inference in a hidden Markov model [87] . This computation relies on a recursive high-dimensional update that is perhaps readily suited to the massively recurrent nature of CA3 . These computations would be consistent with earlier proposals that the hippocampus itself is suited for solving shortest path problems [88] . In regard to motor planning , this paper has described a forward and backward inference procedure which allows an agent to solve an open-loop control problem . This produces a control trajectory that is a set of ballistic commands that cannot be updated in light of future information regarding the state of the system . It is possible to augment the generative model to include extra links from states to actions , so that the agent instead learns a policy - a mapping from states to actions , as in [78] . This would then provide a solution to the closed-loop ( feedback ) control problem . However , it may be the case that the mammalian brain solves the closed-loop problem in two stages . First , the computational power of recurrent networks in CA3 could be used to implement forward and backward inference to solve the open-loop problem . Estimated trajectories would then be replayed to ventral striatum during quiet wakefulness or slow wave sleep . This is consistent with an earlier model [89] and the observation of ripple activity propagating to this region [90] . These replays would then be used to train up a habitual dorsal striatal decision making system ( see [11] for a review of habitual versus flexible/deliberative systems and their anatomy ) . This is also consistent with proposals that for known environments , navigational control is gradually transferred from a flexible inferential system to a habitual system based on a hippocampo-striatal mapping [14] . Such a hippocampo-striatal model has previously been proposed by Foster et al . [29] . This paper has described how the various aspects of spatial cognition can be implemented using inference in a statistical model . It has not , however , addressed the broader cognitive control issues such as how internally generated goals are produced or when to switch between localisation versus model selection versus decision making modes . A recent computational framework [22] , called Information Foraging ( IF ) , however , does address some of these issues . This approach requires that agents compute the information that will be gained by making spatial decisions , which in turn requires the agent to have a probabilistic model of its environment . Thus , it would be possible for both IF and the Forward-Backward ( FB ) model to both use the same underlying probabilistic model , with perhaps IF deciding when to run an iteration of FB . This paper has proposed how model-based control may be implemented using spatial models implemented in hippocampal circuits . But it has not addressed how the control of decision making is arbitrated between , for example , model-based and model-free controllers . An influential proposal here [15] is that such arbitration is based on the confidence with which each system can make a decision . Thus , model-based and model-free systems can be combined by weighting each decision with their relative confidence . The ‘Mixed Instrumental Controller’ [19] also makes use of both types of decision making system . The model-based system incurs a fixed computational penalty reflecting the fact that model-based decisions require time to reach . If the estimated benefit of a model-based decision does not exceed this penalty then control is given to the model-free controller . The next and final section of this discussion summarises the specific predictions of the model proposed in this paper . To put these predictions in context we now briefly review two sets of empirical findings . These are , firstly , the observations of ‘theta sequences’ [91] which are sequential patterns of place cell firing occurring whilst rats move about in their environment and theta activity is recorded in hippocampus . The second set of observations are , again , sequential patterns of place cell firing but now occurring during sleep or quiet wakefulness and when Sharp Wave Ripples ( SWRs ) ( henceforth ‘ripples’ ) [24] are recorded in hippocampus . The phenomenon of phase precession refers to the observation [92] , [93] that place cells fire at gradually earlier phases of the hippocampal theta rhythm as rats move through their place fields . This is consistent with the notion of ‘theta sequences’ in which place cells fire in sequence within a theta cycle . Theta sequences have since been measured across cell-populations [91] . Additionally , theta sequences which sweep forward in advance of a rat's current location have been observed and are especially noteworthy at decision points in maze navigation . For example Johnson and Redish [67] recorded the activity of neural ensembles in the dorsal hippocampal CA3 region of awake behaving rats running in a T-maze . They found that as rats reached a decision point , representations swept predominantly forward from the current location , first down the right path and then the left . This activity did not occur in both forward directions simultaneously: the representation first encoded one arm and then the other . Finally , Gupta et al . [4] have shown that theta sequences represent distances further ahead of a rat during acceleration and further behind during deceleration , and that these sequences represent the environment in ‘chunks’ . A key feature of theta sequences is that they are time-compressed , occurring at about 5 to 10 times the speed of actual behaviour [91] , [93] , [94] . That is , were a rat to run through an environment at a typical speed , it could activate the same sequence of place cells , but would do so 5 to 10 times more slowly . We now turn to the discussion of ripple activity . In humans , episodic memories are thought to be encoded by the Medial Temporal Lobe ( MTL ) memory system . Information regarding these memories can then be transferred to neocortex [95]–[97] and a proposed mechanism of this transfer is the replay of episodes during later waking or sleep [27] so that neocortical synaptic plasticity can then act to strengthen cortico-cortical connections . This replay activity has been observed primarily in rodents using spatial navigation tasks [98] during ripples in Slow Wave Sleep ( SWS ) [99] and quiet wakefulness . There is evidence that this pattern replay is related to consolidation and transfer , as disrupting ripples impairs performance in a spatial memory task [100] . Place cell sequences observed during awake ripples have been observed to be played backwards . This is known as reverse replay . Foster and Wilson [76] , for example , recorded from cell ensembles in dorsal CA1 hippocampus in awake behaving rats and detected reverse replays after a rat had run the length of a 1D track . Similar reverse replays that start immediately after navigation have been observed on other 1D tracks [101] , a linear path through a 2D environment [102] , a 2D open-field environment [103] , and a two choice T-maze [104] . Place cell sequences observed during awake ripples have also been observed to be played forwards [101] . This is known as forward replay . Replay activity during ripples is also time-compressed , with sequences being replayed within the duration of a single ripple ( 50–250 ms ) . This corresponds to a compression factor of about 15 to 20 relative to the original behaviour [102] . The above forward and backward replays are also known as ‘local replays’ or ‘locally initiated replays’ so as to distinguish them from another phenomenon known as ‘remote replay’ or ‘remotely initiated replay’ . This occurs when a rat replays an experience of one place whilst being physically located in another . In one experiment [105] , rats were exposed to two different environments which had the same physical structure ( allocentric layout ) but differed in their set of visual cues . Replays of trajectories in one maze were observed whilst the rat was located in the other . Remote replay has also been observed [102] , [104] where rats replayed activity corresponding to remote parts of the same environment . As is the case with local replays , remote replays can be forward or backward in time [104] . In general , replay activity during ripples can be forward or backward , whereas theta sequences are always forward . Jadhav et al . [106] have interrupted awake ripples during performance of a navigation task with alternating goals in a W-shaped maze . Ripple disruption was found to affect decision making on the outbound leg of the task , which required linking of past information with current location . However , it did not affect the inbound leg which required no such memory component therefore providing evidence that awake ripples support spatial working memory . Finally , Dragoi and Tonegawa [107] have observed ‘preplay’ activity . Here , the sequence of place-cell firing during a novel spatial experience occurred on a significant number of occasions during the resting or sleeping period prior to that experience . They propose that this activity organises hippocampal assemblies into dynamical structures ready for subsequent associations with sensory episodes . This section summarizes the predictions of our model ( the ‘FB model’ ) . We indicate where these predictions are unique to the proposed model and where they are shared by others . We have shown that the various computations underlying spatial cognition can be implemented using statistical inference in a single probabilistic model . Inference is implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time . We have proposed a mapping of the above computational processes onto lateral and medial entorhinal cortex and hippocampal regions CA3-CA1 . This proposed mapping is consistent with recent findings in rat electrophysiology , and other proposals that one function of the hippocampus that is preserved across mammalian species , is that it integrates spatial and non-spatial information . We have also proposed that these computations are reflected in recent findings of pattern replay in the mammalian brain . Specifically , that theta sequences reflect decision making , theta flickering reflects model selection , and remote replay reflects route and motor planning . Many of the underlying hypotheses can be tested using existing data .
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The ability of mammals to navigate is well studied , both behaviourally and in terms on the underlying neurophysiology . Navigation is a well studied topic in computational fields such as machine learning and signal processing . However , studies in computational neuroscience , which draw together these findings , have mainly focused on specific navigation tasks such as spatial localisation . In this paper , we propose a single probabilistic model which can support multiple tasks , from working out which environment you are in , to computing a sequence of motor commands that will take you to a sensory goal , such as being warm or viewing a particular object . We describe how these tasks can be implemented using a common set of lower level algorithms that implement ‘forward and backward inference over time’ . We relate these algorithms to recent findings in animal electrophysiology , where sequences of hippocampal cell activations are observed before , during or after a navigation task , and these sequences are played either forwards or backwards . Additionally , one function of the hippocampus that is preserved across mammals is that it integrates spatial and non-spatial information , and we propose how the forward and backward inference algorithms naturally map onto this architecture .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"circuit",
"models",
"mathematics",
"computational",
"neuroscience",
"statistics",
"statistical",
"methods",
"biology",
"computational",
"biology",
"nonlinear",
"dynamics",
"neuroscience",
"learning",
"and",
"memory"
] |
2013
|
Forward and Backward Inference in Spatial Cognition
|
Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment . Therefore , therapeutic vaccines represent an attractive alternative control strategy . The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium ( rSh28GST ) in healthy volunteers , and to determine its immunogenicity . Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide ( Alum ) as adjuvant ( n = 8 ) , or Alum alone as a comparator ( n = 8 ) , twice with a 28-day interval between doses . A third dose of rSh28GST or Alum alone was administered to this group at day 150 . In view of the results obtained , another group of healthy volunteers ( n = 8 ) received two doses of 300 µg of rSh28GST , again with a 28-day interval . A six-month follow-up was performed with both clinical and biological evaluations . Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production , the capacity of sera to inhibit enzymatic activity of the antigen , and in vitro cytokine production . Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration . Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects . No hematological nor biochemical evidence of toxicity was detected . Immunological analysis showed that rSh28GST was immunogenic . The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST . rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response . Together with previous preclinical results , the data of safety , tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis .
Schistosomiasis , the second major human parasitic infection after malaria , remains a major health problem in many developing countries , mainly among children and it is estimated that this chronic disease is responsible for 300 000 deaths per year . During Schistosoma haematobium infection , morbidity and mortality are associated with worm fecundity and the deposition of schistosome eggs in tissues , especially in the genital and urinary tracts . More than 20 years after its introduction , the most effective intervention for the control of schistosomiasis remains the use of chemotherapy by praziquantel ( PZQ ) [1] but , it is generally agreed that this shows numerous limits . Indeed , rapid re-infection following treatment is commonly observed in most endemic areas [2] . Thus , efficient drug delivery requires a substantial infrastructure to regularly cover endemic areas , which makes chemotherapy an expensive approach [3] . In addition , although there is not yet clear evidence of the existence of PZQ-resistant strains , a decreased susceptibility to the drug has been suspected in several countries [4] , [5] . The lack of efficient treatment emphasizes the need for more specific and long-term approaches against schistosomiasis . A vaccine strategy may therefore play a crucial role in the control of this parasitic disease . Among several vaccine candidates [6] , the 28 kDa glutathione S-transferase antigen ( 28ShGST ) has been well characterized ( from molecular cloning to crystallisation ) [7] , [8] . Immunization of rodents [9] , [10] , monkeys [11] , [12] or cattle [13] , [14] with 28GST , followed by experimental infection led to a reduction in worm burden and/or a significant decrease in parasite fecundity , establishing this antigen as a promising vaccine candidate [15] , [16] . In addition , field studies have shown that resistance to re-infection in humans could be associated with the presence of acquired anti-28GST antibodies able to inhibit the parasite GST enzymatic activity [17][18] . Moreover , studies have demonstrated the role of Th2-type responses in anti-schistosome protective immunity in human infection [19] . Toxicological studies were conducted in animal models to support the clinical use of the recombinant Sh28GST ( CERB , France and Pasteur Institute of Lille , France; unpublished results ) . In compliance with Good Laboratory Practice standards , a rabbit model was selected because it produces immune responses to the protein , and the full dose intended for human use can be subcutaneously administered to rabbits . In these conditions , a 4-week study was performed where each rabbit was tested with 9 injections distributed over the lumbar area ( 2 with aluminium hydroxide ( Alum ) , 2 at a 100-µg dose of rSh28GST in Alum , 2 at a 300-µg dose , 2 at a 1000-µg dose and a final mock injection ) . A transient local inflammatory reaction after injection was common ( erythema , swelling or small induration at the site of injection ) and comparable to that seen with Alum alone . In another toxicity study using 3 subcutaneous injections in rats ( 40-µg dose of rSh28GST in Alum , a 200-µg dose in Alum or Alum alone ) , animals were monitored for morbidity , mortality , general health and signs of toxicity , food consumption , body weight , clinical observations and also for respiratory , circulatory , dermatological , autonomic and central nervous system parameters . Hematology , blood chemistry , and immunological analyses were also performed . Major tissues and organs were histologically examined on necropsy . In these conditions , no significant clinical , anatomical or biological alterations were found . Finally , a lack of significant alterations was also demonstrated in toxicity studies performed in dogs . All these findings taken together justify the decision to initiate a phase 1 clinical trial to evaluate the safety and tolerability of the rSh28GST in healthy adult male volunteers . The secondary objective of this randomized phase 1 study was to assess the immunogenicity of rSh28GST .
According to requirements of the French Health authorities regarding first-in-human clinical trials that should avoid the inclusion of children and women with childbearing potential except under specific conditions , the present phase 1 study was conducted in young healthy Caucasian male adults . All subjects had to meet the study inclusion criteria within 21 days prior to treatment , which included written informed consent , age from 18 to 30 , absence of any medical history of schistosomiasis , biological parameters ( haematological , biochemical , renal and hepatic ) within the normal range as well as a normal clinical examination . Moreover , subjects were not smokers and were exempt of any inflammatory or immunological pathology such as atopic diseases , evidence of inflammation or acute infection ( including positive serology to viral hepatitis B and C or HIV ) , any immunological deficiency , any clinically relevant alcohol or drug use ( cannabis , opiates , cocaine , amphetamines , benzodiazepines , nicotine , barbiturates , meprobamates or antidepressant drugs according to a urinary drug and metabolites screen ) or any other medication use within 2 weeks before the study , any vaccination within the last 6 months and had no antibodies against the Sh28GST protein . This phase 1 randomized controlled study was designed to investigate the safety and tolerability of rSh28GST antigen injected subcutaneously ( deltoid muscle ) with Alum as adjuvant . In a first step , the study was performed on healthy male volunteers according to a double-blind design . Subjects selected at random received either two 100 µg doses of rSh28GST together with Alum as vaccine adjuvant ( n = 8 ) or two doses of Alum in a 0 . 5 ml solution as a comparator ( n = 8 ) with a 28-day interval ( Day 0 [D0] and Day 28 [D28] ) between doses in both groups ( figure 1 ) . These subjects also received a third administration of 100 µg Sh28GST or Alum alone on day 150 [D150] . As a dose escalation trial , a third group of healthy volunteers received two 300 µg doses of rSh28GST at a 28-day interval according to an open-label design ( figure 1 ) . All biological analyses were carried out blind to treatment conditions . Batches of rSh28GST were produced and purified from recombinant Saccharomyces cerevisiae culture ( TGY73 . 4 - pTG8889 strain ) under Good Manufacturing Practice ( GMP ) conditions by Eurogentec S . A . ( Belgium ) . The rSh28GST clinical batch ( # B98H11 ) was conserved lyophilized ( 124 µg per vial for the administrated dose of 100 µg; 352 µg per vial for the administered dose of 300 µg ) by Sterilyo ( France ) under GMP conditions . The lyophilized preparation was re-suspended extemporaneously using 0 . 6 ml of apyrogenic and sterile aluminium hydroxide solution 0 , 2% ( Al2O3 0 . 2%; Al ( OH ) 3 3%; NaCl 9 g/L; ammonium carbonate buffer 10 mM , pH7 . 8 ) ( Alum from Superfos , Denmark; batch #14093 ) and administered in a volume of 0 . 5 ml . Following each administration , participants were kept under constant observation during 4 hours and further evaluated at D1 , D21 , D28 , D29 , D49 , D120 , D150 , D165 and D180 . Clinical evaluations consisted of measurement of vital signs and assessment of the local injection site and general signs or symptoms . Local signs and symptoms included pain or tenderness , swelling , induration and erythema at the site of injection . Assessment of general tolerability was obtained through a complete physical examination including an examination of general appearance , body weight and rectal temperature , head , eyes , ears , nose and throat , neck , skin , cardiovascular and respiratory system , abdominal system , nervous system , lymphatic area , blood pressure , pulse and respiratory rates . Recordings of 12-lead ECG were also obtained allowing analysis of ventricular rate , rhythm , PR interval , QRS duration and QT/QTc . A number of general signs and symptoms were specially considered including fever ( rectal temperature ≥38°C ) , headache , nausea , vomiting , myalgia , arthralgia , irritability/fussiness and drowsiness , loss of appetite and sleep disturbances . In addition to these predefined signs and symptoms , all adverse events were reported throughout the study . Adverse events were graded by severity following FDA guidance [22] and judged for potential association with vaccination . Briefly , grade 1 adverse events were easily tolerated , causing minimal discomfort and not interfering with daily activities; grade 2 adverse events were sufficiently discomforting to interfere with normal activities , grade 3 adverse events prevented normal daily activities while grade 4 were potentially life threatening [22] . Biological tolerability was assessed at baseline and at D1 , D21 , D28 , D29 , D49 , D120 , D150 , D165 and D180 using clinical laboratory tests including haemoglobin , haematocrit , packed cell volume , mean corpuscular volume , mean cell haemoglobin , mean cell haemoglobin concentration , white blood cells and platelet counts; coagulation tests such as activated partial thromboplastin time ( PTT ) , prothrombin time and International Normalized Ratio ( INR ) ; serum biochemistry such as sodium , potassium , creatinine , urea , alanine amino transaminase ( ALT ) , aspartate amino transaminase ( AST ) , gamma glutamine transferase ( gamma-GT ) , bilirubin , uric acid , total protein , alkaline phosphatases , calcium , C-reactive Protein ( CRP ) and fasting glucose; urinalysis including pH , protein , glucose , ketones , urobilirubin , blood and leucocytes . For all these laboratory tests , toxicity grading was assigned using local laboratory values or previously published normal reference ranges [23] . Specific antibody titers in individual sera were determined by ELISA . rSh28GST ( 10 µg/ml ) was coated on 96-well plates ( Nunc , Roskilde , Denmark ) 2h30 at 37°C . Plates were blocked with phosphate-buffered saline containing 0 . 5% gelatin ( Merck , Darmstadt , Germany ) . Then , six serial dilutions of individual sera ( first dilution for IgG1 1/100; IgG2 , IgG3 and IgA 1/20; IgG4 and IgE 1/10 ) were added and incubated . Each corresponding biotinylated mAbs to human Ig isotype ( Southern Biotechnology , Birmingham , AL , USA ) was incubated ( 2 h at 37°C ) at a 1/1000 dilution for IgG , IgG1 , IgG2 , IgG3 , IgG4 , IgE and IgA , and 1/1500 for IgG1 detection . Peroxidase-conjugated streptavidin ( 1/1500; 30 min at 37°C ) was then added ( Amersham , Les Ulis , France ) . Colorimetric development was performed with ABTS ( Sigma , St . Louis , MO , USA ) in 50 mM citrate buffer ( pH = 4 ) and absorbance ( OD ) was measured at 405 nm . Titers were defined as the highest dilution yielding an absorbance value three times above the background ( wells containing buffer instead of serum in the same plate ) . Vaccinated individuals were considered as positive responders when their titer was greater than three fold the standard deviation above the mean titer value of all individuals at D0 ( vaccine and comparator group ) . Cytokine production was evaluated in whole blood diluted in RPMI 1640 medium ( Gibco , Courbevoie , France ) to obtain 2×106 mononuclear cells per ml . The blood preparation ( 1 ml ) was then plated in duplicate culture in 24 flat-bottomed plates ( Nunc ) and stimulated with 1 ml of rSh28GST antigen ( 20 µg/ml ) . Supernatants were harvested on day 3 for analysis of cytokine production . Quantitative ELISA for human IL-2 , IL-4 , IL-5 , IL-10 , IL-12 , IL-13 and IFN-γ detection were performed in supernatants using EASIA Kits ( Immunotech , Marseille , France ) and human TGF-β1 by ELISA System Kit ( Promega , Madison , WI , USA ) . Cytokine concentrations were expressed as pg/ml or IU/ml ( for IFN-gamma ) after subtracting the amount detected in unstimulated control cultures . Cytokine production was considered stimulated when the value was more than three times the standard deviation above the mean value . To evaluate the capacity of induced antibodies to inhibit GST activity , 20 µl of rSh28GST solution ( 4 µg/ml in 50 mM potassium phosphate at pH 6 . 5 ) was incubated with 20 µl of human serum for 1 h at 37°C in Immulon 3 Plates ( Nunc , Roskilde , Denmark ) . The enzymatic reaction was carried out using 1-chloro-2 , 4 , dinitrobenzene ( Sigma , St . Louis , MO ) substrate as described elsewhere [18] . Enzymatic reaction intensity was measured by OD at 340 nm . Every enzyme-inhibition test was performed with appropriate controls ( enzyme without serum and tested serum alone ) . The percentage of inhibition was calculated by the ratio of GST activity after serum incubation to the GST activity control . Percentage inhibition above 10% was considered significant . The sample size for this first-in-human study was not powered on the basis of statistical hypothesis testing since as is customary for phase 1 studies , hypothesis testing is not part of the primary objective . The target number of subjects was expected to minimize the risk by not exposing too large a population to treatment , while providing an ample size to gain information on safety and tolerability . The sample size of 8 subjects for each group ( both rSh28GST and control ) ensured at least an 85% probability of observing ≥1 subject with a specific adverse event if the background incidence rate of that adverse event was ≥10% . This sample size also ensured a ≥80% probability of observing common placebo-associated adverse events , defined as those events with a population incidence ≥15% . Descriptive statistics are presented using mean and standard deviation or median and range as required . Baseline laboratory values were defined as the pre-treatment values obtained during the baseline screening visit . Statistical analysis for the immunological studies was performed using the Wilcoxon signed rank test to assess the difference in immunological parameters between days using SPSS v15 for Windows . Differences were considered significant at p<0 . 05 .
A total of 31 healthy male volunteers underwent initial evaluation for the study . Among these , 7 were eliminated using exclusion criteria such as allergy or hypersensitivity , tobacco use or elevated immunoglobulins . Finally , 24 volunteers were randomized . They were 18 to 29 years old ( 23±3 years ) , height from 170 to 191 cm ( 178±6 cm ) and weight from 52 to 82 kg ( 71±8 kg ) . All other clinical and routine laboratory data were considered in normal ranges although 1 subject in the comparator group and 3 subjects in the 300 µg group had a slight increase in bilirubin according to local laboratory values ( main baseline biological data in table 1 ) . All volunteers were followed until the end of the study . All local , general and biological adverse events reported in the present study are detailed in table 2 .
This phase 1 study demonstrates the safety , tolerability and immunogenicity of rSh28GST in Alum as a first vaccine candidate against schistosomiasis . These results allowed us to conduct further clinical trials in endemic regions ( manuscripts in preparation ) . The objectives of these studies were again to assess the safety , tolerability and immunogenic profile of the recombinant rSh28GST in Alum in healthy school children , and also in adults and children infected with S . haematobium . A clinical trial evaluating the efficacy of the vaccine candidate is currently in progress in a large cohort of infected schoolchildren .
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Therapeutic vaccines represent an attractive tool in the fight against schistosomiasis . Pre-clinical immunization studies with the schistosome enzyme 28 kDa glutathione S-transferase ( 28GST ) has been shown to significantly reduce schistosome egg production and subsequent pathology . The objective of this study was to assess the safety and immunogenicity of the recombinant 28GST of Schistosoma haematobium ( rSh28GST ) in healthy adult volunteers . After three administrations of 100 µg or two of 300 µg , no serious adverse events were reported in the days or weeks after each administration . Some mild adverse events were noted , including minor reactions at the injection site reported for four subjects receiving rSh28GST , but there was no hematological or biochemical evidence of toxicity . Immunological analysis showed that rSh28GST induced a consistent immune response characterized by antibodies endowed with the capacity to inhibit 28GST enzymatic activity . Present data provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"vaccines",
"medicine",
"vaccination",
"clinical",
"immunology",
"immunity",
"parasitology",
"immunology",
"biology",
"microbiology",
"vaccine",
"development",
"immune",
"response"
] |
2012
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Safety and Immunogenicity of rSh28GST Antigen in Humans: Phase 1 Randomized Clinical Study of a Vaccine Candidate against Urinary Schistosomiasis
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Alveolar echinococcosis ( AE ) is caused by infection with the larval stage of the tapeworm Echinococcus multilocularis . An increasing understanding of immunological events that account for the metacestode survival in human and murine AE infection prompted us to undertake explorative experiments tackling the potential of novel preventive and/or immunotherapeutic measures . In this study , the immunoprotective and immunotherapeutic ability of recombinant EmP29 antigen ( rEmP29 ) was assessed in mice that were intraperitoneally infected with E . multilocularis metacestodes . For vaccination , three intraperitoneal injections with 20μg rEmP29 emulsified in saponin adjuvants were applied over 6 weeks . 2 weeks after the last boost , mice were infected , and at 90 days post-infection , rEmP29-vaccinated mice exhibited a median parasite weight that was reduced by 75% and 59% when compared to NaCl- or saponin–treated control mice , respectively . For immunotherapeutical application , the rEmP29 ( 20μg ) vaccine was administered to experimentally infected mice , starting at 1 month post-infection , three times with 2 weeks intervals . Mice undergoing rEmP29 immunotherapy exhibited a median parasite load that was reduced by 53% and 49% when compared to NaCl- and saponin–treated control mice , respectively . Upon analysis of spleen cells , both , vaccination and treatment with rEmP29 , resulted in low ratios of Th2/Th1 ( IL-4/IFN-γ ) cytokine mRNA and low levels of mRNA coding for IL-10 and IL-2 . These results suggest that reduction of the immunosuppressive environment takes place in vaccinated as well as immunotreated mice , and a shift towards a Th1 type of immune response may be responsible for the observed increased restriction of parasite growth . The present study provides the first evidence that active immunotherapy may present a sustainable route for the control of AE .
Alveolar echincoccosis ( AE ) , caused by the metacestode larval stage of the fox tapeworm Echinococcus multilocularis , is one of the most severe helminthic diseases worldwide . Human AE affects the liver in more than 98% of the cases [1 , 2] , and metacestodes grow and proliferate continuously and infiltratively , forming hepatic lesions that consist of parasite tissue , which is intermingled with host connective tissue and immune cells . Development of metacestodes affects liver homeostasis and causes irreversible granulomatous liver fibrosis [3–9] . Similar to malignant tumours , metastasis formation into other organs can take place at a later stage of infection [10] . Radical surgical removal of hepatic lesions is the optimal treatment option , but is feasible in only about 30% of the patients [11] . In advanced stages of AE , surgery is often incomplete due to the diffuse infiltration of metacestode tissue into non-resectable structures or sites [12] . The currently available chemotherapy is based on benzimidazole derivatives only , i . e . albendazole ( ABZ ) and mebendazole ( MBZ ) that target E . multilocularis β-tubulins [13] . However , despite that benzimidazole-based therapy has clearly increased the life expectancy of affected patients [12 , 14] , its mode of action remains parasitostatic rather than parasitocidal [15] . In vitro studies have shown that ABZ has only restricted parasitocidial activity on E . multilocularis metacestodes [16 , 17] . Recently , it has been shown that stem cells of the E . multilocularis larval are expressing a particular β-tubulin isoform that is resistant to ABZ [18] , which may partially explain the limited effectiveness of ABZ in the complete killing of the parasite [19 , 20] . From the immunological perspective , modulatory effects of ABZ on the host immune system has not yet been investigated . Thus , it may be that ABZ contributes to an increase of the anti-AE immune response [21] . For AE-patients who do not respond to or do not tolerate the benzimidazoles , no alternative drugs are available so far [22] . These limitations of the presently widely applied anti-AE chemotherapy have called for new or alternative or complementary or supportive therapy options , such as immunotherapy ( e . g . complementary to chemotherapy ) . Because of the high clinical and economic impact of human AE [23] on each patient , the development of protective and effective vaccines as a preventive measure against infection may represent an attractive alternative strategy . Vaccine candidates for inducing preventive protective immunity have been described so far , including e . g . recombinant Em 14-3-3 antigen that protects mice by 97% against E . multilocularis egg infection ( primary AE ) but not against challenge with E . multilocularis metacestodes ( secondary AE ) [24 , 25] . EG95 is an E . granulosus antigen that exhibited high-level protection against egg infection in sheep [26 , 27] . Immunization of mice with the homologous E . multilocularis EM95 antigen resulted in protection rates ranging between 78 . 5 and 82 . 9% [27] which was lower than that obtained using the EG95 antigen in sheep [26 , 28] . Recently , seven members of a tetraspanin transmembrane protein family ( TSP1 to TSP7 ) have been shown to exhibit varying protective effects against primary AE . In infected BALB/c mice [29] , the highest and lowest rates of lesion reduction were 87 . 5% and 37 . 6% , achieved by vaccination with TSP1 and TSP7 , respectively [29 , 30] . The survival strategy of E . multilocularis is fundamentally based on its ability to induce anergy / immune tolerance in the host , by exerting potent and selective immunomodulatory activity [2] . The infection with E . multilocularis triggers an immune response that is characterized by an increasing imbalance between an initial rather cellular ( Th1 ) and a subsequently prevailing humoral ( Th2 ) immune response [2 , 3] . In the experimental mouse model , during the initiation of the infection , ( early stage AE ) both Th1 ( IFN-γ ) and Th2 ( IL-4 ) related cytokines are present; but an initial dominance by Th1 cytokines and chemokines has been evidenced [5 , 6] . Subsequently ( middle stage ) , an increase of mRNA levels of IL-4 , IL-5 , CCL8 , CCL12 , and CCL17 ( Th2 cell-associated cytokines and related chemokines ) is developing [6] . An increasing anergic immune status is evolving during late stage of infection , with an increase of Th2-oriented cytokine patterns , and an increase of immune-down regulating processes modulated by Tregs [6 , 7] . High transcription levels of IL-10 and TGF-β go in line with this anergic late-phase AE immune-status [3] . Indeed , the current evidence suggests that protection against E . multilocularis infection is basically associated with the maintenance of a Th1-oriented cellular immune response , while an increasingly dominating Th2 profile has been associated with a rather progressive form of AE in humans [31 , 32] , although full metacestode proliferation capacity only occurs when cell-mediated immunity completely fails such as experienced with AIDS [33] . In a similar context , it has been recently shown that individuals under immunosuppressive therapy for cancers , autoimmune diseases or subsequent to liver-transplantation are at significant risk for E . multilocularis infection associated with a delayed diagnosis . In those patients , progression of AE occurs faster than in non- immunocompromised patients [34] . It has been suggested that E . multilocularis actively governs the immunological orientation of the host through up-regulation of immunosuppressive cytokines , mainly IL-10 [35] and TGF-β [7 , 36] . This may occur via metabolites , and immunomodulatory proteins such as EmTIP secreted by E . multilocularis during the very early stage of metacestode development [37] may accordingly be key protagonists . The laminated layer ( LL ) represents the most outer component of the E . multilocularis larvae , and LL-associated carbohydrate antigens such as Em2 ( G11 ) and Em492 [31 , 38 , 39] were shown to exhibit suppressive effects on concanavalin A—mediated proliferation of spleen cells from E . multilocularis-infected mice . Thus components of the LL could be involved in modulating the Th1/Th2 balance by driving it from a Th1-domination to a rather Th2-oriented control characterized by anergy promoted via Tregs and other immunomodulating parameters . The working hypothesis for the present study was based on the work published by Harraga et al . [40] that a support of the host to maintain , or re-orientate , its immune response at a Th1-level may contribute to restrict or inhibit metacestode growth . Immunotherapy can either be active ( or antigen-specific ) or passive ( non-specific ) . In non-specific immunotherapy , immune molecules are administrated to infected individuals to modulate an existing , but non-protective , immunity towards an adequate and effective response . Cytokines that promote differentiation of Th1 cells such as IL-12 [41] and IFN-α-2a [40 , 42] have been assessed for passive immunotherapy of AE in experimentally infected mice . Passive cytokine therapy based on rIFN-γ was also evaluated in human and murine AE [43–45] . Although Th1 cytokine-mediated therapies resulted in partial inhibition of the parasite growth in mice and in clinical stabilization of AE in human patients , such treatments do not generate immunologic memory and may lead to non-specific inflammation , which in turn can cause severe adverse effects . An alternative approach , referred to as active immunotherapy , is to administer potential target antigens , and thus to elicit a specific immune response and immune memory . A high potential for such therapeutic vaccines directed against various pathogens has been reported , including rabies virus [46] , Mycoplasma pulmonis [47] , Mycobacterium tuberculosis [48] , Leishmania major [49] , and Schistosoma mansoni [50] . These studies demonstrated that administration of antigen doses ( alone or in combination with additional chemotherapy in the case of rabies [46] and Schistosoma [50] could clear or greatly reduce an ongoing infection by the respective pathogen . It is now believed that therapeutic vaccines against chronic infectious diseases may overcome the potential impairment of immune responses due to an established infection [51] . However so far , no studies focused on creating and assessing therapeutic vaccines for the treatment of AE . The P29 protein has been identified in the larval stage of both E . granulosus [52 , 53] , and E . multilocularis [54] . Prior studies suggested a possible role of E . granulosus P29 ( EgP29 ) as developmentally regulated component of the E . granulosus metacestode [55] . Vaccination of mice with bacterially produced recombinant EgP29 ( rEgP29 ) was shown to lead to significant protective immunity , resulting in 96 . 6% protection against challenge infection with E . granulosus protoscoleces [56] . In this study , we examined the antigenicity and immunogenicity of recombinant E . multilocularis P29 ( rEmP29 ) , and applied rEmP29 either as a vaccine ( prevention of infection ) or as an active immunotherapy ( treatment of infection ) .
Female BALB/c mice , 8–10 weeks old were obtained from Charles River GmbH , Germany . All mice were housed und handled under standard aseptic animal laboratory conditions according to the Swiss Animal Welfare regulations ( license No . Be108/08 ) . For all experiments , animals were matched for age and weight . Experimental groups of 6 animals each were used . The E . multilocularis isolate H95 [57] was used in this study . Metacestodes were maintained in BALB/C mice by serial transplantation passages as previously described [58] . For experimental infections , vesicle suspensions were prepared as described earlier [59] , and mice were intraperitoneally ( i . p . ) injected with 100 μl vesicle suspension ( corresponding to approximately 50 metacestodes ) . The recombinant E . multilocularis P29 ( rEmP29 ) protein was expressed and purified as previously described . [60] . Recombinant Neospora caninum microneme protein 1 ( rNcMIC1 ) , used here as an irrelevant control antigen expressed and purified under identical conditions , was produced as previously described [61] . Purified rEmP29 and rNcMIC1 were dialysed against PBS for two days at 4°C and kept at -80°C for subsequent use in serological assays . Prior to the use in cell cultures or mouse experiments , recombinant antigens were purified by affinity chromatography employing a Detoxi-gel affinity pack column ( Pierce , Rockford , IL USA ) to remove endotoxins . Protein concentration was determined by the Bio-Rad Bradford protein assay ( Bio-Rad Laboratories GmbH , Germany ) . No measurable endotoxins were detected using Pierce LAL Chromogenic Endotoxin Quantitation test ( Thermo Scientific , Rockford , IL , USA ) conducted according to the manufacturer's protocol . Three experiments were performed . In Experiment 1 , mice were vaccinated with rEmP29 and the immune response towards vaccination was investigated . In experiment 2 , mice were vaccinated and then experimentally infected by intraperitoneal ( i . p . ) injection of E . multilocularis vesicles to evaluate the immunoprotective potential of rEmP29 ( experiment 2 ) . In Experiment 3 , mice were infected by ( i . p . ) injection of E . multilocularis metacestode vesicles , and animals were treated at one month post-infection by ( i . p . ) injection of rEmP29 antigen in order to assess the immunotherapeutic potential of rEmP29 . For the present mouse experiments , saponin was chosen as adjuvant since it appears as the most widely used adjuvant in previous research studies on vaccination against E . multilocularis [24] or E . granulosus [27 , 62] . In addition , Quillaja saponins are potent adjuvants that enhance both cellular and humoral immune responses [63 , 64] . For experiment 1 ( assessment of immunogenicity of rEmP29 ) , 18 mice were divided into 3 groups . On days 1 , 14 , and 28 , post-vaccination , all mice from group 1 ( rEmP29-Sap ) were i . p . injected with 20 μg rEmP29 plus 50 μg saponin adjuvant ( Sigma-Aldrich , Buchs , Switzerland ) , formulated in sterile saline solution ( 0 . 09% NaCl ) such as to yield a final volume of 100μl . Mice from group 2 were administered 50 μg saponin in saline solution ( adjuvants control = Sap ) , and mice from group 3 received only sterile saline solution ( infection control = NaCl ) . On day 42 , all mice were euthanized , sera and spleen were collected and analysed for antibody and cytokine responses . For experiment 2 ( assessment of immunoprotective potential of rEmP29 ) , 24 animals were divided into 4 groups . On days 0 , 14 , and 28 ( Table 1 ) , all mice from group 1 ( rEmP29-Sap/Inf ) were i . p . injected with 20 μg rEmP29 plus 50 μg saponin adjuvant , formulated in sterile saline solution ( 0 . 09% NaCl ) in a final volume of 100μl . Mice from group 2 were administered 50 μg saponin in saline solution ( Sap/Inf = adjuvants control ) , and mice from group 3 received only sterile saline solution ( NaCl/Inf = infection control ) . In addition , another control group ( group 4 ) was introduced , mice of which received 100 μl saline solution containing 20 μg rNcMIC1 plus 50 μg saponin ( rNcMIC1-Sap/Inf control ) . Two weeks after the last immunization , all mice were challenged by i . p . injection of 100 μL E . multilocularis vesicle suspension . At 3 months p . i . , all mice were euthanized , followed by careful removal of the metacestode tissue from the peritoneal cavity . The parasite mass was immediately determined on a Mettler AE160 scale ( Mettler Toledo AG , Greifensee ) . Spleen and blood samples were also taken for further analyses . Spleens were taken and placed in Hanks' balanced salt solution on ice . Blood samples were allowed to clot at room temperature for 30 min and then were centrifuged for 10 minutes at 3000 rpm . For experiment 3 ( assessment of immunotherapy with rEmP29 ) , 24 mice were infected i . p . with 100 μl E . multilocularis vesicle suspension . At 1 month post infection ( p . i . ) , mice from group 1 ( Inf/rEmP29-Sap ) were i . p . injected with 20 μg rEmP29 plus 50 μg saponin adjuvant , emulsified in sterile saline solution ( 0 . 09% NaCl ) for a final volume of 100μl . Mice from group 2 were administered 50 μg saponin in saline solution ( Inf/Sap = adjuvants control ) , mice from group 3 received only sterile saline solution ( Inf/NaCl = infection control ) , and mice from group 4 were treated with 100 μl saline solution containing 20 μg rNcMIC1 plus 50 μg saponin ( Inf/rNcMIC1-Sap-control = irrelevant antigen control ) . These treatments were repeated 14 and 28 days later ( days 87and 101 , p . i . , Table 1 ) . At 3 months p . i . , mice were euthanized and parasite mass was assessed as in experiment 2 . Spleen and blood samples were also collected for further analyses , as described below . Infections , euthanasia and sample collection time points were synchronized between mouse experiment 2 and 3 ( Table 1 ) . An additional control group ( non-infected ) consisted of 6 female mice , animals were maintained in the same conditions till the end as outlined for experiments 2 and 3 . This control group was also sacrificed and blood and spleens were taken for the same further analyses . For experiment 1 ( see above ) , spleen cells were collected by mincing spleen tissue and passing it through sterile 40μm-mesh stainless steel sieves . Erythrocytes were depleted using red blood cell lysis solution ( Miltenyi Biotec , Germany ) for 10 minutes , and the residual spleen cells containing amongst others T-cells , dendritic cells , B-cells and macrophages , were then suspended in RPMI 1640 complete culture medium , ( Gibco BRL , Basel , Switzerland ) including 10% heat-inactivated fetal calf serum ( FCS; Biochrom , Berlin , Germany ) , 0 . 05 mM 2-mercaptoethanol ( Sigma-Aldrich , Buchs , Switzerland ) , 2 mM L-glutamine ( GibcoBRL ) , and 100 U of penicillin plus 50 mg of streptomycin per ml ( GibcoBRL ) . Cell suspensions were distributed in polystyrene 96-well flat bottom sterile plastic plates ( Greiner Bio-One; HuberLab , Aesch , Switzerland ) at 2×105 cells/100μl/well , and spleen cells were stimulated with rEmP29 at 0 . 1μg/2μL/well ( 1 μg/mL ) . Wells containing cell suspensions that were left unstimulated or stimulated with Conavalin ( Con A triggers T- lymphocyte proliferation [65] ( Sigma-Aldrich , Buchs , Switzerland ) with a concentration of 0 . 2 μg/μL/well 2 μg/mL were included as negative control and as an internal positive stimulation control , respectively . Experiments were performed in triplicates and cultures were maintained in a 37°C humidified chamber containing 5% CO2 for 72 hours . Spleen cells proliferation was assessed using the BrdU Cell Proliferation Assay kit ( Calbiochem , Weidenmattweg , Switzerland ) according to manufacturer's protocol . In experiment 2 and 3 ( see above ) , spleen cells were identically isolated , and single cell suspensions were either left unstimulated ( maintenance in culture medium ) or they were stimulated with Con A ( 2 μg/mL ) and assessed using the BrdU Cell Proliferation Assay kit . All experiments were performed in triplicates and the proliferative response was expressed as a stimulation index ( SI ) calculated using the equation: SI = median absorbance of cells stimulated with Con A or rEmP29/median absorbance of unstimulated cells . Serum levels of rEmP29 antigen-specific IgG , IgG1 , IgG2a antibodies induced by vaccination in experiment 1 were measured by direct rEmP29-ELISA . IgG1 and IgG2a antibody concentrations were used as markers of Th2 and Th1 immune responses , respectively . Serum IgG concentrations against rEmP29 and Em2 ( G11 ) in mice from experiments 2 and 3 were determined by ELISA as previously described [66] . Briefly , antigens were coated onto 96-well microplates at a concentration of 1μg/mL for rEmP29 and 0 . 57μg/mL of carbohydrate for Em2 ( G11 ) -antigen [66] . Plates were incubated at 4°C for overnight . After three washes with washing buffer ( 1 . 5 mM KH2PO4 , 10 mM Na2HPO4 , 150 mM NaCl , 2 . 5 mM KCl , pH 7 . 4 ) , the plates were blocked with blocking solution ( washing buffer supplemented with 1% horse serum ) for 30 min at 37°C and subsequently incubated with the mouse sera diluted at 1:100 in blocking buffer for 30 min at 37°C . AP-conjugated goat anti-mouse IgG , IgG1 or IgG2a ( Sigma-Aldrich , Buchs , Switzerland ) were used as the secondary antibody to detect bound antibodies . Finally , immune complexes were revealed by incubating with orthophenylene diamine ( Sigma-Aldrich , Buchs , Switzerland ) and 0 . 15% H2O2 for 30 min . The reaction was stopped by addition of 50 μL of 1 M NaOH to each well , and the absorbance at 405 nm was measured with a Microreader ( model 550 , Bio-Rad ) . All samples were run in triplicates . For experiments 1 , 2 and 3 , total RNA was prepared from approximately 30 mg of mouse spleen tissue , which was disrupted and homogenized using a FastPrep-24 homogenizer and lysic matrix tubes ( both MP Biomedicals , Illkirch Cedex , France ) . RNA was isolated using the RNeasy mini kit with on-column DNA digestion ( Qiagen , Basel . Switzerland ) , according to the manufacturer’s instructions . RNA purity and quantity were calculated using the Nanodrop 1000 spectrophotometer . Equal amounts of six RNA samples derived from mice of the same group were pooled . The cDNA synthesis was performed with 1μg total RNA using M-MLV Reverse Transcriptase RNase H Minus ( Promega , Zurich , Switzerland ) according to the manufacturer’s instructions . Comparative quantification of IL-4 , IL-2 , IL-10 , and IFN-γ mRNA in murine spleens was performed by real time PCR using β-actin as housekeeping gene for the qRT-PCR . Primers were purchased from Sigma-Genosys and are all described in Table 2 . Real-time PCRs were performed using FastStart Essential DNA Green Master Kit ( Roche , Rotkreuz , Switzerland ) , and 2 μL of diluted cDNA in the presence of 0 . 2 μM of each specific primer . Quantitative PCR was performed using the Rotor-Gene 6000 ( Corbett Life Science ) . Samples ( including cytokines and β-actin ) were run in triplicates . The programme included a hold at 95°C for 15 min , 50 cycles each of denaturation at 95°C for 15 s , annealing at 55°C for 30 s , extension at 72°C for 30 s . Melting curves were generated by heating the samples from 50°C to 90°C . Normality of distribution was checked with Shapiro-Wilk test in the software R version 3 . 0 . 1 ( R core team , A Language and Environment for Statistical Computing , R Foundation for Statistical Computing , Vienna , 2013 ) . One-way analysis of variance ( ANOVA ) was applied for comparisons of the parasite load among mouse groups followed by Bonferroni-adjusted P values calculated by Pairwise T-Test in R . A P-value of <0 . 05 was considered significant . Data was visualized by boxplot in Microsoft Office Excel 2010 . For the spleen cell proliferation assay , ELISA and cytokines expression , data were expressed as median ± standard error ( S . E . ) and examined for statistical significance with the Student's t-test . P-values of less than 0 . 05 were considered to be statistically significant .
To determine the antibody responses and the subclass distribution of serum IgG antibody in mice immunized with rEmP29 , serum levels of IgG , IgG2a and IgG1 were analysed . As shown in Fig 1 , intraperitoneal immunization of mice with rEmP29 in combination with saponin resulted in a respectively specific anti-rEmP29 IgG antibody production . Furthermore , both tested IgG subclasses ( IgG1 and IgG2α ) were induced in rEmP29-Sap-immunized mice , with the IgG2a median OD value being significantly higher than that of IgG1 ( P<0 . 001 ) . To examine the cellular immune response elicited by the rEmP29 vaccination , spleen cells proliferation was studied in vitro ( Fig 2 ) . In response to the rEmP29 specific antigen stimulation , spleen cells from rEmP29-vaccinated mice exhibited a significantly higher proliferation rate when compared to spleen cells from mice treated with saponin ( P = 0 . 002 ) or saline solution ( P = 0 . 006 ) . Spleen cells from all animals responded to the polyclonal stimulant Con A as a positive proliferation control ( Fig 2 ) . Thus , priming with the rEmP29 vaccine led to specific proliferation of spleen cells , suggesting subsequent expansion of antigen-specific T cells . To characterize the cytokine expression profile in response to recombinant rEmP29 vaccination , we examined the mRNA levels of IFN-γ , IL-2 , IL-4 and IL-10 in spleen cells by real-time RT-PCR . As shown in Fig 3A and 3B , immunization of mice with rEmP29 or with adjuvants alone ( Sap ) resulted in a significantly higher IL-4 ( P < 0 . 001 ) and IFN-γ ( P < 0 . 001 ) mRNA expression level when compared to spleens from saline-treated mice ( NaCl ) ( Fig 3A and 3B ) . IL-2 mRNA expression in spleen cells from mice immunized with rEmP29-Sap or saponin alone was significantly higher ( P < 0 . 001 ) than that one observed in the control non-immunized animals ( NaCl group ) ( Fig 3C ) . Similarly , IL-10 mRNA-level was significantly higher in rEmP29-Sap- and Sap-vaccinated mice when compared to the mouse group receiving only saline solution ( NaCl group ) . However , the magnitude of IL-10 and IL-2 mRNA levels rising in rEmP29-Sap-immunized mice was lower than in mice inoculated with adjuvants alone ( Fig 3D ) . Recombinant EmP29 antigen was evaluated as a vaccine against E . multilocularis in the murine model of secondary AE ( Fig 4A , Immunization and then infection ) . The efficacy was assessed by monitoring the parasite burden in the peritoneal cavity . In non-vaccinated saline treated mice , experimental infection resulted in a median parasite weight of 10 . 66 ± 1 . 68 g , while saponin treatment ( 6 . 57 ± 2 . 18 g ) and vaccination with rNcMIC1 ( 10 . 70 ± 4 . 03 g ) had no significant impact . In contrast , vaccination of mice by rEmP29 resulted in a significantly reduced median parasite bio-mass ( 2 . 68 ± 2 . 08 g ) , which was 75% , 59% and 75% lower when compared to NcMIC1-vaccinated , saponin-treated , or saline treated groups , respectively ( Fig 4A ) . To determine whether the course of an already established secondary E . multilocularis infection in mice could be altered by immune stimulation with rEmP29 antigen , three doses of rEmP29 formulated in saponin adjuvants were administered during the early phase of ( after 1 month ) ( Fig 4B , Infection and then immunization ) . Mice that were infected and received only the saline solution developed a high median parasite load ( 5 . 37 ± 2 . 72 g ) . Similarly , mice that were treated with the adjuvant alone also developed high infection intensities ( 4 . 9± 2 . 31 g ) . Mice treated with rNcMIC1 antigen emulsified in saponin showed the highest median parasite burden ( 6 . 95 ± 3 . 81 g ) . In contrast , mice that were treated with three injections of rEmP29-saponin formulation on days 73 , 87 and 101 ( Table 1 ) post-infection exhibited a significantly lower parasite load ( 2 . 51± 2 . 07 g ) . In two mice the parasite loads ( 0 . 13 and 1 . 08 g ) were largely inferior to the median . Thus immunotherapy with rEmP29 resulted in a reduction of the median metacestode weight of 53% , 49% and 64% , as compared to the NaCl- , saponin- and rNcMIC1-saponin-treated control mice , respectively . These differences were significant only upon comparison of rEmP29 and rNcMIC1 treated groups . In order to characterize humoral immune responses , sera of the different experimental groups in experiments 2 and 3were analysed by rEmP29 ELISA . All samples from E . multilocularis infected mice contained antibodies directed against rEmP29 protein , and the significantly highest anti-rEmP29 IgG-levels were detected in those animals that had been vaccinated with rEmP29-antigen and in mice that were treated with rEmP29 after being infected ( Fig 5 ) . As shown in Fig 5 , serum levels of the specific IgG response against the Em2 ( G11 ) antigen was significantly lower in rEmP29 vaccinated mice , when compared to the corresponding groups immunized with saline solution only , saponin alone or with rNcMIC1-saponin . Similarly , anti-Em2 ( G11 ) -specific IgG antibody levels in infected mice that were treated with rEmP29 were inferior to those in infected and treated animals with rNcMIC1 or left untreated . However , animals treated with saponin alone showed similar levels of anti-Em2 ( G11 ) -specific IgG as compared to rEmP29-sap treated mice . The proliferative response of spleen cells to ConA-mitogen is an important overall indicator of immune fitness . The proliferative responses of spleen cells to ConA stimulation from all groups ( experiments 2 and 3 and including non-infected mouse group ) are presented in Fig 6 , by using the value of 100% as a reference proliferation of spleen cells ( derived from non-infected mice ) . The proliferation indices of spleen cells from the four infected mouse groups of experiment no . 2; NaCl/Inf , Sap/Inf , rNcMIC1-Sap/Inf and rEmP29-Sap/Inf were 41% , 32% , 30% and 47% , respectively and were thus considerably ( P < 0 . 001 ) lower than those observed in non-infected control groups ( 100% ) ( Fig 6 ) . In mouse experiment no . 3 , spleen cell proliferation rates triggered by ConA were significantly ( P < 0 . 001 ) reduced ( 44% , 50% and 41% in Inf/NaCl , Inf/rNcMIC1-Sap and Inf/rEmP29-Sap mouse groups , respectively ) . However , the proliferation of spleen cells in infected animals that were treated with saponin alone was similar ( 97% ) to the non-infected control group ( 100% ) ( Fig 6 ) . The cytokine expression profiles in response to recombinant rEmP29 vaccination were investigated by comparing IFN-γ , IL-2 , IL-4 and IL-10 mRNA levels in the spleen of the different treatment groups with cytokine mRNA levels in non-infected control mice by real time RT-PCR ( Fig 7 ) . Significantly higher levels of IL-4 mRNA expression were detected in all 8 groups from experiments nos . 2 and 3 , when compared to non-infected mice ( Fig 7A ) . In terms of IFN-γ expression , in rEmP29- vaccinated or-treated mice , IFN-γ mRNA levels were significantly elevated when compared to the corresponding groups treated with saline only ( NaCl/Inf and Inf/NaCl groups ) ( Fig 7B ) . In addition , saline-treated mice in infected animals exhibited similar levels of splenic IFN-γ mRNA expression as non-infected mice ( Fig 7B ) . Furthermore , the IL-4 to IFN-γ mRNA expression ratio was calculated for each animal group and displayed as a graph ( Fig 7C ) . Transcription of IL-4 mRNA in spleen cells from secondary AE-infected mice ( NaCl/Inf and Inf/NaCl ) was 16 times higher than that of IFN-γ ( Fig 7C ) . In infected BALB/c mice that were vaccinated ( rEmP29-Sap/Inf ) or treated ( Inf/rEmP29-Sap ) with rEmP29 , the ratio of IL-4/IFN-γ mRNA was lower than those of different control groups from experiments no . 2 and 3 ( NaCl/Inf , Sap/Inf , rNcMIC1-Sap/Inf , Inf/NaCl , Inf/rNcMIC1-Sap ) . Nevertheless , treatment of infected mice with saponin ( Inf/Sap ) alone resulted in a similar IL-4/IFN-γ mRNA-ratio as found in animals that received rEmP29-Sap at 1 month post-infection . In experiment no . 2 , as shown in Fig 7D , infected mice ( NaCl/Inf and Inf/NaCl ) had significantly higher IL-10 mRNA expression levels than controls ( non-infected ) ( P < 0 . 001 ) . In addition ( Fig 7D ) , IL-10 mRNA transcription levels were significantly lower in rEmP29 vaccinated and infected mice ( rEmP29-Sap/Inf ) , when compared to the following groups: NaCl/Inf , Sap/Inf and rNcMIC1-Sap/Inf . Similarly , immunotherapy treatment ( experiment no . 3 ) of infected mice with rEmP29 in conjunction with the saponin adjuvant resulted in significantly reduced IL-10 transcription levels when compared to different control groups: Inf/NaCl , Inf/Sap and Inf/rNcMIC1-Sap ( Fig 7D ) . The highest transcription level of IL-10 mRNA was observed in spleen cells from rNcMIC1-sap vaccinated mice ( rNcMIC1-Sap/Inf group ) . All infected groups ( experiment nos . 2 and 3 ) presented significantly higher expression levels of IL-2 mRNA when compared to controls ( non-infected ) ( Fig 7E ) . Infected mice that were immunized ( experiment no . 2 ) or treated ( experiment no . 3 ) with rEmP29 displayed a significantly lower amount of mRNA coding for IL-2 , correspondingly compared to the following groups: only infected mice ( NaCl/Inf and Inf/NaCl ) , infected mice that were either immunized or treated with adjuvant ( Sap/Inf and Inf/Sap ) or immunized with rNcMIC1-saponin ( rNcMIC1-Sap/Inf group ) as shown in Fig 7E .
This is the first study describing an antigen-based immunotherapy for experimentally induced secondary murine AE , based on the bacterially produced recombinant rEmP29 antigen . We also present data that demonstrates an explorative experiment tackling the efficacy of rEmP29 antigen as a vaccine candidate against experimental ( secondary ) E . multilocularis infection in mice . In addition , spleen cells recall-responses upon antigen stimulation and real time PCR-based cytokine mRNA expression analyses suggested that both , vaccination and immunotherapy , overrode to some extent the Echinococcus-mediated immunomodulation at the host-parasite interface . EmP29 was selected as a target antigen for two reasons: ( i ) EmP29 exhibits high expression levels in both E . multilocularis [54] and E . granulosus [52 , 53] metacestodes , indeed it was previously suggested that P29 could have an important role in developmental regulation of the E . granulosus metacestode [55]; ( ii ) recombinantly expressed E . granulosus P29 ( rEgP29 ) vaccination provides effective protection of mice against challenge infection with E . granulosus protoscoleces [56] . Three injections of rEmP29-antigen formulated in saponin adjuvants prior to experimental infection lead to a significantly reduced ( 75% ) parasite mass formation as compared to non-vaccinated control mice . In addition , intraperitoneal injection of rEmP29 antigen during the chronic phase of AE resulted in a partial control of parasite growth , and yielded a reduced the median parasite mass by 53% as compared to non-treated mice . In contrast to the result obtained in our study , with rEmP29 applied as preventive vaccine against secondary AE , immunization of mice with the recombinant Em14-3-3 ( E14t ) in combination with a saponin adjuvans , did not show any reduction in the parasite load when compared to non-vaccinated animals [24] . This discrepancy between rEmP29 and E14t cannot yet be explained , but hypothetically the effect may be related to distinct biological function or by different antigenic and immunogenic properties of the reagents . So far , the core biological function of EmP29 in the Echinococcus biology is still unresolved . An NCBI BLAST search showed that the amino acid sequence of EmP29 ( GenBank , accession no . AAD53328 . 1 ) exhibited 100% homology to E . granulosus endophilin B ( GenBank , accession no . CDJ21798 . 1 ) [18] . However , EmP29 shares only a low degree of identity ( 22% ) with human endophilin B1 ( S1 Fig ) . In eukaryotes , endophilin B1 is essential for synaptic transmission [67] , and inhibition of its expression has been shown to produce profound defects in synaptic vesicle endocytosis [68] . However , the mechanism by which endophilin B promotes endocytosis has remained controversial . Therefore , the future functional characterization of EmP29 will be of great interest to understand the cellular pathways and functional activities associated with EmP29 . To induce experimentally murine secondary AE , E . multilocularis metacestodes are injected intraperitoneally into susceptible mice . Thus at the starting point of an experimental immunotherapy ( one month p . i . ) , it is conceivable that metacestodes are already well established within the host , including a solid protection by the parasite-derived laminated layer [69 , 70] . In our experiments , the third and final injection of rEmP29 antigen was administered at 1 month before the end of the experiment . Thus , we do not know yet how the parasite growth-potential develops at later time points . Therefore , further animal studies are planned , during which different experimental starting- and end-points will be assessed . We hypothesize that the effectiveness of immunotherapy is largely dependent on the time point of the initiation of treatment in relation to the infection stage . For example in pythiosis in horses , caused by Pythium insidiosum , a successful immunotherapy depends on the chronicity of the lesions prior to immunotreatment . Therefore , all horses with lesions less than 15 days old were cured by an early P . insidiosum-antigen , while those with chronic pythiosis ( more than 2 months duration ) eventually died [71 , 72] . Other important parameters also require further assessment , such as antigen dosages , adjuvants , frequency of applications and inoculation routes , all of which would lead to an optimization of the immunotherapeutic potential of rEmP29 antigen as exploratively documented in the present study . In a similar line of plans , a future evaluation of the immunotherapeutic and immunoprotective potentials of rEmP29 will have to focus on the primary egg infection model that mimics the natural infection in humans more properly . In human AE , specific IgG antibody levels against Em2 ( G11 ) and recEm18 correlate to some extent with the clinical status of the patient [66 , 73] . In our study , we found that IgG concentrations against Em2 ( G11 ) were significantly lower in rEmP29 vaccinated mice in the pre- or post- infection status , when compared to the corresponding control mouse groups immunized or treated with saline solution , saponin alone or with mice that were immunized with rNcMIC1-saponin before challenge . In our study , administration of adjuvant alone prior to the infection , resulted in a non-specific but still significant ( P = 0 . 002 ) reduction of the median parasite weight of 38% when compared to mouse groups that received only saline solution . It was reported in a previous study that administration of saponin alone also gave a limited non-specific protection against secondary AE in mice . Similarly , other adjuvants such as CpG oligodeoxynucleotides [30] ( CpG ODN ) , Freund’s complete/Incomplete adjuvants ( CFA/IFA ) [30] , Gerbu [74] or cholera toxin subunit B ( CTB ) [75] have also been shown to exhibit non-specific protection against larval or adult stages of E . multilocularis . In our study , non-specific and significant protection against secondary AE with saponin alone was observed when mice were treated before ( pre-infection treatment; exp . 2 ) but not after the infection ( post-infection treatment; exp . 3 ) . In the same line , it was shown that prophylactic treatment of cotton rat ( Sigmodon hispidus ) with BCG ( Bacillus Calmette-Guerin ) entirely and non-specifically inhibited the establishment of E . multilocularis metacestodes in the intraperitoneal cavity [76 , 77] , however , application of BCG at two weeks post-infection did not limit the development and the proliferation of the parasite[76] . Until now there is no accurate scientific explanation for this phenomenon , however , in addition to the fact that adjuvants generally increase host immunity against infectious diseases , one reason for such a non-specific protection phenomenon may be that saponin or other adjuvants display a direct effect on E . multilocularis metacestodes or adult worms . In the present study , we found that the IL-4/IFN-γ mRNA ratios in the spleen of vaccinated mice , or animals treated with rEmP29 saponin formulations , were lower than in spleens of mice treated with saline ( infection controls ) . In addition , corresponding IL-10 and IL-2 expression levels were lower in vaccinated and treated mice compared to saline-treated mice . Thus , higher levels of IFN-γ and reduced expression of IL-10 and IL-2 mRNA might be associated with reduced growth of the parasite in vaccinated and treated mice , and a Th1-oriented response could contribute to the restricted parasite growth of E . multilocularis within its intermediate host . This is supported by elderly published data where the essential role of cellular immunity in controlling E . multilocularis infection in humans as well as in mice was confirmed [31 , 32] . In AE , CD4 ( + ) CD25 ( + ) regulatory T cells , have been studied intensively since they are the main IL-10 secreting cells [78 , 79] and their deficiency abrogates parasite-tolerance . Accumulating evidence from correspondingly selected knockout mice suggested that IL-2 is crucial for the homeostasis and function of CD4 ( + ) CD25 ( + ) regulatory T cells in vivo [79–85] . This synergic functional relationship between IL-10 and IL-2 may explain the simultaneous down regulation of mRNA levels of both cytokines observed in our study . The recombinantly expressed rNcMIC1 was used as negative control , since no homologue was found in E . multilocularis [18] . Recombinant NcMIC1was produced and purified under the same conditions as rEmP29 . The antigenic but not the immunosuppressive properties of NcMIC1 were reported [61] , but since our results revealed that rNcMIC1 harbors a significant immunomodulatory activity by enhancing IL-10 production; we do not recommend its usage as irrelevant control for future research on Echinococcus immunology studies . To compare the activity level of the immune system between infected mice ( independently of the vaccination or immunotherapy status ) and non-infected animals , we measured the proliferative activity of spleen cells in response to the mitogen ConA . In line with findings on E . multilocularis [31 , 86] and E . granulosus [87] reported earlier , spleen cells of all infected groups exhibited a significantly decreased responsiveness compared to cells from non-infected mice . Spleen cells from E . multilocularis-infected mice treated with saponin at 1 month p . i . exhibited a full responsiveness to ConA-stimulation ( 99% ) . Conversely , spleen cells from mice receiving i . p . injection of saponin prior to infection showed a significantly reduced proliferation activity of 32% when compared to non-infected mice . We conclude that the administration of saponin prior to infection had an immuno-stimulating activity by itself , but that this activity was subsequently abolished by the strong immunosuppressive response mediated by the parasite at 3 months p . i . . Upon saponin administration starting at 1 month p . i . , the immunosuppression exerted by E . multilocularis on the host immune system was most likely still moderate , and could not overcome the adjuvant effect . It was previously shown that saponin significantly promoted the ConA , lipopolysaccharide [88] and phytohemagglutinin [89] induced spleen cells proliferation . In conclusion , we showed that both , vaccination and active immunotherapy employing rEmP29 antigen had a significant inhibitory effect on secondary infection with E . multilocularis metacestodes in mice , and yielded thus a reduced parasite infection intensity when compared mock-treated control animals . Further studies will aim to assess alternative antigens and also other classes of adjuvants that could improve the immunotherapeutical potential of rEmP29 . Furthermore , as vaccination provided partial protection against secondary infection , it will be important now to assess the protection mediated by rEmP29 against primary egg infection .
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Current medical management of AE that relies on surgery and continuous benzimidazole administration is of limited effectiveness . Therefore , alternative preventive and therapeutic tools need to be explored . Here , we demonstrate that vaccination with recombinant antigen EmP29 ( rEmP29 ) , prior or after secondary infection of BALB/c mice , resulted in a significant reduction of the median parasite weight when compared to different control groups . We then characterized the transcription level of splenic IL-4 and IFN-γ cytokines as hallmarks for AE-anti-protective humoral immune reaction ( Th2 ) and for AE-effective ( restrictive ) cellular response ( Th1 ) , respectively . Results revealed that vaccinated mice in pre- or post-infection situation exhibited the lowest IL-4/IFN-γ mRNA ratios . In addition , those groups showed also significantly low levels of IL-10-encoding mRNA coding ( immunosuppressive cytokine ) , as well as IL-2 . These findings suggest that reduction of parasite load in rEmP29-vaccinated mice ( in pre- or post-infection status ) might be triggered by a decline of the immunosuppressive environment and a change of the host immune reaction towards a Th1-re-oriented cell-mediated immune defense . A similar non-specific effect appears also to be yielded by the immunostimulating adjuvants . This study provides the first insight into the potential benefits of antigen-specific immunotherapy as new treatment option of AE .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Prevention and Immunotherapy of Secondary Murine Alveolar Echinococcosis Employing Recombinant EmP29 Antigen
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Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure , dynamics and function . Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures . Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance ( NMR ) Residual Dipolar Couplings ( RDCs ) . Using this ensemble , we probe two proposed relationships between properties of protein ensembles: ( i ) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures , and ( ii ) a relation between dynamics of an individual protein and the conformational variability explored by its natural family . We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs , encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family . Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution . More practically , the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics .
It has long been known that a protein's native state is best represented as an ensemble of conformations rather than as a single structure [1] . Conformational ensembles provide a detailed structural picture of protein dynamics . As motions are crucial for many aspects of protein function , such as molecular recognition [2]–[4] and catalysis [5]–[10] , an ensemble description of proteins is also useful for improving applications of molecular modeling such as protein-small molecule [11] and protein-protein docking methods [12] , [13] as well as protein design [14]–[19] . Two related concepts characterizing and interpreting properties of protein conformational ensembles have been proposed: The first suggests a correspondence between the conformational heterogeneity present in crystal structures and the native-state dynamics of proteins observed in simulations and using nuclear magnetic resonance ( NMR ) measurements . Several studies provide support for this idea . Zoete et al . concluded that the conformational changes present in a large number of crystal structures of HIV-1 protease reflect the inherent flexibility of the protein [20] . Vendruscolo and coworkers showed [21] that side chain relaxation order parameters , reflecting motions on the picosecond to nanosecond time scale [22]–[28] , could be described using ensembles of crystal structures of the same protein or proteins with high sequence identity . Similarly , modeling “Backrub” motions , a type of conformational change inspired by alternate side chain and backbone conformations observed in high-resolution crystal structures [29] , has led to improvements in modeling NMR side chain relaxation order parameters [30] , side chain conformations [31] , [32] and structural changes upon mutation [31] . Lange et al . [4] showed that ensembles derived from ensemble-average-restraint molecular dynamics ( MD ) simulations of ubiquitin using Residual Dipolar Coupling ( RDC ) data describing picosecond to millisecond motions [33]–[41] encompassed conformations similar to those of ubiquitin in different crystal structures alone and in complex with different partner proteins . These findings support the idea that conformational states pre-existing in solution are selected upon binding . Strong experimental evidence for this conformational selection model was also provided earlier by Wright and colleagues [42] validating previous theoretical suggestions [43] , [44] . The second concept proposes a link between the dynamics of a single protein and the conformational variability explored within its family of homologous proteins . This link was suggested based on the similar conformational variability observed in an MD simulation of myoglobin and in structures of different members of the globin family [45] . Similarly , Gaussian network models have suggested common dynamical features of proteins in the same family [46] , [47] . Recently , Lee and colleagues proposed that side chain dynamics measured by NMR relaxation are conserved across members of the PDZ domain family [48] . Several studies extended the notion of a relationship between the dynamics of a single protein and properties of its homologs to the sequence level , showing that modeled sequences consistent with a single protein structure had characteristics in common with a multiple sequence alignment of the protein's natural family [49] . Further investigating the relation between protein dynamics and family sequence variability , other work suggested that sequence diversity [32] and overlap between modeled and evolutionarily observed sequences could be increased by incorporating conformational flexibility of the protein backbone [14]–[16] , [50] , [51] . To combine the two concepts outlined above , here we ask whether conformational ensembles reflecting variability observed in protein crystal structures of a single sequence can be simultaneously related to experimentally determined native-state solution dynamics of an individual protein , and to the conformational and sequence variability of the protein's family . To address these questions , we investigate two related hypotheses using ubiquitin as a model system: First , we test whether ensembles generated using the Backrub motional mechanism ( “Backrub ensembles” ) , a model inspired by heterogeneity observed in experimental protein structures [29] , capture properties of ubiquitin solution state dynamics derived from amide backbone RDC measurements in 23 alignment media [35] . The motions modeled using the Backrub mechanism are related to those described by the 1D-Gaussian Axial Fluctuation ( GAF ) approach , which has been used to model residual dipolar coupling ( RDC ) measurements [52] . Furthermore , we compare the structural variation in modeled Backrub ensembles to that seen in a set of 46 crystal structures of ubiquitin [4] . Second , we test whether the conformational heterogeneity present in Backrub ensembles that are consistent with the solution dynamics of an individual ubiquitin sequence resembles the structural diversity observed in the UBQ subfamily [53] . Furthermore , we predict sequences compatible with ubiquitin Backrub ensembles using computational protein design as implemented in Rosetta [54] and test whether these sequences are similar to the sequences of the UBQ subfamily . Supporting our hypotheses , we find Backrub ensembles that are simultaneously consistent with native-state dynamics reflected in RDC measurements , the conformational variability observed in ubiquitin complex structures , and the conformational and sequence diversity of ubiquitin homologs . As an additional validation of our approach , we show that Backrub ensembles give similar agreement with the RDC data as ensembles generated from RDC-restrained MD simulations [4] , and support previous observations of ubiquitin core flexibility [21] and binding by conformational selection [4] . Notably , we discover that a common set of Backrub sampling parameters are simultaneously able to best fit the RDC data and allow sampling of sequences most similar to those of the ubiquitin family . Our method to model Backrub ensembles and sequences consistent with these ensembles may thus be useful for providing insights into the relationship between native state dynamics and sequence diversity and for characterizing evolutionary sequence changes . These results also support the idea that Backrub ensembles will be useful for engineering new protein functions through experimental selection from computationally designed libraries [55] , [56] that contain sequences accommodated by exploiting intrinsic native-state dynamics .
We set out to investigate the hypothesized relations between conformational changes reflecting observed heterogeneity in protein crystal structures , native-state protein dynamics and evolutionarily sampled conformational and sequence diversity in two steps ( Figure 1 ) . First , to test relation 1 , we generated ensemble descriptions of ubiquitin dynamics using the Rosetta scoring function and several parameterizations of the Backrub motional model ( described below ) without using experimental restraints . Subsequently we selected ensembles according to their agreement with RDC measurements ( Test 1 ) . This approach is significantly different from many of the methods applied earlier to find ensembles compatible with NMR restraints [4] , [57] , [58] , which incorporated experimental data directly in the refinement process . Similar to previous work [4] , we compare the resulting Backrub-generated conformational ensembles with an ensemble of 46 crystal structures of ubiquitin ( Test 2 ) . Second , we use the insight gained from the comparison of Backrub ensembles with characteristics of solution-state dynamics to evaluate relation 2 ( Figure 1 ) . We investigate whether Backrub ensembles that sample the conformational space available on the RDC timescale have similar conformational variability to that explored by ubiquitin homologs ( Test 3 ) . Moreover , we test whether sequences consistent with Backrub ensembles fitting RDC measurements of a single ubiquitin sequence , as predicted by computational protein design using Rosetta [54] , show overlap with the sequences of the natural UBQ subfamily [53] ( Test 4 ) . To test relation 1 , our approach first uses unrestrained conformational sampling with the Backrub motional model to generate a large set of initial conformations , starting from the ubiquitin crystal structure ( Protein Data Bank ( PDB ) code 1UBQ ) . We use a Monte Carlo protocol consisting of rotamer changes and Backrub moves . Backrub moves involve selection of a random peptide segment , followed by a rigid body rotation of all atoms in that segment about an axis defined by the endpoint C-alpha atoms [31] . The peptide segment length is chosen at random to be either 2 or 3 residues ( denoted in the following as “maximum segment length of 3”; Figure 2A ) or between 2–12 residues ( “maximum segment length of 12”; Figure 2B ) . 10 , 000 Backrub-Monte-Carlo simulations are run to generate 10 , 000 possible conformations in an initial set ( see Methods for details ) . The Backrub motional mechanism thus directly accounts for correlated motions of continuous peptide segments of up to length 3 or 12 . Applying these moves repeatedly in randomly chosen regions of the protein using Monte Carlo sampling allows for correlated motions of residues distant in sequence yet close in tertiary structure . Correlations between side-chain and backbone dynamics have also been observed in numerous NMR studies , such as for Ribonuclease H on the relaxation time scale [59] , [60] and on the RDC time scale for ubiquitin [61] and Protein G [38] . Subsequently we select ensembles from the resulting structures based on their agreement to the RDC measurements as measured by the Q-factor ( Figure 2C ) , defined as: An ensemble selection approach similar to the one described above has been successfully applied to model relaxation order parameters using snapshots from MD trajectories [62] . In the following sections , “RDC-optimized” ensembles are defined as those undergoing the Q-factor optimization process described in Figure 2C and “non-RDC-optimized” ensembles are generated by choosing random ensembles of 50 structures . To validate our approach , we compare the Backrub-generated conformational ensembles to reference methods such as snapshots from an MD simulation in explicit solvent [63] and a set of representations of the dynamics commonly used to interpret the motional information present in RDC measurements . One such representation uses the ‘model-free’ formalism , which provides five parameters describing the movement of each residue [35] , [64]–[66] . Another approach is ensemble-average-restrained ( EAR ) molecular dynamics , in which an ensemble of molecules ( the “EROS” ensemble ) is optimized with respect to a molecular mechanics force field potential in combination with ensemble-average restraints on the NMR measurements , including RDCs [4] . We reason that sampling methods that result in low Q-factors more closely approximate the conformational space relevant to motions on the RDC timescale than other models that describe the experimental data less well . We first tested whether Q-factors of Backrub ensembles selected according to the strategy described in Figure 2C decreased as the ensemble size was increased ( 2 , 3 , 5 , 10 , 20 , 50 and 100 structures per ensemble ) . This behavior would be expected if our description captures dynamical information contained in the measurements . Figure 3A shows the Q-factors of RDC-optimized ensembles of varying size generated with a Backrub maximum segment length of 12 and a simulation temperature of kT = 1 . 2 ( see Methods ) . There is a clear trend that the Q-factors of RDC-optimized ensembles decrease as the ensemble size increases . This trend indicates that adding more structures allows a better representation of the RDC measurements and further suggests that these ensembles are representative of conformations that are populated on the timescale of the experiments ( even though the Monte Carlo simulations are agnostic to timescale ) . This result is not simply explained by inclusion of more degrees of freedom and overfitting , as cross-validation analysis supports an optimal ensemble size of around 50 ( Table S1 ) . We use this ensemble size in the experiments below . The RDC-optimized Backrub ensemble described above has a Q-factor of 0 . 086 over regions of regular secondary structure ( see Methods ) and was found by comparing motional models using different Backrub sampling parameters . The first Backrub parameter we varied was the maximum segment length ( as described above and illustrated in Figure 2A and B , the longest peptide segment rotated about an axis defined by the segment endpoint C-alpha atoms ) . The Backrub conformational change observed in ultra-high resolution X-ray structures consisted of concerted 2- and 3-residue Backrub moves [29]; thus we first tested a maximum segment length of 3 . In a previous study [30] , we showed that ensembles of structures generated using this maximum segment length improved predictions of side-chain relaxation order parameters . To test the relevance of larger-scale changes , we also tested a maximum segment length of 12 , which included moves of all intermediate segment lengths from 2–12 . To measure the effect of varying the amplitude of motion , we tested a range of temperatures for the Metropolis Monte Carlo simulations from kT = 0 . 3 to 4 . 8 . Each simulation was run for 10 , 000 steps . The resulting mean pair-wise root mean squared deviations ( RMSDs ) to the ubiquitin X-ray structure 1UBQ of the Backrub ensembles spanned the range of 0 . 2 Å to 0 . 5 Å for the maximum segment length of 3 simulations , and spanned the range of 0 . 3 Å to 3 . 2 Å for the maximum segment length of 12 simulations ( see Methods for details ) . Figures 3B shows the RDC-optimized ensembles of size 50 with lowest Q-factor for different initial Backrub starting sets of 10 , 000 structures with maximum segment length of 12 and different simulation temperatures . Simulation temperatures of kT = 0 . 3 , 0 . 6 , 1 . 2 , 2 . 4 and 4 . 8 gave mean pair-wise RMSD values to the ubiquitin X-ray structure 1UBQ of 0 . 3 Å , 0 . 5 Å , 0 . 9 Å , 2 . 1 Å and 3 . 2 Å , respectively . For the maximum segment length of 12 , the lowest Q factor is 0 . 086 at kT = 1 . 2 and for the maximum segment length of 3 the lowest Q factor is 0 . 089 at kT = 2 . 4 ( see Table S2 for results for all parameters ) . To compare these two ensembles , we performed cross-validation with four RDC datasets of N-C′ couplings and four datasets of H-C′ couplings ( see Methods for details ) . The resulting Rfree values for these ensembles were 18 . 0% and 21 . 3% , respectively ( Table S1 ) . Thus the ensemble generated using a maximum segment length of 12 appears to be a better representation of the dynamics in the RDC measurements; we focus on this ensemble in the analyses below . The structural variability of the ensemble is illustrated in Figure 4A . The average NH order parameter in regular secondary structure elements is 0 . 76 , the same as that computed for the model free analysis ( 0 . 77 ) described in Lakomek et al . , but lower than that computed for the EROS ensemble ( 0 . 83 ) [4] , [35] . We compared the Q-factor of the RDC-optimized Backrub ensemble to the Q-factors from various other ubiquitin ensembles ( Figure 3C ) : the Self-Consistent RDC-based Model-free ( SCRM ) description ( an analytical description of the RDCs with five parameters per residue that does not provide an explicit all atom structural representation of the motions ) [35] , an ensemble of 46 X-ray structures of ubiquitin alone and in different complexes ( henceforth called the ubiquitin X-ray ensemble ) as used in reference [4] , three sets of NMR structures ( 1D3Z , 1UD7 , and 1G6J ) , three molecular dynamics ( MD ) ensemble-average-restraint ( EAR ) ensembles ( 1XQQ , 2NR2 , EROS PDB code 2K39 ) [4] , [57] , [58] , and snapshots from a 100-nanosecond MD simulation [63] . We also examined the root mean squared error in the RDCs as a measure of quality of fit , and the results were similar ( Figure S1A ) . The RDC-optimized Backrub ensemble has lower Q-factors than ensembles generated using other methods , except for the SCRM description [35] and the EROS ensemble , both of which were fit with the same dataset of RDC measurements as the Backrub ensembles . Not surprisingly , the SCRM Q-factor is the lowest because it is an analytical description fit to the RDCs . The EROS ensemble was created with an approach where the RDCs are incorporated into the potential function of an ensemble MD simulation and this approach gives very low Q-factors . An analysis of structural quality measures of Backrub and other conformational ensembles is given in Text S1 and Figure S2 . The RDC-optimized Backrub ensembles also have similar Rfree values from cross-validation: 18 . 0% , 16 . 1% , 20 . 0% , 17 . 8% , and 23 . 3% , respectively for the RDC-optimized Backrub ensemble , the EROS ensemble , the 1D3Z structures , the ubiquitin X-ray ensemble and the ensemble of MD snapshots ( Table S1 ) . One important criterion with which the various ensembles of ubiquitin can be assessed , as mentioned above , is whether an ensemble matches the RDCs better than any single structure within it . If this is the case , dynamical information contained in the experimental measurements can be interpreted by analyzing the conformational variability in the ensemble . The RDC-optimized Backrub ensemble , the MD-EAR ensembles ( 1XQQ , 2NR2 and EROS PDB code: 2K39 ) and , the ubiquitin X-ray ensemble and the ensemble of MD structures have improved Q-factors over the best single structure ( Figure 3C ) . The two MD-EAR ensembles that were fit to relaxation NMR measurements ( 1XQQ and 2NR2 ) have small fractional improvement in Q-factor , suggesting that the dynamic information present in the RDCs may be different from the information present on the shorter time scale relaxation measurements; this observation is supported by the different pattern of order parameters observed between these two classes of measurements [35] . The Backrub and the EROS ensembles show the largest fractional Q-factor improvement . Note that this does not contradict the fact that Backrub moves were able to improve modeling of faster time-scale picosecond-nanosecond side-chain motions [30]; the Backrub ensembles used in our previous work were not selected for agreement with the RDCs and the simulation temperature used was lower , resulting in smaller motional amplitudes . The three sets of NMR structures ( 1D3Z , 1UD7 , and 1G6J ) do not show an improvement in the Q-factor over the best single structure . For the 1D3Z NMR structures , a subset of the RDCs were used in the refinement and , as a result , the Q-factor ( Q = 0 . 107; calculated over all 23 datasets used in this paper ) is lower than for the other NMR structures . The Q-factor of the lowest single 1D3Z NMR structure indicates that the 1D3Z NMR structure is a good representation of the average structure . We also used the strategy described in Figure 2C to generate RDC-optimized ensembles consisting of structures from the various ubiquitin ensembles ( Figure S1B ) . The Q-factor decreased substantially for the ubiquitin X-ray ensemble ( 34% lower Q-factor ) , the MD-EAR ensembles , ( 41% , 49% and 31% decrease in Q-factor for 1XQQ , 2NR2 , and EROS , respectively ) and the ensemble of snapshots from the 100-ns MD simulation ( 64% decrease ) . These findings are consistent with the results above that all ensemble types except the three sets of NMR structures provide insight into the RDC dynamics . The Q factors of the RDC-optimized ensembles of ubiquitin X-ray structures ( Q = 0 . 089 ) and the MD snapshots ( Q = 0 . 069 ) are quite similar to the Q factors of the best RDC-optimized Backrub ensemble . This latter result suggests that the 100 ns explicit water MD simulation , although short in comparison to the RDC timescale , may allow regions of ubiquitin to locally sample conformations in agreement with the RDC measurements; this is consistent with the observation from other studies that relatively short MD simulations capture a significant fraction of the motions measured by RDCs [67] , [68] . Longer timescales or analyses of multiple trajectories may be needed to sample combinations of these conformations throughout the ubiquitin structure . This idea was suggested by Henzer-Wildman et al . [8] to explain the ability of adenylate kinase to sample substates in nanoseconds along the open-closed trajectory that exchanged on the order of micro- to milliseconds . To characterize the conformational variability of different regions of the protein in our ensembles , we calculated C-alpha difference distance matrices ( see Methods and Figure S3A ) [45] . These matrices show the motion of each residue with respect to all other residues . For clarity , we collapse these matrices onto a single dimension that represents the average C-alpha difference distance with respect to other residues in the protein ( Figure S3B ) . This metric is sensitive to motions relative to those of other residues in the ensemble , as opposed to C-alpha RMSD , which is sensitive to changes relative to one conformation in the ensemble . Figures 4B and 4D show these C-alpha difference distance values mapped onto the ubiquitin structure ( see Methods ) . Supporting relation 1 , the pattern of motion of the ubiquitin X-ray ensemble and the RDC-optimized Backrub ensemble show substantial similarities . In both these ensembles the most flexible regions are the C-terminal end of the helix and the N-terminal end of beta strand 2 . This result is consistent with the suggestion of Lange et al . [4] that the native state dynamics of ubiquitin encompass the conformational flexibility found in crystal structures of ubiquitin bound to different partners , supporting a conformational selection model for binding . Moreover , the patterns of motions of the RDC-optimized Backrub ensemble are similar to the EROS and the MD ensembles despite their different amplitudes . In addition , RDC-optimized and non-RDC-optimized ensembles are similar to each other with respect to the average C-alpha difference distance matrices shown in Figure 4B . Text S1 and Figure S4 give a more detailed comparison of RDC-optimized and non-RDC-optimized conformational ensembles . We also investigated the differences between the RDC-optimized Backrub and the ubiquitin X-ray ensemble flexibilities in light of the errors in the calculated RDC values in these regions ( Figure 4B and Figure S3C ) . The differences in flexibility of these ensembles are mainly around the C-terminus of beta strand 1 and the alpha-helix . In the C-terminal tail of beta strand 1 , residue 6 has some of the highest errors in the Backrub ensemble . Since the flexibility is low in this region in both the X-ray ensemble and the EROS ensemble , the Backrub model may overestimate the flexibility . In the helix , the relative amplitude of flexibility is also higher in the Backrub ensemble than in the X-ray ensemble; however , the pattern of flexibility is quite similar ( see Figure S3C ) . Interestingly , the helix C-terminal residues in the X-ray ensemble show less agreement between experimental and back-calculated RDCs ( Figure S3C ) , implying that the high flexibility in this region for the Backrub ensemble is likely to be a better representation of the RDC data . This observation agrees with the amplitude and pattern of flexibility in this region of the EROS ensemble . In addition , we observe correspondingly higher flexibility in the helix in a structural alignment of members of the ubiquitin family ( Figure 4D ) , as discussed further below ( Test 3 ) . As a final point of comparison , we applied a Gaussian network model ( GNM ) [69] . These models have been used to describe slow motions in proteins . Figure 4C shows the GNM computed B-factors mapped onto the ubiquitin structure , displaying conformational variability similar to the other methods and the X-ray ensemble , although some differences compared to the X-ray ensemble are apparent , such as along the alpha-helix and in beta strand 2 . We showed above that our RDC-optimized Backrub ensemble ( i ) gives similar Q-factors to reference ensembles including an RDC-restrained MD ensemble ( EROS ) [4] , a ubiquitin X-ray ensemble and an ensemble of snapshots from a 100-nanosecond MD trajectory [63] and ( ii ) has similar regions of structural variability ( Figure 4B ) . As an additional point of comparison and validation of our approach , we asked whether the RDC-optimized Backrub ensemble also supports other structural and functional insights derived from previous ensemble descriptions of ubiquitin . Lindorff-Larsen et al . [58] as well as Richter et al . [57] used MD simulations with side chain and backbone relaxation order parameters as restraints . These ensembles displayed liquid-like flexibility of side chains buried in the protein core . The RDC-optimized Backrub ensemble also has this property , with buried or near buried residues 13 , 23 , 44 , 61 , and 67 correctly modeled as flexible with calculated order parameters close to their respective values from NMR relaxation experiments . As shown in Figure 5 , Ile 13 chi2 , Ile 44 chi2 , and Leu 67 chi2 have modeled order parameters within 0 . 04 of the experimental values . Ile 13 chi 1 and Ile 61 chi2 have modeled order parameters that are substantially lower than the experimental values but these differences can be due to the short timescale of the relaxation measurements compared to the longer timescale of the RDCs fit by the RDC-optimized Backrub ensemble . ( See Figure S5 for comparison to more side chains analyzed in [58] . ) Side chain order parameters derived from the 100 ns MD simulation discussed earlier are also shown in Figure S5 for comparison . In several cases , the side chain order parameters from the MD simulation are higher than those obtained from the relaxation experiments , possibly due to sampling limitations at the side chain level . Exceptions are the modeled order parameters for L15 chi2 and I61 chi2 , which are significantly lower than the measured relaxation order parameters ( this may be because the timescale of the MD simulation is longer than the rotational correlation time of the molecule ) . Ubiquitin has several hotspots shown to be important in recognition of different binding partners: Ile 44 , Asp 58 , and His 68 . These were identified as rigid in the order parameters of the EROS ensemble [4] . Residues Ile 44 and His 68 are also among the most rigid in the Backrub ensemble according to analysis by order parameters and C-alpha distance difference value ( Figure S4G and S3B , respectively ) . Likewise the secondary structure residues observed to be most flexible by order parameters calculated from the EROS ensemble are those in the N-terminus of strand 2 which our analysis also observes to be quite flexible . We find flexible regions in the C-terminus of the alpha helix that is reflected in the C-alpha distance difference value of the EROS ensemble but not in its order parameter . Our results above provide support for the hypothesis of a correspondence between the properties of Backrub-derived conformational ensembles , solution-state dynamics reflected in NMR measurements and a conformational ensemble of 46 experimental crystal structures of ubiquitin . To broaden this result and shed light more generally on a link between protein dynamics and evolution , we next ask whether there is also a correspondence between the dynamics of a single protein sequence and the conformational variability explored in its protein family to accommodate sequence changes during evolution ( relation 2; Figure 1 ) . In order to test this relation , we first compare the conformational variability present in the RDC-optimized Backrub ensemble with that observed in a structural alignment of 20 members of the UBQ subfamily ( Test 3 ) . Second , we extend this comparison from structural variation to sequence variation by comparing sequences modeled on Backrub ensembles to the sequences of the natural UBQ subfamily ( Test 4 ) . To test the correspondence of the conformational variability of an individual protein and that of its family , we constructed an ensemble from the available structures of proteins in a multiple sequence alignment of the UBQ subfamily ( see Methods for details ) [53] . We performed a multiple structure alignment of this 20-member UBQ subfamily ensemble using MAMMOTH-mult [70] resulting in 66 positions that aligned in all proteins ( see Methods ) . These aligned positions had at most 85% and an average of 21% pair-wise sequence identity . We calculated the C-alpha average distance difference matrix for these aligned positions and Figure 4D shows the average values for each residue in the matrix mapped onto the 1UBQ structure , as described for Test 2 . The resulting UBQ subfamily ensemble shows high variability in the C-terminus of the helix and in the N-terminus of beta strand 2 , which is strikingly similar to the regions of high flexibility in the RDC-optimized Backrub ensemble . Thus , we find similar conformational variability in the structures of the ubiquitin homologs and in an ensemble fit to the solution state dynamics of ubiquitin . This correspondence in pattern of flexibility holds despite the different motional amplitudes of these ensembles: 2 . 0 Å and 0 . 9 Å pair-wise RMSD to the 1UBQ X-ray structure , respectively , for the UBQ subfamily ensemble and the RDC-optimized Backrub ensemble . We proposed in hypothesis 2 and showed above that there are similarities in the conformational variability of a single protein and that of its homologs . Here we extend this idea to ask whether the sequences compatible with a structural ensemble describing the dynamics of a single protein are similar to the sequences of the natural family members . We first tested whether there is a difference between the sequence spaces consistent with the RDC-optimized and non-RDC-optimized Backrub ensembles . We performed computational protein design with Rosetta [54] using simulated annealing of rotamer conformations and amino acid identities on each backbone in an ensemble to determine low-scoring sequences compatible with that ensemble . All positions were allowed to vary to any amino acid and 1000 low-energy sequences were generated for each ensemble . In the following , we use the term ‘sequence space’ to describe the high-dimensional space of possible sequences of a protein . To compare the sequence space coverage of the various ensembles , we used the BLOSUM62 matrix [71] to calculate the distances between all pairs of sequences . This resulted in a distance matrix of size NxN ( where N is the number of sequences compared ) representing a sequence space of dimensionality N . To visualize the relative sequence space coverage of different sets of sequences we collapsed this sequence space into two dimensions using multidimensional scaling , retaining the two dimensions containing the most variation in sequence distances ( see Methods ) . The sequence spaces sampled by the RDC-optimized and non-RDC-optimized Backrub ensembles with optimal Backrub parameters ( maximum segment length of 12 and kT = 1 . 2 ) are very similar ( Figure 6A ) . This is consistent with the idea that the Backrub method captures a significant portion of near-native protein motions , even without directly incorporating the RDC information into the model . In the following , we use results for non-RDC-optimized ensembles; the results are similar for RDC-optimized ensembles . Next we compared the 2-D sequence space of designs on various non-RDC-optimized Backrub ensembles to the sequence space of designs on the ubiquitin X-ray ensemble . Different non-RDC-optimized Backrub ensembles of maximum segment length of 12 with varying amplitude ( kT = 0 . 3 , 1 . 2 and 4 . 8 ) sample largely separate sets of sequences ( Figure 6B ) . Sequences move further away from the sequences sampled using the fixed backbone with increasing amplitude of motion in the ensemble . Notably , the Backrub sampling parameters used to generate ensembles which sample a range of sequences most similar to the 46-member ubiquitin X-ray ensemble are the same parameters that gave the lowest Q-factor ( maximum segment length of 12 with kT = 1 . 2 ) , supporting the hypothesis that the Backrub ensembles are sampling similar conformational heterogeneity to the ensemble of ubiquitin X-ray structures ( Test 2 ) . Sequences obtained from the MD ensemble are likewise most similar to the kT = 1 . 2 amplitude ensemble ( Figure S6C and D ) , although spanning a somewhat larger region of sequence space . Finally , to test whether there exists a link between the conformational heterogeneity of solution dynamical ensembles and the sequence space compatible with these ensembles ( Test 4 ) , we compared the 2-D sequence space of designs on various Backrub ensembles to the sequence space of the UBQ subfamily of the ubiquitin αβ roll subfold ( Figure 6C ) . The subfamily sequences we used came from a high quality manually curated alignment of 36 homologues created using 3D structural analysis [53] . As shown in Figure 6C , the sequences in these naturally occurring proteins represent a subset of the sequence space of the non-RDC-optimized Backrub ensemble ( maximum segment length of 12 with kT = 1 . 2 ) . In contrast , the UBQ subfamily sequences barely or do not at all overlap with the sequences from design simulations using the fixed backbone , or the kT = 0 . 3 and kT = 4 . 8 ensembles . We obtain similar results when considering core residues only ( Figure S6B ) . The sequence logo representations in Figure 7A–H for residues in buried core regions ( see Methods ) support the correspondence between the sequence diversity in Backrub ensembles and the natural family . The predominant amino acid in the UBQ subfamily is generally recapitulated in the non-RDC-optimized Backrub ensembles of maximum segment length 12 with kT = 0 . 3 and kT = 1 . 2 ( e . g . positions 5 , 27 , 43 , 50 , 56 , 61 , and 69 ) . One notable exception is that the designed sequences fail to recapitulate the frequently observed glutamine at position 41 . Kiel et al . [53] use this position as the main indicator in categorizing subgroups of the UBQ subfamily because its presence correlates with the structure of a nearby loop . The side chain amide nitrogen atom of Gln 41 forms a buried hydrogen bond with the backbone of residue 36 , which may be responsible for structural specificity of the loop conformation that we are not accounting for in the design simulations . Several positions , such as residues 21 , 25 , 45 , 55 , 61 , 65 , and 68 , have high sequence entropy in the natural family . The Backrub ensemble designs recapitulate high sequence entropy for these residues . Especially for residues 45 , 55 , 61 , and 65 the high entropy underscores one of the uses of flexible backbone design , as with a fixed backbone or low temperature Backrub ensemble only a few amino acid types predominate at those positions failing to capture the substantial natural sequence plasticity within the family . We also generated designs compatible with the trajectory of the 100-ns MD simulation , which showed similar results to the RDC-optimized Backrub ensemble overall , but with higher sequence entropy for several positions ( as reflected also in Figure S6 ) . Taken together , our results thus indicate that the conformational sampling methods we use here to match RDC dynamics produce variability similar to the conformational heterogeneity of X-ray ensembles ( both using different ubiquitin structures as well as structures from the UBQ subfamily ) and may lead to significant overlap between sequences consistent with modeled ensembles and the sequence space covered by the natural family . Additionally , it appears from the similarity of sequences from RDC-optimized and non-RDC-optimized ensembles that the RDCs have led us to determine optimal Backrub sampling parameters ( Figure 3B ) that can be used prospectively to make modeling predictions .
In this work , we describe the application of the Backrub motional model to create ensembles of structures consistent with RDC measurements and to sample the conformational and sequence space of the UBQ subfamily . The main new aspect of our work is that we link the conformational dynamics of a single sequence , as reflected by both RDC data and Backrub ensembles , to conformational diversity observed in crystal structures of ubiquitin and its family , and to evolutionary sampled sequence diversity . We achieve this by applying computational protein design to select low-energy sequences consistent with Backrub ensembles . The fact that low-Q factor Backrub ensembles sample a similar sequence space to that of the ubiquitin X-ray ensemble extends results by other groups demonstrating the correspondence of solution-state dynamics and crystallographic heterogeneity [21] , [35] . In addition , we find that this designed sequence space consistent with optimal Backrub ensembles encompasses the sequence space of the UBQ subfamily , providing evidence for the idea suggested by Davis et al . [29] that the Backrub motional mechanism may facilitate amino acid changes during evolution . We find that RDC-optimized ensembles created with only certain Backrub sampling parameters were able to reach the lowest Q-factors , indicating that the conformational space sampled by these Backrub parameters is the most similar ( compared to other parameters ) to the conformations giving rise to the RDC measurements . However , while we see significant improvements in Q-factors during the selection protocol , we also find substantial similarities between RDC-optimized and non-RDC-optimized Backrub ensembles in patterns of C-alpha RMSD , order parameters and designed sequence space . This somewhat surprising observation could mean that the selection procedure primarily optimizes for subtle differences in NH-vector orientations ( Figure S7 ) , while other dynamical features that are commonly characterized ( such as the anisotropy of motions ) are essentially indistinguishable between RDC-optimized and non-RDC-optimized Backrub ensembles . Analysis by cross-validation shows an improvement in Rfree for RDC-optimized over non-RDC-optimized ensembles , indicating that other aspects of the peptide plane orientation are better represented in the RDC-optimized ensembles . Notably , there are defined Backrub parameters that simultaneously give the best agreement with the RDC data ( after selection ) and the best sequence space overlap with the natural family , irrespective of whether we apply selection or not . This could indicate that it is primarily the mechanism and amplitude of motions that are important , and that , as long as the amplitude is in the correct range defined by the appropriate sampling parameters , the Backrub motional model can sample relevant motions without requiring RDC data . Hence , the Backrub motional model may be useful ( i ) to predictively sample conformations similar to ensembles of bound conformations and ( ii ) to use with design to sample the sequence space of the natural family . Such sampling of sequences likely to be accommodated by a given protein fold may help improve engineering of new protein structures , functions and interactions . For example , coupling backbone ensemble generation and sequence design may be useful to computationally predict sequence libraries enriched in functional members [56] . There are several potential limitations of the Backrub method , as applied here . As we implement Backrub in a Monte Carlo protocol , the timescale of conformational transitions is not taken into account . Also , the method used here limits the backbone conformational space sampled to those conformations accessible with the Backrub mechanism , a restriction which can be alleviated for example with the addition of small phi/psi changes to the method or by using analytical methods for local loop closure [72] , which is a superset of the Backrub move . Nevertheless , Backrub changes have an interesting similarity to the 1D-Gaussian Axial Fluctuation ( GAF ) analytical model , a simple motional model that has been used with success to fit RDCs [52] . A dipeptide Backrub move ( a tripeptide Backrub move is shown in Figure 2A ) is similar to motions described by the 1D-GAF model; thus the Backrub Monte Carlo protocol , which includes moves of longer peptide segments incorporated into a Monte Carlo scheme , can be viewed as a extension of the analytical GAF model to discrete structural ensembles . As necessitated by the scarcity of proteins with sufficient RDC data , we limit our study here to one protein and further work is needed to extend modeling of protein native state dynamics and tolerated sequence space to more proteins . However , the usefulness of the Backrub mechanism for modeling protein motions is supported by several studies [29]–[32] , [73] . Our studies on ubiquitin provide an interesting benchmark case for future analyses of the correspondence of individual and family variation . Analysis of the generated ubiquitin Backrub ensembles allows several fundamental insights on the relationship between structure , function , sequence and dynamics . The ubiquitin core flexibility and a binding mechanism by conformational selection have been pointed out previously [4] , [58] . Furthermore , our study allows characterization of differences between computationally predicted and evolved protein sequences that may lead to testable hypotheses on effects not modeled in the simulations , such as evolutionary pressures to conserve functional residues . An example is the discrepancy between the predictions and the naturally occurring glutamine residue at position 41 in ubiquitin . A likely explanation why our design simulations fail to predict this preference for glutamine is that we are not taking into account avoidance of certain non-native conformations due to evolutionary pressure enforcing structural specificity . In conclusion , we have tested a method for sampling conformational diversity using Backrub conformational changes and shown that it can generate ensembles consistent with millisecond-timescale measurements of protein dynamics . This method is computationally more efficient than molecular dynamics-based methods , allowing it to be applied to a variety of protein modeling tasks such as sequence design . Notably , we find that the method recapitulated many of the structural properties of the RDC-optimized Backrub ensembles even when the RDC measurements were not incorporated in the sampling procedure , despite the fact that the RDCs were necessary to determine the amplitudes of motion in the Backrub ensembles . We additionally find that the sequence diversity tolerated by non-RDC-optimized Backrub ensembles is similar to that of both the ubiquitin X-ray ensemble and the UBQ subfamily X-ray ensemble . This result needs to be tested on more proteins and , if validated , should be useful in making prospective predictions to numerous applications , such as protein-protein or protein-small-molecule docking , protein interface design , and enzyme design .
The dataset of RDCs we use here consist of measurements in 23 alignment media as described in Lakomek et al . [35] . For all X-ray structures , explicit hydrogen atoms were added according to standard geometry using Rosetta , and the positions of hydrogens with rotatable bonds were optimized [74] . The 46-member ubiquitin X-ray ensemble used was the same as that of [4] . To generate protein conformational ensembles , we ran “Backrub” Monte Carlo simulations , as described in [30] and [31] . Briefly , this method randomly makes one of three types of moves: ( a ) a rotamer change ( 50% of the time ) , ( B ) a local backbone conformational changes ( Backrub move ) consisting of a rigid body rotation of a random peptide segment about the axis connecting the endpoint C-alpha atoms ( 25% of the time ) , or ( c ) a composite move with a Backrub change and one or two rotamer changes ( 25% of the time ) . After each move , the positions of the C-beta and H-alpha atoms are modified to minimize bond angle strain as described [31] . This results in bond angle changes of the main chain atoms of one to four standard deviations . The mean values and standard deviations are very similar to those computed in a set of 240 high-resolution crystal structures ( better than 1 . 3 Å ) with less than 25% sequence identity culled from the Dunbrack database [75] , except for some perturbation to the N-CA-C angle ( mean and standard deviations are 111 . 5° and 4 . 1° in the Backrub ensembles and 111 . 0° and 2 . 5° in the crystal structure set ) . See Figure S2 for details on the structural quality analysis for all structures and ensembles used in this study . We ran a Backrub Monte Carlo simulation at kT = 0 . 1 from the starting PDB conformation ( using 1UBQ , which has the highest resolution ( 1 . 8 Å ) of the unbound ubiquitin structures; similar results were obtained for maximum segment length of 3 with PDB entries 1UBI and 1CMX and worse Q factors were obtained for PDB entries 1FXT , 1AAR , 1F9J , and 1TBE ) for 10 , 000 steps with a maximum segment length of 3 or 12 , matching the segment length used later . The lowest energy structure from this simulation is used as the starting conformation for 10 , 000 randomly seeded Backrub simulations at one of 5 different temperatures ( kT = 0 . 3 , 0 . 6 , 1 . 2 , 2 . 4 , or 4 . 8 ) run for an additional 10 , 000 steps . The last structure from each of these simulations is used to form the starting set of 10 , 000 structures . From this initial set of 10 , 000 structures , ensembles are selected to match the RDCs by minimizing the Q-factor of the ensemble . First , structures are randomly chosen to create a starting ensemble of a given size ( 2 , 3 , 5 , 10 , 20 , 50 or 100 structures ) , and the Q-factor of the ensemble is calculated ( see below ) . Next , a random structure in this ensemble is chosen and replaced with a randomly chosen structure from the initial ensemble of 10 , 000 structures; then the new Q-factor of the ensemble is calculated . If the new Q-factor is lower than before the replacement , the change is kept , otherwise it is reverted . These structure replacements are repeated until the Q-factor changes by less than 0 . 001 in 5000 steps . By repeating this method 1000 times , 1000 RDC-optimized Backrub ensembles are created . There are a very large number of possible subsets of a given size . For example , there are 4*10^61 different sub-ensembles of size 20 from the initial ensemble of size 10 , 000 , too many to be evaluated . The approach described here does not guarantee that the ensemble with the lowest Q-factor will be found , but it starts from many random starting points to broadly sample the space of possible sub-ensembles and the selection process converges to a low Q-factor solution within 10 , 000 Backrub-generated structures for all Backrub Monte Carlo temperatures ( except kT = 4 . 8; see Figure S8 ) . RDCs are calculated from a single structure and an ensemble of structures as described in [76] . Briefly , we first find the alignment tensor from a structure ( or set of structures ) and the experimental couplings . This is done using the equation T = A−1 Dexp , where T is the alignment tensor , A−1 is the Moore-Penrose inverted matrix of projection angles for the amide bonds ( or averaged projection angles for a set of structures ) , and Dexp is the vector of experimental couplings . The predicted couplings are then calculated with the equation Dcalc = AT where A is the same matrix of projection angles from above and Dcalc is the vector of calculated couplings . Q-factors were calculated for all RDC measurements with the equation: Errors between experimental and predicted RDCs were calculated with: Loop residues ( i . e . those with DSSP [77] secondary structure type not H , E , G or I ) are excluded from the analysis in both tensor determination and back-computation of RDCs and Q-values . The non-loop residues used in all analyses in this paper are ubiquitin residues 2–7 , 12–16 , 23–34 , 38–45 , 48–49 , 57–59 , and 66–71 . There are several sources of error in our analysis to consider when assessing the significance of the results . First , there is error in the RDC measurements due to experimental uncertainty . The uncertainty in these values is estimated to be 0 . 3 Hz [35] . To calculate the resulting uncertainty in the Q-factor , we added Gaussian-distributed noise of mean amplitude 0 . 3 Hz to the RDC measurements ( see section below ) in 1000 Monte Carlo trials . This resulted in a value of Qexperimental_error = 0 . 036 . A second source of error results from not finding the ensemble with the lowest possible Q-factor from a given initial structure set . We estimated this error by repeating the selection procedure many times and evaluating the variance in the resulting Q-factors . We take explicit steps to minimize this error by enforcing two convergence criteria on the optimization: 1 ) ensemble selection is not finished until 5000 steps have passed without a change in Q of more than 0 . 001 , and 2 ) enough RDC-optimized ensembles are generated from random starting structures such that the difference in the Q-factors of the best and 10th best RDC-optimized ensemble is not more than 0 . 005 . Thus , this Qoptimization_error is on the order of 0 . 005 . A third important source of error is due to insufficient sampling of conformational space with the Backrub Monte Carlo protocol and the 10 , 000 structures that we use to select ensembles from . We estimated this Qsampling_error by running the structure generation protocol at each temperature 10 times , thus creating 10 sets of 10 , 000 Backrub-generated structures at each temperature . The standard deviations of the minimum Q-factors over these 10 sets of 10 , 000 structures are 0 . 0151 , 0 . 0104 , 0 . 0025 , 0 . 0039 , and 0 . 0049 for kT = 0 . 3 , 0 . 6 , 1 . 2 , 2 . 4 and 4 . 8 , respectively for a maximum segment length of 12 . The standard errors of the mean of these values are 0 . 0048 , 0 . 0033 , 0 . 0008 , 0 . 0012 , and 0 . 0015 , respectively . Gaussian-distributed noise was added to the experimental RDCs with 1000 Monte-Carlo samples . The RDC uncertainty of each measurement was 0 . 3 Hz [35] , which was used as the standard deviation of the Gaussian noise function . The resulting Qexperimental_uncertainty is 0 . 036 with a standard deviation of 0 . 00102 over the 1000 samples . Order parameters were calculated with the equationwhere x , y and z are the coordinates of the normalized unit vectors representing the amide bond vector orientations [78] . For the Backrub ensemble , these values were then scaled by 1/1 . 12 = 0 . 89 to account for librational effects that cannot be sampled by the Backrub method when considering only one type of RDCs [79] . We used the 100-nanosecond AMBER trajectory of ubiquitin in TIP4Pw/e water from Wong and Case [63] . The protein was allowed to equilibrate over the first 4 . 32 nanoseconds , and snapshots were taken from the following 100 nanoseconds at 10-picosecond intervals . This resulted in 10 , 000 structures , which were used to calculate an overall Q-factor for the trajectory . In addition , we applied the selection scheme in Figure 2C on these 10 , 000 snapshot structures to select ensembles with optimized Q-factors . To estimate the sequence space compatible with different structures and ensembles , we used Rosetta computational protein design to generate 1000 low-energy sequences for each single structure or 20 sequences per ensemble member for ensembles of size 50 . To find a low-scoring sequence , each design simulation consists of 20 rounds of Monte Carlo simulated annealing with the number of steps in each round equal to the number of rotamers created for the simulation . The backbone of each structure or ensemble member is kept fixed during the design simulations and all positions were allowed to vary to any of the 20 naturally occurring amino acids , adding extra conformers at one standard deviation around the mean rotamer for chi 1 and 2 dihedral angles . The scoring function used was the Rosetta all-atom scoring function [54] , which is dominated by a Lennard-Jones potential , a geometry-dependent hydrogen-bonding potential [74] and an implicit solvation potential [80] . Distances between sequences were calculated as in [50] . Briefly , these distances were calculated as the sum of the substitution costs ( using the BLOSUM62 matrix after normalizing it to range from 0 to 1 ) [71] for the positions that aligned in all sequences: residues 1–9 , 12–24 , 26–35 , 40–53 , 55–63 , 65–71 . After calculating the distances between all pairs of sequences within each ensemble and between pairs of ensembles , we used metric multidimensional scaling in R [81] to reduce the dimensionality of the space to the two dimensions spanning the most sequence distance . The procedure was repeated with the sequences of core residues only , where core residues were defined by counting the number of neighbor residues with C-beta atoms within 10 Å of the C-beta atom of the residue of interest ( or C-alpha atoms for glycine ) . The cutoff value used ( greater than or equal to 18 ) was chosen so that approximately one third of the residues fell into the core category ( excluding the flexible C-terminus ) , resulting in 21 buried positions: residues 3 , 5 , 17 , 21 , 23 , 25 , 26 , 27 , 30 , 41 , 43 , 45 , 50 , 55 , 56 , 59 , 61 , 65 , 67 , 68 , and 69 . First , for each structure , we calculated the matrix of distances between all C-alpha atoms . Then , for each pair of structures , we calculated the distance difference matrix as the absolute value of the difference of the distance matrices of the structures . These distance difference matrices were averaged to give the C-alpha difference distance matrix of the ensemble [45] . Theoretical B-factors were calculated by applying the online Gaussian Network Model ( oGNM ) tool at http://ignm . ccbb . pitt . edu/GNM_Online_Calculation . htm [69] to PDB structure 1UBQ using 1 node per residue and a cutoff of 10 Å for amino acid pairs . To create a structural ensemble for the UBQ subfamily we took the highest resolution X-ray structure for each protein listed in Table 1 of Kiel et al . [53] ( or the first structure of an NMR ensemble if no X-ray structure was available ) . We removed structures that had 100% sequence identity to other structures in the ensemble . We performed a multiple structural alignment using MAMMOTH-mult [70] and removed PDB id 1WIA because it was missing residues that aligned with part of the helix in the native ubiquitin sequence; all other structures had residues that aligned with all the residues in the secondary structure regions of ubiquitin . The resulting ensemble consisted of 20 structures: 1XD3 chain B , 1BT0 chain A , 1EUV chain B , 1IYF , 1J8C , 1LM8 chain B , 1M94 , 1NDD chain A , 1OQY , 1P1A , 1TGZ chain B , 1V5O , 1V5T , 1V86 , 1WE6 , 1WE7 , 1WGD , 1WGG , 1WH3 , and 1WM3 chain A . To create the C-alpha distance difference matrix we used the 66 positions that aligned in all 20 structures , which were ( using 1UBQ numbering ) : 1–7 , 9–16 , 18–34 , 36–46 , 48–55 , 57–64 , 66–72 . We performed cross-validation by using the alignment tensor calculated from the NH RDC datasets to calculate RDCs for four datasets of NC′ RDC couplings and four datasets of HC′ couplings . These “free” data were not included in the selection process and are reported as Rfree factors , as calculated by Lange et al . [4] . for the N different types of experiments with ni measurements each and Q-factor Qi . For RDC-optimized Backrub ensembles , the Rfree values are averaged over the five lowest-Q factor ensembles .
|
Knowledge of protein properties is essential for enhancing the understanding and engineering of biological functions . One key property of proteins is their flexibility—their intrinsic ability to adopt different conformations . This flexibility can be measured experimentally but the measurements are indirect and computational models are required to interpret them . Here we develop a new computational method for interpreting these measurements of flexibility and use it to create a model of flexibility of the protein ubiquitin . We apply our results to show relationships between the flexibility of one protein and the diversity of structures and amino acid sequences of the protein's evolutionary family . Thus , our results show that more accurate computational modeling of protein flexibility is useful for improving prediction of a broader range of amino acid sequences compatible with a given protein . Our method will be helpful for advancing methods to rationally engineer protein functions by enabling sampling of conformational and sequence diversity similar to that of a protein's evolutionary family .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"computational",
"biology/macromolecular",
"structure",
"analysis",
"biophysics/theory",
"and",
"simulation",
"computational",
"biology/macromolecular",
"sequence",
"analysis",
"computational",
"biology",
"computational",
"biology/molecular",
"dynamics"
] |
2009
|
A Correspondence Between Solution-State Dynamics of an Individual Protein and the Sequence and Conformational Diversity of its Family
|
The class II trans-activator CIITA is a transcriptional co-activator required for the expression of Major Histocompatibility Complex ( MHC ) genes . Although the latter function is well established , the global target-gene specificity of CIITA had not been defined . We therefore generated a comprehensive list of its target genes by performing genome-wide scans employing four different approaches designed to identify promoters that are occupied by CIITA in two key antigen presenting cells , B cells and dendritic cells . Surprisingly , in addition to MHC genes , only nine new targets were identified and validated by extensive functional and expression analysis . Seven of these genes are known or likely to function in processes contributing to MHC-mediated antigen presentation . The remaining two are of unknown function . CIITA is thus uniquely dedicated for genes implicated in antigen presentation . The finding that CIITA regulates such a highly focused gene expression module sets it apart from all other transcription factors , for which large-scale binding-site mapping has indicated that they exert pleiotropic functions and regulate large numbers of genes .
Most mammalian transcription factors are believed to activate numerous genes having diverse functions . Direct support for this pleiotropic nature of transcription factor function has been provided by techniques permitting the unbiased mapping of transcription factor binding sites in substantial segments of genomic DNA , whole chromosomes or entire genomes . These large scale location techniques rely on sequencing or microarray ( chip ) analysis of genomic DNA sequences that are enriched by chromatin immunoprecipitation ( ChIP ) experiments . To date , all transcription factors for which these methods have been applied were found to bind to numerous target genes , typically in the order of several hundred to several thousand per genome [1]–[5] . The past few years has witnessed a controversy about whether this general rule of pleiotropic function is also valid for the Major Histocompatibility Complex class II ( MHC-II ) gene transactivator CIITA ( NM_000246 ) . CIITA was first established to be a key regulator of MHC-II genes because it was found to be mutated in an in vitro generated B cell line lacking MHC-II expression [6] . Mutations in the CIITA gene were next shown to be one of the causes of the Bare Lymphocyte Syndrome ( BLS ) ( MIM number: 600005 ) , a hereditary immunodeficiency disease characterized by the virtually complete absence of MHC-II expression and a significant reduction in MHC class I ( MHC-I ) expression [6] , [7] . In accordance with these genetic findings , CIITA was subsequently shown to be a non-DNA-binding transcriptional co-activator that is essential for expression of the genes encoding the α and β chains of all classical and non-classical MHC-II molecules ( HLA-DR , HLA-DP , HLA-DQ , HLA-DM , HLA-DO in humans ) as well the gene encoding the Invariant chain ( Ii ) , an accessory molecule controlling intracellular transport and peptide loading of MHC-II molecules [7]–[9] . CIITA was also found to contribute , albeit to a lesser extent , to the transcription of MHC-I genes [7]–[12] . All clinical and immunological abnormalities documented in BLS patients can be explained by defects in MHC expression [7] . The same is true for knockout mice carrying mutations in the CIITA gene [7] , [9] . The absence of other overt phenotypes suggested that CIITA is highly dedicated for the transcription of MHC genes . It therefore came as a major surprise when a growing number of reports suggested that CIITA can affect the expression of numerous genes involved in diverse functions within and outside the immune system . Microarray experiments identified the gene encoding Plexin-A1 , which was reported to be activated by CIITA in mouse DC [13] , and over 40 genes that were suggested to be up-regulated by CIITA in human B cells and interferon-γ ( IFNγ ) induced cells [14] . The genes encoding IL-4 and Fas ligand were proposed to be repressed by CIITA in mouse T cells [15]–[18] . Those encoding cathepsin E and IL-10 were suggested to be repressed by CIITA in mouse B cells and/or DC [19] , [20] . The genes encoding collagen type I α2 , tymidine kinase and cyclin D1 were proposed to be repressed by CIITA in IFNγ induced cells [21] , [22] . Finally , microarray experiments identified 16 genes of diverse functions that were proposed to be down-regulated by CIITA in human B cells [14] . Taken together , these reports suggested that CIITA has widespread functions extending beyond its well established role in the control of MHC expression . The notion that CIITA exerts pleiotropic functions was at odds with the highly specific defects observed in BLS patients and CIITA-deficient mice . To address this discrepancy we set out to define the complete set of CIITA target genes by performing genome-scale ChIP-chip experiments . Our results demonstrate that CIITA is remarkably dedicated for the regulation of genes implicated in MHC-II and MHC-I mediated antigen presentation . Outside of the well established MHC-II , MHC-I and Ii genes , only nine new target genes were identified , seven of which are known or likely to function in processes related to antigen presentation . The finding that CIITA regulates such a highly specialized genetic module sets it apart from all other transcription factors for which large scale mapping of binding sites has been performed .
Approaches for identifying novel target genes of CIITA were developed on the basis of its expression and mode of action ( Figure 1 ) . The pattern of CIITA expression dictates the cell type specificity of MHC-II expression ( Figure 1A ) [23] . B cells and immature dendritic cells ( iDC ) are MHC-II positive because they express CIITA . The CIITA gene is instead silenced in mature DC ( mDC ) [24] . Most non-hematopoietic cells are MHC-II negative because they lack CIITA . The latter can however be induced to activate CIITA expression by stimulation with IFNγ . CIITA is recruited to its known target genes through protein-protein interactions with a transcription factor complex that assembles on a characteristic enhancer composed of four sequences called the S , X , X2 and Y boxes ( Figure 1A ) [7]–[9] . Regulatory factor X ( RFX ) - a trimeric factor containing three subunits called RFX5 ( NM_000449 ) , RFXAP ( NM_000538 ) and RFXANK ( NM_134440 ) - is an essential component of this transcription factor complex [25]–[29] . CIITA recruitment is abolished in RFX-deficient cells [30]–[33] . Like defects in CIITA , mutations in the RFX5 , RFXAP and RFXANK genes give rise to the BLS disease ( MIM number: 601863 , 601861 , 603200 ) . Based on the above , we devised genome-wide ChIP-chip approaches according to four different experimental designs ( Figure 1B ) . In all four approaches , ChIP samples obtained with CIITA-specific antibodies were used to prepare probes that were hybridized in conjunction with control probes to microarrays carrying the promoter regions of 27434 human genes . The four strategies differed with respect to the cell type used to prepare the ChIP samples and the control probes to which they were compared . In the first strategy , CIITA-ChIP probes derived from the wild type B cell line Raji were compared with CIITA-ChIP probes derived from RJ2 . 2 . 5 , a CIITA-deficient mutant of Raji . In the second strategy , CIITA-ChIP samples prepared from iDC were compared with CIITA-ChIP samples from mDC . In the third strategy , CIITA-ChIP probes derived from Raji were compared with input genomic DNA from Raji . In the fourth stratetgy , CIITA-ChIP probes derived from Raji were compared with CIITA-ChIP probes prepared from a cell line ( SJO ) derived from an RFX5-deficient BLS patient . In all four experimental settings , there is a robust enrichment of known CIITA target sequences , such as the HLA-DRA promoter , in the test samples relative to the control samples ( Figure 1C ) . To validate our screening strategies we examined binding of CIITA to the promoters of well established target genes , including MHC-II , Ii and MHC-I genes ( Figure 2 , Table 1 , Figure S1 ) . Clear binding of CIITA – visualized as peaks in the test/control signal ratios - was observed in Raji B cells and iDC at positions corresponding to the S-Y enhancers of these genes . Despite some variability in the width and height of the peaks , binding of CIITA at the correct position was observed reproducibly in nine independent experiments using all four screening strategies . Binding signals were strong; test/control signal ratios typically ranged from a minimum of 6 to over 30 depending on the target gene and the experiment . Binding of CIITA was never observed at control genes that are not regulated by CIITA ( Figure 2 , Figure S2 ) . The sensitivity of our ChIP-chip approach was high . The false-negative rate was only ∼5% at well-established target genes ( Table 1 ) . Furthermore , robust peaks were observed in all or most experiments even at the HLA-DOB , HLA-DPB , HLA-DMB and HLA-DQA genes ( Table 1 ) despite the fact that CIITA binding signals observed at these genes in classical ChIP experiments are typically 10–20 fold lower than those observed at the prototypical HLA-DRA gene [32] . Certain peaks exhibit a dip in CIITA binding at a position that coincides with the S-Y enhancer ( Figure 2 ) . These dips are likely to be artifacts because they generally concerned only a single oligonucleotide on the array , were not observed in all experiments and were not observed in experiments using high density microarrays ( see discussion ) . To identify potential new targets of CIITA , we developed a procedure to screen for the presence of reproducible peaks in the test/control signal ratios obtained in multiple Raji/RJ2 . 2 . 5 and iDC/mDC comparisons ( see Materials and Methods ) . Candidate genes were assigned a score on the basis of peak height , width and reproducibility . A score of 3 was assigned to the candidate genes at which peak quality and reproducibility were similar to those observed for known target genes ( Table 1 ) . A score of 2 was assigned to candidate genes for which peak quality and/or reproducibility were promising but significantly lower than for known target genes ( Table 1 ) . Finally a score of 1 was assigned to possible but unlikely candidates exhibiting only weak and poorly reproducible peaks ( Table S1 ) . The number of likely candidates was surprisingly low: scores of 3 and 2 were assigned , respectively , to only twelve and twenty genes . To validate bona fide new target genes we screened the most promising candidates for binding of CIITA in Raji B cells by quantitative ChIP experiments ( Figure 3A ) . All twelve score 3 candidates , sixteen score 2 candidates and a selection of the best score 1 candidates were tested . The primers used for real-time PCR analysis of the ChIP experiments were designed within the regions at which peaks were observed in the ChIP-chip experiments . Four score 2 candidates were not tested because suitable primers could not be designed . Binding of CIITA was confirmed for eight score 3 candidates ( RAB4B , TRIM26 , FLJ45422 , KIAA0841 , RFX5 , ZNF672 , TPP1 and MYBPC2 ) and only one score 2 candidate ( PSMD3 ) . Weaker binding was observed at two additional score 2 genes ( BRD2 and TRIM14 ) , but these were not studied further because signals were only 2–3 fold above background . No significant binding was evident at the remaining score 3 and score 2 genes , or at any of the tested score 1 genes ( Figure 3 , Table 1 , Table S1 ) . The finding that most of the score 3 candidates , only one of the score 2 candidates and none of the tested score 1 candidates are true targets of CIITA demonstrates the validity of our scoring procedure . These results also demonstrate that the specificity of our approach is greatest if peak reproducibility is chosen as the most critical parameter for predicting target genes ( Table 1 ) . Thus , among genes at which peaks are present in at least four out of five experiments ( MHC-II and score 3 genes ) less than 20% were found to be false positives , whereas the false positive rate increased dramatically among genes at which peaks were detected at only three out of five experiments ( score 2 genes ) . Representative ChIP-chip profiles corresponding to binding of CIITA in Raji are provided in Figure 3B for the nine validated target genes . Peaks similar in quality to those found at the MHC-II , MHC-I and Ii genes are observed near the transcription initiation site of all nine genes ( compare Figure 3B with Figure 2 and Figure S1 ) . To further confirm the specificity of our quantitative ChIP experiments we compared occupation of the new targets by CIITA between Raji and its CIITA-deficient derivative RJ2 . 2 . 5 . As observed for the control HLA-DRA and Ii genes , binding of CIITA is reduced to non-specific background levels in RJ2 . 2 . 5 at all nine new target genes ( Figure 3C ) . To address the possibility that numerous true target genes might have been missed by our data analysis procedure we used two additional independent methods to analyze the same datasets . First , the windowing and threshold program ACME ( Algorithm for Capturing Microarray Enrichment ) [34] was used for peak detection , and significant peaks present in at least four out of five experiments were identified . In addition to MHC-II and related genes , this procedure identified only 17 potential new target genes ( data not shown ) . Quantitative ChIP experiments performed with 11 of these showed that only 3 represented true CIITA targets ( RFX5 , RAB4B , PSMD3 ) . The other 8 were found to be false positives that had been assigned scores of 3 ( 3 genes ) , 2 ( 2 genes ) and 1 ( 3 genes ) by our initial procedure . The remaining 6 candidates were not tested because visual inspection of their ChIP-chip peaks revealed that they were of very low quality and highly likely to correspond to false positive hits . Importantly , 6 of the validated new CIITA target genes ( TRIM26 , FLJ45422 , KIAA0841 , ZNF672 , TPP1 and MYBPC2 ) were not picked up . As a second alternative approach we developed an unsupervised clustering procedure ( see Material and Methods ) . This method grouped potential candidates into 8 groups on the basis of peak reproducibility and log2 signal ratios ( data not shown ) . The two groups corresponding to the most likely targets contained only 12 and 29 genes respectively . 12 of these were MHC-II genes and 6 were among the new validated targets ( RAB4B , TRIM26 , KIAA0841 , RFX5 , ZNF672 and MYBPC2 ) . Among the remaining 23 genes , 2 were found to be false positives by ChIP experiments and 22 were eliminated as good candidates by visual inspection of the peaks . Finally , 3 of the newly validated target genes ( FLJ45422 , TPP1 and MYBPC2 ) were again not singled out as likely candidates . In conclusion , neither alternative approach was superior to our original method with respect to specificity or sensitivity . More importantly , the alternative methods did not identify a large number of likely targets that were missed by our original procedure . Strong and reproducible peaks were also evident at the new target genes in ChIP-chip experiments comparing ChIP probes from iDC and mDC ( Figure 4A , Table 1 ) . We therefore performed quantitative ChIP experiments to measure binding of CIITA to the new targets in iDC . For eight of the new targets , significant binding was observed in iDC ( Figure 4B ) . As observed for the control HLA-DRA and Ii genes , this binding was strongly reduced in mDC ( Figure 4B ) . CIITA can be induced in most CIITA negative cells by stimulation with IFNγ ( Figure 1A ) . We therefore performed quantitative ChIP experiments to determine whether binding of CIITA to the new targets is induced by IFNγ in a melanoma cell line exhibiting well documented IFNγ induced CIITA expression [35] . IFNγ induced occupation by CIITA was evident at all nine new target genes ( Figure 4C ) . As observed for the control HLA-DRA and Ii genes , this occupation by CIITA is induced rapidly , reaching maximal levels by 6 hours of stimulation ( Figure 4C ) . At all nine new target genes , strong and reproducible peaks were evident in ChIP-chip experiments comparing CIITA-ChIP probes from Raji and SJO cells ( Figure 5A , Table 1 ) . This suggested that recruitment of CIITA to the new genes is – as established for other known target genes – strictly dependent on binding of RFX . To verify this we performed quantitative ChIP experiments comparing binding of CIITA between Raji , RFX5-deficient SJO cells and RFXANK-deficient BLS1 cells . As observed for the control HLA-DRA and Ii genes , binding of CIITA is completely lost in SJO and BLS1 cells , indicating that it requires an intact RFX complex ( Figure 5B ) . To document binding of RFX to the new target genes directly , we performed quantitative ChIP experiments with Raji , RJ2 . 2 . 5 and SJO cells . As shown previously for well established CIITA regulated genes [30] , [31] , binding of RFX to the new target genes is strong in Raji , unaffected in RJ2 . 2 . 5 , but completely abolished in SJO ( Figure 5C ) . Finally , RFX was also found to bind to the new target genes in iDC ( Figure 5D ) and IFNγ induced cells ( data not shown ) . The analysis of RFX5 , RFXAP and RFXANK mRNA abundance by quantitative RT-PCR demonstrated that the level of RFX expression is very similar in Raji B cells and iDC ( data not shown ) . This is consistent with the observation that the strength of CIITA binding observed in ChIP-chip experiments at validated target genes is very similar between Raji and DC ( compare Figures 3B and 4A ) . The finding that recruitment of CIITA to the new target genes is dependent on binding of RFX prompted us to search for sequences resembling the S-Y enhancer . In all MHC-II genes , the S , X , X2 and Y boxes of the S-Y enhancer are highly conserved with respect to their order , orientation and spacing ( Figure 6A ) [7]–[9] . A similar tightly-constrained arrangement of S , X , X2 and Y sequences was evident in six of the new target genes ( RAB4B , FLJ45422 , ZNF672 , MYBPC2 , TPP1 and PSMD3 ) at positions lying within the region to which CIITA is recruited ( Figure 6A ) . Homology to a complete S-Y motif was less evident in the remaining three new target genes ( TRIM26 , KIAA0841 and RFX5 ) . Although these three genes do contain a well conserved X/X2 region , no obvious S element is evident and only two of them ( TRIM26 and RFX5 ) have a Y-like sequence ( Figure 6A ) . This prompted us to determine whether NF-Y actually binds to the new S-Y enhancers . Quantitative ChIP experiments performed with an NF-Y antibody confirmed that the new S-Y enhancers , including two of the imperfect ones , are indeed bound by NF-Y ( Figure S3 ) . The only gene to which binding of NF-Y was detected only very weakly is the one ( KIAA0841 ) lacking a potential Y-like sequence . To determine whether the new S-Y motifs can function as RFX and CIITA dependent enhancers we generated reporter gene constructs in which the S-Y motif of the HLA-DRA promoter was replaced with the S-Y motifs from the new target genes ( Figure 6A ) . The activity of these chimeric constructs , the control HLA-DRA construct and a construct driven by a minimal promoter lacking an S-Y module were assessed in Raji , RJ2 . 2 . 5 and SJO cells ( Figure 6B ) . In Raji , the constructs containing the new S-Y motifs exhibited activities that were at least 20-fold greater than the basal activity of the minimal promoter and attained levels ranging from 25% to 40% of the activity of the HLA-DRA construct . This expression was abolished in RJ2 . 2 . 5 and SJO cells . These results confirm that the new S-Y motifs function as transcriptional enhancers regulated by RFX and CIITA . To confirm that the X box is a critical element of the new S-Y enhancers we performed reporter gene assays with constructs having a mutated X box ( Figure 6C ) . The X box mutation strongly decreased activity of the new S-Y enhancers ( Figure 6C ) . We have recently reported similar results for the new S-Y motif of RAB4B [32] . To confirm that the novel target genes are regulated by CIITA we quantified their endogenous mRNA abundance by quantitative RT-PCR in RJ2 . 2 . 5 cells and in RJ2 . 2 . 5 cells complemented with a CIITA expression vector ( Figure 7A ) . Expression of eight of the new target genes ( RAB4B , TRIM26 , FLJ45422 , KIAA0841 , RFX5 , ZNF672 , MYBPC2 and TPP1 ) was significantly reduced in RJ2 . 2 . 5 . This reduction was not as strong as that observed for classical targets of CIITA , such as HLA-DRA . However , it was similar to or stronger than the reduction observed for HLA-C . The new targets thus resemble MHC-I genes in that their expression is modulated by , but not strictly dependent on , CIITA [7]–[11] . We next performed real-time RT-PCR experiments with melanoma cells to determine whether expression of the new target genes is induced by IFNγ ( Figure 7B ) . mRNA abundance for four of the new target genes ( RAB4B , FLJ45422 , MYBPC2 and RFX5 ) was increased by IFNγ according to a time course similar to that observed for the control HLA-DRA and HLA-C genes . Although induction of the new target genes was not as strong as for HLA-DRA , it was similar in magnitude to the induction of HLA-C . The new targets thus behave like MHC-I genes in that they exhibit a significant level of basal expression prior to IFNγ induced CIITA expression [7]–[12] .
We have used four ChIP-chip approaches to establish a list of genes regulated directly by CIITA in B cells and DC . In addition to MHC-II , MHC-I and Ii genes , only nine new bona fide target genes were discovered . The current number of genes that have been demonstrated to be regulated directly by binding of CIITA does therefore not exceed twenty five . At all of these genes , the recruitment of CIITA is strictly dependent on binding of RFX to an X box sequence situated near the promoter . In most cases , this X box is situated in a characteristic S-Y enhancer . One of the new target genes encodes the RFX5 subunit of RFX , indicating the existence of a positive feedback loop for enhancing CIITA recruitment . Finally , seven of the nine new target genes are known or likely to be implicated in cellular processes contributing to antigen presentation ( see below ) . Taken together , these results show that CIITA is dedicated for the regulation of a remarkably compact and highly specialized gene expression module devoted to antigen presentation . This is consistent with the fact that all phenotypes documented in BLS patients can be attributed to defects in antigen presentation . Furthermore , the finding that CIITA recruitment is strictly dependent on RFX at all validated target genes is in agreement with the observation CIITA deficient patients can not be distinguished phenotypically from RFX-deficient patients . Robust and reproducible binding of CIITA was observed at all previously known and newly validated target genes in nine independent ChIP-chip experiments . No other bona fide target genes were identified by any of the four strategies . Genes at which signals were weaker and less reproducible turned out to be false positives . This suggests that most target genes of CIITA have been identified . It is nevertheless possible that certain target genes have been missed . There could be additional targets in specific cell types . In this respect it may be relevant that the COL1A2 promoter was not found to be occupied by CIITA in B cells or DC ( Figure S4 and Figure S5 ) although earlier ChIP experiments had suggested that CIITA regulates this gene in IFNγ induced cells [22] . It is also possible that certain targets were missed for technical reasons . For example , the relevant CIITA associated sequences could be refractory to PCR amplification , hybridize inefficiently or have been excluded by the NimbleGen array design program . Finally , additional target genes could be controlled by distant CIITA-dependent enhancers that are situated outside of the 5 kb promoter regions present on the NimbleGen arrays . To address the latter possibility we performed ChIP-chip experiments using a high density array of our own design carrying the entire extended human MHC as well as other selected regions of interest , including several of the target genes identified here ( see Materials and Methods ) . ChIP-chip experiments performed with Raji and DC confirmed binding of CIITA to all target genes present on the array but did not - with the exception of known enhancers found in the vicinity of MHC-II genes [36] , [37] - lead to the identification of any novel intergenic binding sites ( Table S2 , Figure S6 ) . This contrasts with the high density of STAT1 , p53 and NF-kB binding sites found by ChIP-chip in chromosome 22 , many of which are situated far from transcription start sites . Intergenic CIITA-binding sites do thus not appear to be frequent , although we can of course not exclude their existence in other regions of the genome . The functions of two of the new target genes are unknown . KIAA0841 encodes a protein containing no characteristic sequence motifs providing clues to its function . ZNF672 encodes a transcription factor belonging to the Kreuppel zinc-finger family [38] . However , the genes and functions that ZNF672 might regulate are unknown . For the remaining seven new target genes there is a known or potential link within antigen presentation by MHC-II or MHC-I molecules . TRIM26 and FLJ45422 are situated with the class I region of the MHC . For RAB4B and RFX5 , a key role in antigen presentation has already been established . RFX5 plays a pivotal role in activating MHC-II expression because it encodes the largest DNA-binding subunit of RFX and is essential for recruiting CIITA to its target genes [30]–[33] . RAB4B encodes an isoform of the small GTPase RAB4 . RAB4 is associated with early and recycling endosomes , and regulates recycling of membranes and proteins from these compartments back to the cell surface [39] . These recycling processes play important roles in various antigen presentation processes , including MHC-II restricted presentation of peptides derived from antigens internalized by receptor-mediated uptake in B cells , cross-presentation of endocytosed antigens by MHC-I molecules in DC and the presentation of intact proteins by DC to the antigen receptors of B cells [40]–[44] . RAB4 has been implicated directly in the MHC-II restricted presentation of antigens internalized by receptor-mediated uptake in B cells [44] . For MYBPC2 , TRIM26 , PSMD3 , TPP1 and FLJ45422 , a role in antigen presentation is suggested by the nature of the protein and/or the cellular processes in which they function . FLJ45422 encodes a protein of unknown function exhibiting similarity to MHC-I molecules . TPP1 encodes a lysosomal protease [45] that could influence the generation of peptides presented by MHC-II molecules . PSMD3 encodes a regulatory subunit of the proteasome , a large protease complex implicated in the generation of peptides presented by MHC-I molecules [46] . MYBPC2 encodes an immunoglobulin superfamily member [47] that can bind to myosin and filamentous actin , and modifies the actin-stimulated ATPase activity of myosin . Although MYBPC2 is expressed abundantly in muscle and is best known for its role in muscle contraction [48] , it is also expressed at lower level in other cell types and its tight regulation by CIITA suggests that it could have additional functions in antigen presenting cells . Importantly , the actin cytoskeleton and/or actin-based myosin motors have been implicated in MHC-II trafficking and receptor-driven antigen presentation in B cells , the formation of immune synapses between antigen presenting cells and T cells , and antigen capture and presentation by MHC-I and MHC-II molecules in DC [49]–[51] . Finally , TRIM26 encodes a member of the tripartite motif ( TRIM ) family of ubiquitin E3 ligases [52] . Members of this family are implicated in diverse biological processes . They promote the ubiquitination of specific substrate proteins , thereby controlling their abundance by proteasome mediated degradation or their activity , intracellular trafficking or subcellular localization by proteasome-independent mechanisms . It is tempting to speculate that TRIM26 may regulate either the generation of specific antigenic peptides by the proteasome , or the abundance , activity or subcellular localization of specific proteins implicated in antigen presentation . The CIITA gene is frequently silenced by epigenetic mechanisms in tumors . It has been proposed that the loss of MHC-II expression and/or a reduction in MHC-I expression resulting from the silencing of CIITA might allow tumors to evade immune surveillance [23] , [53] . Our finding that CIITA is remarkably specific for genes implicated in antigen presentation is consistent with the hypothesis that the association between silencing of CIITA and tumorigenicity reflects a reduction in the antigen presentation capacity of the tumor cells . Although there is a well established link between the loss of MHC-I expression and escape from immune surveillance [54] , it remains unclear how direct MHC-II mediated antigen presentation by tumor cells contributes to anti-tumor responses in vivo . Therefore , an alternative possibility that has to be kept in mind is that defective CIITA expression could represent a selective advantage for tumors because it contributes to the expression of non-MHC genes . In this respect , certain of the new target genes may be relevant . For instance , an altered transcription program due to reduced ZNF672 expression , deregulated vesicular traffic due to reduced RAB4B expression , perturbed intracellular actin-based transport due to reduced MYBPC2 expression , and altered ubiquitin-dependent degradation or regulation of specific proteins due to lower TRIM26 expression , could all contribute to the development of tumors . Two of the new target genes , RAB4B and TPP1 , had been suggested to be regulated by CIITA in earlier studies . Both were among the genes that were found by microarray experiments to be downregulated in CIITA-deficient cells [14] . RAB4B was also singled out by a bioinformatic screen designed to identify genes containing S-Y motifs [32] . In contrast , more than 70 other genes suggested previously to be regulated by CIITA [13]–[22] were not found to be direct targets in our ChIP-chip experiments ( Figure S4 ) . For several of the most interesting candidates we were moreover unable to confirm binding of CIITA to their promoters by classical ChIP experiments ( Figure S5 ) . Finally , in a previous study using CIITA-deficient and CIITA-transgenic mice no direct control of these genes by CIITA could be documented [55] . The influence of CIITA on the expression of these genes is therefore likely to be mediated by indirect mechanisms . For several genes , an indirect mechanism involving sequestration of the general co-activator CBP by CIITA has been proposed [16] , [17] , [21] . Certain of the genes could be regulated by one of the two transcription factors - RFX5 and ZNF672 - shown here to be controlled by CIITA . Finally , there is growing evidence that ubiquitination and regulatory subunits of the proteasome can play key roles in transcriptional regulation [56] . The modulation of TRIM26 and PSMD3 expression by CIITA could thus have indirect impacts on the transcription of certain genes . The remarkably focused role of CIITA emphasized here contrasts with results derived from large-scale binding studies for other transcription factors . Good illustrations are provided by Foxp3 , Stat1 , cMyc and p53 . Over 1000 Foxp3 target genes were identified by scanning for binding sites in the promoter regions of 16'000 mouse genes [1] . Large scale mapping of Stat1 binding sites in chromosome 22 or selected ( ENCODE ) regions of the human genome have pointed to hundreds if not thousands of target genes [2] , [3] . More than 300 cMyc target sites were identified in chromosomes 21 and 22 [4] . Finally , p53 binds to at least 500 target sites in the human genome [5] . Similar large numbers of targets have been reported for all other transcription factors for which large-scale binding studies have been reported . The high degree of specificity observed here for CIITA is thus unprecedented . Why CIITA presents this unique degree of specificity is unknown . One explanation may reside in the finding that CIITA recruitment appears to be strictly dependent on the assembly of a well-defined multifactor enhanceosome complex on a relatively large ( 65–70 bp ) composite regulatory module ( the S-Y motif ) that is tightly constrained with respect to its sequence content and architecture [32] . Such S-Y modules are likely to be much less frequent in the genome than binding sites for individual transcription factors . A second explanation may lie in the fact that CIITA has quite an unusual origin for a nuclear transcription factor . It is the only transcriptional activator belonging to the mammalian nucleotide-binding domain and leucine-rich repeat ( NLR ) containing family , a large group of proteins exerting cytoplasmic functions implicated in cell death , inflammation and innate immunity [57]–[59] . The ability to activate transcription of a specific set of genes in the adaptive immune system may represent a recently evolved specialization acquired by an ancestral NLR protein originally having a completely different cytoplasmic function .
Raji , BLS1 , SJO and RJ2 . 2 . 5 B cells , RJ2 . 2 . 5 cells complemented with an expression vector encoding CIITA isoform III [32] and Me67 . 8 melanoma cells [35] were cultured in RPMI + Glutamax medium complemented with 10% fetal calf serum and antibiotics . Me67 . 8 cells were induced with 200U/ml IFNγ ( Invitrogen ) . Human monocyte-derived DC were generated and matured with LPS as described [24] . ChIP experiments were performed as described using antibodies specific for CIITA and RFX [30] , [31] or NF-Y ( Diagenode ) . Results were quantified by real-time PCR using the primers listed in Table S3 . PCR was performed using the iCycler iQ Real-Time PCR Detection System ( Biorad ) and a Sybr-Green-based kit for quantitative PCR ( iQ Supermix Biorad ) . CIITA-ChIP samples were verified by quantitative PCR to assess the enrichment of HLA-DRA sequences . DNA extracted from the ChIP samples were blunted for 30 minutes at 72°C with 3U of Pfu polymerase ( Promega ) and phosphorylated with T4 Polynucleotide kinase ( New England Biolabs ) . 120 pmoles of adaptors , consisting of annealed oligonucleotides A ( 5′-GCGGTGACCCGGGAGATCTGAATTC-3′ ) and B ( 5′-GAATTCAGATC-3′ ) , were ligated to the DNA by overnight incubation at 16°C with 2000U of T4 DNA ligase ( New England Biolabs ) . Two rounds of PCR amplification with oligonucleotide A were performed using 1 . 25U of Taq polymerase ( New England Biolabs ) and 0 . 025U of PfuTurbo polymerase ( Stratagene ) . The cycle used was: 1 X ( 2′ at 55°C , 5′ at 72°C , 2′ at 95°C ) , 28 X ( 1′ at 95°C , 1′ at 60°C , 2′ at 72°C ) , 5′ at 72°C . 4 µg of each DNA were purified and sent to NimbleGen for probe preparation and hybridization to arrays carrying the 5 kb promoter regions ( approximately −4 kb to +1 kb relative to the transcription start site ) of 27434 human genes , or to a custom array of our own design . The latter carries all unique sequences from the entire extended human MHC ( 7 . 7 Mb on chromosome 6 , genomic coordinates 26 . 1 Mb to 33 . 8 Mb on hg17 ) as well as a number of other selected regions ( total of 0 . 9 Mb ) , including several of the target genes identified here . These genomic regions are covered at high density with overlapping Tm-matched oligonucleotides ( ∼50 bp long ) spaced such that their 5′ ends are situated ∼10 bp apart . Data sets from five independent experiments ( three Raji/RJ2 . 2 . 5 and two iDC/mDC comparisons ) were analyzed with SignalMap software ( NimbleGen ) . Positive peaks in the test/control signal ratios were calculated using a 500 base-pair sliding window and a cut-off that was deliberately set at a very low value ( 5% of maximum ) to minimize the risk of eliminating weak peaks . This generated a large number of potential peaks ( 11262 , 13322 and 15980 in the three Raji/RJ2 . 2 . 5 experiments , 13017 and 11795 in the two iDC/mDC experiments . The following procedure was then used to identify peaks present reproducibly in the five experiments . First , all peaks found in the five experiments were merged into a single list and sorted according to their genomic midpoint coordinates . A sliding window of five consecutive peaks was then used to identify genes exhibiting a peak in each of the five experiments . For these genes , the distance was calculated between the midpoints of the first and fifth peak and the list of these genes was then resorted according to these calculated peak proximities . Since the likelihood that peaks detected in different experiments correspond to the same binding site will increase with increased peak proximity , candidates with the smallest distance values between peaks were considered to be the most likely candidates . Similar algorithms were used to identify peaks present in only four or three of the five experiments . Finally , to avoid the risk of eliminating target genes because of a threshold difference between B cells and DC , all genes exhibiting closely superposable peaks in only the three Raji/RJ2 . 2 . 5 experiments or only the two iDC/mDC experiments were retained as candidates . Peaks for approximately 500 candidate genes identified by this procedure were re-examined visually and assigned a score based on reproducibility , width and strength of the signals . A score of 3 was assigned to genes where strong signals ( log2 ratios exceeding 2 ) were spread over at least 400 base-pairs and were present in at least four experiments . A score of 2 was assigned to genes where strong signals were present in at least three experiments , or when weaker or narrower peaks were present in four or five experiments . A score of 1 was assigned to genes where three experiments exhibited weak signals or when signals were detected in only two experiments . A score of 0 was assigned to genes that did not meet the above criteria . To simplify representation of the results in the Figures , negative test/control signal ratios were set at log2 = 0 . Two alternative approaches were also used to analyze the ChIP-chip data . One was a published method [34] . The second consisted of the following unsupervised approach . Peaks from the three Raji/RJ2 . 2 . 5 and two iDC/mDC experiments were pooled together , sorted by chromosome , midpoint coordinate and experiment , and considered to correspond to the same binding site if their distance was < 750 bp . Peaks that were only positive in 1 or 2 experiments were excluded from the following steps . To identify groups in the remaining peaks , they were represented as 4 bit binary vectors with the first 2 bits representing the number of peaks with significant log2 ratios and the last 2 bits representing the number of positive experiments . Log2 ratios were considered significant if their values were above 0 . 66 , which corresponds to the quantile for a probability of 0 . 95 calculated on the complete set of log2 ratios determined over all experiments and peaks . Since the binary variables are asymmetric , a distance matrix between any pair of peaks represented by the rows of the binary matrix is calculated using the Jaccard distance [60] . This distance measure ranges from 0 ( closely related peaks ) to 1 ( unrelated peaks ) . The resulting distance matrix was then used to determine the number of groups ( partitions ) using the PAM ( Partitioning Around Medoids ) algorithm [61] . Based on the above distance matrix , this algorithm calculates all possible partitions ranging from 2 to n-1 subgroups . For each partition the overall average silhouette width [62] is calculated and the partition that maximizes it is considered optimal . S-Y motifs were amplified by PCR and cloned in the HLA-DRA luciferase plasmid described previously [31] . The X box was altered to AAGCTACCACTCGT by site directed mutagenesis as described [32] . This mutation has a major impact on the activity of known S-Y enhancers . Transfections were done by electroporation . Dual luciferase reporter gene assays were performed according to instructions from the manufacturer ( Promega ) . RNA extraction and cDNA synthesis were done as described [31] . Quantification was done by real-time PCR using the primers listed in Table S4 . Results were normalized using 18S rRNA . Results were confirmed with several primer pairs .
|
Most mammalian transcription factors and transcriptional co-activators are believed to regulate the activities of numerous genes fulfilling multiple functions . This pleiotropic role has recently been confirmed directly for several individual factors by large-scale mapping studies aimed at generating comprehensive catalogues of their binding sites in the genome . Until now , all transcription factors , for which such studies have been performed , were found to regulate hundreds or even thousands of genes . We demonstrate , here , that the transcriptional co-activator CIITA ( class II transactivator ) is an exception to this rule . CIITA is a key regulator of the immune system because it controls the transcription of genes coding for Major Histocompatibility Complex ( MHC ) class II molecules , which are cell-surface molecules that present peptide antigens to T lymphocytes . To address the possibility that CIITA might exert more widespread functions , we have performed extensive genome-wide searches to establish a comprehensive list of CIITA-regulated genes . Surprisingly , we found that CIITA regulates only a small number of genes , most of which code for proteins implicated directly or indirectly in MHC-mediated antigen presentation . CIITA is thus remarkably dedicated for the regulation of a unique set of functionally related genes constituting a genetic module devoted to a single biological process .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"immunology/leukocyte",
"signaling",
"and",
"gene",
"expression",
"genetics",
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"genomics/gene",
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2008
|
Identification of CIITA Regulated Genetic Module Dedicated for Antigen Presentation
|
Dengue is one of the most significant public health problems in tropical and subtropical countries , and is increasingly being detected in traditionally non-endemic areas . In Bhutan , dengue virus ( DENV ) has only recently been detected and limited information is available . In this study , we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013–2014 . During this period , 379 patients were clinically diagnosed with suspected dengue , of whom 119 ( 31 . 4% ) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR . DENV serotypes 1 , 2 and 3 were detected with DENV-1 being predominant . Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V , closely related to strains from northern India .
Dengue is one of the most common infectious diseases in tropical and sub-tropical regions of the world [1 , 2] . The World Health Organization ( WHO ) estimates 50–100 million infections per year globally; however , other studies have suggested a much higher figure [2] . Southeast Asia and Western Pacific represent about 75% of the global dengue burden [3] , causing a substantial economic cost in these regions [1] . Dengue virus ( DENV ) , the etiological agent of dengue , is divided into four genetically and antigenically different serotypes , DENV-1 to 4 [4] . Although infection by a particular serotype is known to confer long-lasting homotypic immunity , circulating heterotypic antibodies are only able to provide transient cross-protective immunity often leading to severe forms of DF , dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) [5] . Antibody dependent enhancement ( ADE ) and cross-reactive T-cell responses have been postulated to explain the possible mechanisms of disease enhancement [5 , 6] . Other factors such as host immunity and viral genetics may contribute to severe forms of dengue fever ( DF ) , dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) [4 , 5] . In recent years , DENV is increasingly being detected in newer geographical areas . Dengue outbreaks are relatively new in Bhutan , a country that shares borders with India to the south and China to the north . The earliest documented dengue outbreak in the country occurred in 2004 andwas caused mainly by DENV-2 and 3 [7] . This was followed by sporadic dengue cases [8] . Although dengue is a reportable disease in Bhutan , it is believed to be inconsistently reported largely because diagnosis is clinically-based with rapid serological assays employed only in a few locations where laboratory diagnostic kits are available [9] . DENV molecular detection , isolation and other advanced testing have not yet been established in Bhutan . In addition , vector control efforts in Bhutan are mostly focused on malarial vector control , which is assumed to cover up for dengue as well . Dengue vector surveillances are in place where vectors were previously detected but no dengue-specific vector control measures have been implemented in the country [10] . Bhutan shares a 700 km border with India , which continues to report co-circulation of all 4 DENV serotypes with increasing frequency [11] . DENVs isolated in 2004–2006 from Bhutan during its first reported dengue outbreak are thought to have originated in India [7] . Similar transmission was reported in Nepal , a country with similar geographical features as Bhutan , gradually leading to endemicity [12] . Due to limited studies done in Bhutan , there is very little information regarding currently circulating DENV serotypes or their molecular and epidemiological characterization . In this study , we undertook laboratory confirmation of clinically suspected dengue patients from the southern part of Bhutan during 2013–2014 and elucidated the molecular epidemiology of DENV-1 in Bhutan .
Samples were collected by the Public Health Laboratory in Bhutan as a part of routine diagnosis and surveillance; hence , no written consent was obtained from patients . The Ministry of Health ( MOH ) , Bhutan , provided written permission for use of de-identified specimens and data for further evaluation . Approval of the study was provided by the Institutional Review Boards ( IRBs ) of Mahidol University , Thailand ( COE . No . 2014/020/ . 1010 ) , and Walter Reed Army Institute of Research ( WRAIR ) , United States ( WRAIR No . 2155 ) . Acute blood specimens were collected over a period of two years , 2013–2014 , from patients clinically suspected of having dengue . These patients had either visited the outpatient department ( OPD ) or were admitted to Samtse or Phuntsholing Hospitals in two southwest districts ( Samtse and Chukha , respectively ) of Bhutan ( Fig 1 ) . These districts are located at the foothills of the Himalayas where climate is sub-tropical . Both Samtse and Chukha share porous borders with India , where commerce and tourism are common . After the first outbreak of dengue in Phuntsholing town , Chukha district [7]; this area has continued to report dengue cases . Samtse district , which has similar climatic and ecological factors , was chosen as a site for this study along with Chukha . Other districts were not included in this study since they have had no reported cases of dengue infection . Both Aedes vectors ( Ae . Aegypti and Ae . Albopictus ) have been found in both Chukha and Samtse districts . These districts also have reasonable access to the Public Health Laboratory in the capital city , Thimphu , where specimens can be shipped for further evaluation Clinically suspected dengue was defined as fever ( oral , rectal or axillary temperature ≥38°C ) , or history of fever lasting 2 to 7 days of unknown origin with two or more of the following: headache , retro-orbital pain , myalgia , arthralgia , rash , hemorrhagic manifestation and leucopenia [13] . Patient demographic and clinical information was collected by attending clinicians . The clinical diagnosis of dengue by attending clinicians was not further categorized as to disease severity . Laboratory confirmation of dengue was carried out by DENV specific NS1 antigen and IgM ELISA , and nested RT-PCR . All acute serum specimens were tested for DENV infection by NS1 antigen detection , IgM ELISA and nested RT-PCR . Both dengue NS1 antigen and dengue IgM detection were carried out at the Public Health Laboratory , Bhutan using DENV Detect NS1 ELISA ( InBios , Seattle , Washington ) and Dengue Virus IgM ELISA ( Calbiotech ) . Tests were performed according to manufacturer’s instructions . Acute serum specimens were also tested by nested RT-PCR at the Armed Forces Research Institute of Medical Sciences ( AFRIMS ) in Bangkok , Thailand . Viral RNA was extracted from 140 μl of sera using QIAamp viral RNA mini kit ( QIAGEN , Germany ) following the manufacturer’s instructions . Nested RT-PCR was performed using a method modified from Lanciotti et al as previously described [14 , 15] . One step RT-PCR was carried out using AMV reverse transcriptase ( Promega , Madison , WI , USA ) and AmpliTaq DNA polymerse ( Life Technologies , USA ) in the first round PCR . Nested PCR was performed using AmpliTaq DNA polymerase in the second round PCR . All sera positive for DENV by nested RT-PCR were inoculated into freshly prepared mono-layers of C6/36 cells grown in Minimum Essential Medium ( MEM , GIBCO ) containing 10% heat inactivated fetal bovine serum ( HIFBS ) , 1% Glutamine and 1% Penicillin and streptomycin . These cultures were maintained in maintenance medium ( MM ) containing RPMI with 5% HIFBS . A mock-infected C6/36 cell flask was included as a negative control . Cells underwent 3 passages and were observed for cytopathic effect ( CPE ) . Identification of DENV serotypes was carried out by antigen capture ELISA as previously described [16 , 17] . Molecular confirmation of the isolates was performed by extracting DENV RNA from the cell culture supernatant followed by nested RT-PCR . When cell-based DENV isolation was not possible , nested RT-PCR positive sera were inoculated into Toxorhynchitis splendens mosquitoes ( 0 . 3μl/ mosquito ) as previously described [18] . Surviving mosquitoes were head squashed on microscopic slides and screened for flavivirus antigen by immunofluorescent antibody ( IFA ) staining . Virus isolates amplified from cell culture or mosquitoes were used in envelope ( E ) gene sequencing . E gene of DENV was amplified using one-step RT-PCR amplification [19] . Overlapping fragments were amplified using AccessQuick RT-PCR System ( Promega , Madison , WI , USA ) with two sets of primers covering the entire E gene . Amplified products were purified prior to sequencing using QIAquick PCR purification kit ( QIAGEN ) following manufacturer’s instructions . Capillary-based Sanger sequencing was used to obtain E gene sequences ( 1 , 485 bp ) . Base correction for the obtained sequences was performed using Sequencher 5 . 1 . All new sequences were submitted to GenBank ( accession numbers KP849860- KP849892 ) . Maximum likelihood ( ML ) tree was constructed from 33 new Bhutan DENV-1 sequences along with sequences of 56 global DENV-1 and 3 vaccine strains downloaded from GenBank . The tree was constructed using MEGA v . 6 . 0 ( www . megasoftware . net ) [20]; the Tamura Nei ( TN93 ) model was chosen for nucleotide analysis . A sylvatic strain from Malaysia ( accession no . AF425622 ) was used as the outgroup . Bootstrap value was obtained from 1000 replicates . Selection pressure among the Bhutan DENV-1 sequences was determined using the maximum likelihood approach of codon based test of selection available in Mega v . 6 . 0 . Percent identity of nucleotides and amino acids was calculated by Clustal W function available in MegAlign v . 5 . 05 of DNASTAR package . Accession numbers for E gene sequenced in this study: KP849860 , KP849861 , KP849862 , KP849863 , KP849864 , KP849865 , KP849866 , KP849867 , KP849868 , KP849869 , KP849870 , KP849871 , KP849872 , KP849873 , KP849874 , KP849875 , KP849876 , KP849877 , KP849878 , KP849879 , KP849880 , KP849881 , KP849882 , KP849883 , KP849884 , KP849885 , KP849886 , KP849887 , KP849888 , KP849889 , KP849890 , KP849891 and KP849892 . SPSS version 22 was used for statistical analysis . Independent T-test was used to compare means of various attributes . Frequencies/ percentage of clinical symptoms were compared using Pearson chi-square test . A probability value of p < 0 . 05 was considered statistically significant .
A total of 379 acute sera from suspected dengue cases were collected at the district hospitals in Samtse and Chukha ( Fig 1 ) during 2013 and 2014 , of which 119 were laboratory confirmed for DENV infection . In both years , the number of suspected and laboratory confirmed cases peaked during the summer months ( June-September ) , which also corresponds to the monsoon season in Bhutan ( Fig 2 ) . Clinically suspected and laboratory confirmed dengue cases during the summer months accounted for 278/379 ( 73% ) and 83/119 ( 69 . 7% ) respectively , for the entire two years . During the colder months ( November to March ) , only 17/379 ( 4 . 5% ) of all suspected dengue and 8/119 ( 6 . 7% ) of laboratory confirmed dengue cases occurred . There seemed to be a remarkable difference in the proportion of laboratory confirmed dengue in 2013 and 2014 . In 2013 , 100/168 ( 59 . 5% ) of suspected cases were confirmed to be dengue by laboratory methods , accounting for 84% of all laboratory confirmed dengue . Only 19/211 ( 9% ) of suspected cases were laboratory confirmed in 2014 . Age of patients in this study ranged from 2 to 77 years but young adults , 19–35 years , accounted for the largest age group of suspected as well as laboratory confirmed cases; 178/379 ( 47% ) and 68/119 ( 55 . 6% ) , respectively . Both genders were about equally affected ( female to male ratio of 1:1 . 03 ) . Mean age and days of illness ( DOI ) after onset of symptoms until blood collection were calculated separately for total suspected and laboratory confirmed cases ( Table 1 ) . We did not observe any differences among the two groups . Of the 379 suspected dengue patients , 364 visited the outpatient department ( OPD ) and the remaining 15 were either admitted or visited the emergency room . Clinical data was collected from all 379 patients . All patients had fever and most had features of DF such as headache , myalgia and joint pain ( Table 2 ) . Although we were unable to obtain complete information regarding the severity and classification of the disease , hemorrhagic manifestations characteristic of DHF were noted in some laboratory-confirmed dengue cases including petechiae , gastrointestinal bleeding ( e . g . , haematemesis and melena ) , and bleeding from the mucosa and/or other sites ( Table 2 ) . A total of 97/379 ( 25 . 6% ) specimens were positive by NS1 ELISA , 29/ 379 ( 7 . 6% ) specimens were positive by IgM ELISA and 58/379 ( 15 . 3% ) positive by nested RT-PCR . Combined , 119/ 379 ( 31 . 4% ) specimens were positive by combination of these methods . The mean DOI was calculated separately for both NS1 and nested RT-PCR positive cases ( Fig 3 ) . Specimens positive for only NS1 had a DOI of 3 . 6 days , which was significantly longer than the DOI from samples positive for only nested RT-PCR ( 2 . 6 days , p<0 . 05 ) . DOI for cases that were both NS1 and RT-PCR positive was 2 . 9 days . The DOI for IgM positive cases was 4 . 4 days . Nested RT-PCR was performed on all sera collected . Of 58 positive cases , 53 ( 91 . 4% ) were DENV-1 , 3 ( 5 . 2% ) DENV-2 and 2 ( 3 . 4% ) DENV-3; no DENV-4 was detected . Using cell culture and/or mosquito amplification , isolation of DENV was attempted on all 58 sera in order to obtain sufficient sequencing material . Unfortunately , we were unable to isolate any of the DENV-2 and DENV-3 viruses . Nevertheless , 33 viruses ( all DENV-1 ) were successfully isolated and sequenced ML tree was generated using 92 E gene sequences ( 1 , 485 bp ) , including the 33 Bhutan DENV-1 strains reported here , 3 DENV-1 vaccine candidate strains and 56 global DENV-1 strains obtained from GenBank ( Fig 4 ) . All 33 Bhutan sequences group to genotype V , using the classification of Weaver et al [21] , and were located in the same group as sequences from northern India , categorized as clade IX by Dash et al [22] . Within clade IX , 32 of the Bhutan DENV-1 E gene sequences ( GenBank accession no . KP849860-7 and KP849869-92 ) grouped with the northern Indian sub-clade from 2008–2009 , while 1 Bhutan DENV-1 sequence ( GenBank accession no . KP849868 ) grouped with the northern Indian sub-clade from 2010–2011 . None of the vaccine candidate strain sequences included in our phylogenetic tree fell within the same group as the Bhutan DENV-1 . Considering the diversity observed among the Bhutan DENV-1 E-gene sequences , we calculated the percentage of nucleotide and amino acid identity between the two observed sub-clades of Bhutanese specimens . We found 99 . 3–99 . 5% nucleotide identity and 99 . 6–99 . 8% amino acid identity . An I461V amino acid substitution was the only mutation found in the KP849868 sequence that differentiated it from the rest of the Bhutan sequences . This mutation was also found in an Indian sequence from 2010 ( GenBank accession no . JN415486 ) , but in no other sequence within the same clade . Selection pressure analysis using maximum likelihood approach showed that amino acids of Bhutan DENV-1 E region are under negative ( purifying ) selection with test statistics ( dS-dN ) = 2 . 7 bootstrapped with 1000 replicates .
This study demonstrated a higher number of cases during the summer season , especially affecting young adults , 19–35 years of age . Clinical presentation of these patients ranged from classical dengue fever to hemorrhagic manifestations . We established DENV-1 as the dominant of at least three DENV serotypes currently circulating in Bhutan . With a shift in predominant serotype from DENV-3 documented previously to DENV-1 , dengue in Bhutan seems to follow a cyclical pattern as seen in other countries [23] . Phylogenetic characterization of DENV-1 revealed that they belong to genotype V [21] , and were probably imported from India . A distinct seasonality for DENV infection in the 2 southwest districts of Bhutan was observed . The number of cases was found to peak during the hot and humid monsoon season , probably due to increased habitats for mosquito breeding . Studies have shown a correlation between rainfall , temperature and humidity with serologically confirmed dengue [24 , 25] . These climatic and ecological factors in combination with inadequate public services and ineffective vector control are known to contribute to dengue endemicity [24] , all of which may be playing a role in Bhutan . The majority of suspected and laboratory confirmed cases occurred in young adults aged 19–35 years . The same was observed in the previous study in Bhutan [7] where the mean age was found to range from 28 to 32 years of age . While the underlying pathogenesis leading to more symptomatic dengue in adults than in children is not clear , dengue as a disease of adults is supported by increasing DF notifications from many ageing countries [26 , 27] . Ineffective vector control efforts have been proposed as a reason for causing shift in the age group from children to adults in countries like Singapore and Thailand [28 , 29] . However; in the case of Bhutan , the majority of cases being adults suggest that there are still many dengue-naïve or dengue monotypic individuals among the adult population in Bhutan . Whether this mean age pattern changes in the future may depend on ongoing and future intensity of DENV transmission . Considering the difference in sensitivity of NS1 ELISA and RT-PCR methods at various stages of illness [30] , using both methods was useful in detecting cases that were positive by just one of the methods . As expected , results by each method varied and seemed to correlate with the number of days of illness after onset of symptoms . Interestingly , the percentage of NS1 ELISA-positive specimens ( 25 . 6% ) was higher than the percentage of RT-PCR positive specimens ( 15 . 3% ) . It is unlikely that this is due to higher detection sensitivity by the NS1-ELISA method . One possible explanation is the preponderance of patients in this study seeking clinical care after several days of illness , favoring the detection of NS1 , which circulates in the serum for longer periods than viral RNA [31] . The mean DOI among patients from whom virus genome could be detected was shorter than patients from whom only NS1 antigen could be detected ( Fig 3 ) . It is also possible that the commercial NS1 ELISA test , done in the laboratories in Bhutan and not confirmed at AFRIMS , provided a number of false positive results , accounting for a somewhat inflated positive percentage A large numbers of specimens were negative for DENV by the laboratory methods used in this study , especially in 2014 . One possible hypothesis could be that non-specific febrile illness , having fever with rash , can be caused by a number of other infectious diseases that may commonly be mistaken for dengue [32] . For example , an outbreak of chikungunya , which commonly manifests as fever and polyarthralgia , was recently reported in the same geographical location in Bhutan [33 , 34] . Clinical manifestations that were observed at significantly higher frequencies in laboratory confirmed dengue as compared to the suspected dengue cases include petechiae and positive tourniquet test , symptoms that are specific to DENV infection . We have yet to confirm the etiology of the dengue-like illnesses in 2014 . Other factors such as time of sampling , loss in viral titer as a result of long storage and transportation with several inevitable freeze- thaw cycles ( caused by Bhutan’s poor road infrastructure and transportation networks ) , could have contributed to lesser DENV isolation and further sequencing in general . The report of the first dengue outbreak in Bhutan suggested that DENV was imported from India , probably as early as 2004 [7] . DENV-3 was the predominant serotype in both Delhi ( northern India ) during 2003–2006 and Bhutan during 2004–2006 [7 , 11 , 22 , 32] . Since 2006 , DENV-1 prevalence has increased in India [22 , 35 , 36] . Similarly , in this study , DENV-1 was predominant in 2013–2014 , further supporting the notion that dengue epidemiological patterns in Bhutan and India are closely linked with likely exchange of strains between the two countries . Phylogenetic analysis of the 33 Bhutan DENV-1 isolates showed that all belong to DENV-1 genotype V , also known as the cosmopolitan or the American-African genotype because of its diversity [21 , 22] . The Bhutan sequences were found to group with clade IX from northern India , the most recent Indian clade [22] . Inclusion of the Bhutan sequences from our study in the phylogenetic analysis resulted in the divergence of clade IX into two different sub-clades . One sub-clade consisted of sequences from 2008–2009 northern India , and the other of sequences from 2010–2011 northern India . Most of the Bhutan sequences grouped with the 2008–2009 northern Indian sequences , indicating that the 2013–2014 dengue viruses from Bhutan mainly originated from 2008–2009 northern India sub-clade viruses . Only one sequence ( GenBank accession no . KP849868 ) grouped with the 2010–2011 northern India sub-clade . This virus was obtained from a specimen collected in August 2013 in Chukha district and was found to be associated with mild clinical symptoms ( no hemorrhagic manifestations ) . It is difficult to ascertain whether the differences in these two sub-clades may have any effect on the severity of the disease since we had only one virus in the 2010–2011 northern India sub-clade . None of the vaccine candidate strains grouped in the same genotype as the Bhutan DENV-1 sequences . It is unknown whether these discrepancies would have any impact on the efficacy of possible future vaccinations in the region . DENV from Bhutan showed a strong negative ( purifying ) selection pressure which is usually observed in arboviruses [37] . This has been attributed to evolutionary constraints that make the viruses resistant to change , especially since their lifecycles include both vertebrate and invertebrate hosts . There were several limitations to our study . Since we used pre-existing specimens and data , the available information was not complete , especially regarding disease severity , high risk occupational groups and certain clinical laboratory data such as blood counts . We also did not have access to convalescent sera and , therefore , could not confirm the non-acute dengue cases . Furthermore , this study was limited to only two hospitals in Bhutan so the data presented here may not necessarily represent the entire country . Despite its relative isolation , Bhutan is seeing increased urbanization and travel , factors that have led to DENV of all serotypes co-circulating in many other countries [2 , 38] . WHO has at least partially attributed outbreaks in Nepal and Bhutan to increasing global temperatures [10] . Although Bhutan is mostly situated at elevations of greater than a thousand meters above sea level , some areas of Chukha and Samtse districts are located as low as 100 meters above sea level , bearing climates that favor transmission of DENV and other tropical pathogens [39] . More importantly , these two districts share an unrestricted border with India which regularly reports dengue outbreaks of all serotypes [11] , contributing to the DENV presence in Bhutan . In this study , we report a shift in predominant DENV serotype in Bhutan in concert with prevailing patterns in neighboring India , along with epidemiological features of dengue in Bhutan . The southern part of Bhutan has likely become dengue endemic , hence enhanced and continuous dengue surveillance is required to generate more robust epidemiological data and to monitor for changes in the characteristics of circulating DENVs .
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We describe the epidemiological and molecular features of DENV currently circulating in the two southwestern districts of Bhutan , demonstrating a shift in serotype dominance from previous DENV-3 ( 2004–2006 ) to current DENV-1 ( 2013–2014 ) . The presence of the dengue virus in Bhutan is a relatively recent one . Unfortunately , dengue epidemiological and molecular data in this country is scarce . A fever outbreak in 2013 and 2014 saw patients seeking care at medical facilities in two district of southwestern Bhutan bordering with India . Analyses of serum specimens collected from these patients indicated that dengue virus was at least a major source of this outbreak . These specimens were analyzed in the Public Health Laboratory in Bhutan and in AFRIMS , Thailand . With a combination of three different assays , we established that 31% of all cases captured were caused by dengue virus , although the proportion was higher in 2013 than in 2014 . Three different serotypes of dengue virus were found: DENV-1 , -2 and -3 . No DENV-4 was found . We successfully isolated DENV-1 , from which was sequenced the E gene for further analyses . Our analyses revealed that the current DENV-1 in Bhutan probably originated from India .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014
|
Recycling of signaling proteins is a common phenomenon in diverse signaling pathways . In photoreceptors of Drosophila , light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential ( TRP ) and TRP-like ( TRPL ) and generates the visual response . The signaling proteins are located in a plasma membrane compartment called rhabdomere . The major rhodopsin ( Rh1 ) and TRP are predominantly localized in the rhabdomere in light and darkness . In contrast , TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light , from where it can be recycled to the plasma membrane upon subsequent dark adaptation . Here , we identified the gene mutated in trpl translocation defective 14 ( ttd14 ) , which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark . TTD14 is highly conserved in invertebrates and binds GTP in vitro . The ttd14 mutation alters a conserved proline residue ( P75L ) in the GTP-binding domain and abolishes binding to GTP . This indicates that GTP binding is essential for TTD14 function . TTD14 is a cytosolic protein and binds to PtdIns ( 3 ) P , a lipid enriched in early endosome membranes , and to phosphatidic acid . In contrast to TRPL , rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant . The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation . In conclusion , TTD14 is a novel regulator of TRPL trafficking , involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane .
Photoreceptor membrane proteins undergo a carefully regulated turnover that helps to adjust the sensitivity of the receptors and to renew old and possibly worn out proteins . Throughout the lifetime of a photoreceptor cell , new proteins are synthesized and transported to the photoreceptive membrane while other proteins are removed from this membrane and are either recycled or degraded in the lysosome . Major integral membrane proteins of the Drosophila photoreceptive membrane comprise the G protein-coupled receptor rhodopsin and two ion channels , transient receptor potential ( TRP ) and TRP-like ( TRPL ) . Defects in rhodopsin turnover can result in degeneration of photoreceptors in humans and flies [1–3] . The signaling cascade operating in fly photoreceptors is a G protein-coupled , phospholipase Cβ-mediated signaling pathway that is initiated by the absorption of a photon by rhodopsin and results in the opening of TRP and TRPL channels and subsequent influx of sodium and calcium ions . TRP and TRPL are the founding members of the large family of transient receptor potential channels that comprises 28 members in mammals [4–7] . TRP channels function in sensory systems as well as in calcium regulation in non-neuronal cells , for example in kidney or heart cells . In Drosophila , newly synthesized rhodopsin of R1-6 photoreceptor cells ( Rh1 ) and the two light-activated ion channels TRP and TRPL are transported via the secretory pathway from the endoplasmic reticulum ( ER ) to the apical plasma membrane that forms a light-sensitive microvillar compartment , termed rhabdomere . Precise folding and successful transport of Rh1 and TRP channels to the rhabdomere are crucial for photoreceptor function . A number of reports have identified proteins required for the anterograde transport of Rh1 and for its endocytosis [8–19] . These include chaperones , Rab GTPases ( Rab1 , Rab6 , Rab11 ) , Rab-interacting proteins , a COPII-interacting phosphatidic acid phospholipase A1 , and myosin V . Among these proteins , the chaperone XPORT , Rab11 , and the Rab11-interacting guanine nucleotide exchange factor Crag were shown to also be required for TRP trafficking , but not for TRPL trafficking [9 , 15 , 18] . Although Rh1 undergoes a turnover that is enhanced in the light most of the Rh1 is detected in the rhabdomeres in both light- and dark-adapted flies . A significant portion of Rh1 that is removed from the rhabdomere in the light ends up in the lysosome and becomes degraded [20–22] . However , the rhabdomeral Rh1 content does not change significantly under physiological light conditions , indicating that degraded Rh1 is replenished by newly synthesized protein . It has recently been reported that a fraction of internalized Rh1 is not degraded but enters a recycling pathway that requires components of the retromer complex [23] . The retromer is a hetreromultimeric protein complex composed of Vps26 , Vps29 , Vps35 , and sorting nexins [24–27] . It is a principle component of the retrograde transport from endosomes to the trans-Golgi network or for recycling of proteins to the plasma membrane for many recycling membrane proteins , including Wntless [28] , the β2 adrenoreceptor [29] , the Drosophila adherens junction protein Crumbs [30] , and vertebrate AMPA receptor subunits [31–33] . Wang et al . [23] showed that Vps26 and Vps35 are required for Rh1 recycling and that mutations in these retromer proteins cause retinal degeneration . The Drosophila ion channel TRPL is a recycling photoreceptor membrane protein that undergoes light-dependent translocation between the rhabdomere , where it is located in dark-adapted flies , and a storage compartment in the cell body , to which it is transported upon illumination within several hours [34] . The removal of TRPL from the rhabdomere depends on activation of the phototransduction cascade and the resulting Ca2+ influx through TRP channels [35] . It has been described as a two-step process in which TRPL first ( within 5–10 minutes ) is transported to the base of the microvilli and adjacent stalk membrane via lateral membrane transport and then ( within several hours ) becomes internalized by a vesicular transport mechanism [36–38] . The majority of internalized TRPL does not enter the lysosomal pathway but is stored in the cell body and recycled back to the rhabdomere when the flies are transferred from light to darkness [34 , 37] . In order to identify components required for TRPL transport , we previously performed a genetic screen for TRPL translocation-defective mutants [39] . This screen used a TRPL-eGFP reporter gene to monitor TRPL localization in intact flies and was based on FRT/FLP-driven mitotic recombination enabling the generation of homozygous mutant eye clones of otherwise lethal genes . In the present study , we mapped the mutant ttd14 and identified the mutated gene . The encoded protein TTD14 is a cytosolic GTP-binding protein that interacts with phospholipids and functions in the internalization and recycling of TRPL during light-triggered TRPL translocation between cell body and rhabdomere . In addition , the ttd14 mutant results in photoreceptor degeneration and larval lethality indicating a vital role of ttd14 in other contexts .
The recessive mutant ttd14 is homozygous lethal during the larval stage and was obtained from a genetic screen of chromosome arm 2R that used ethyl methanesulfonate as a mutagene and was based on FRT/FLP-driven mitotic recombination of the chromosome arm to obtain homozygous mutant eye clones [39] . In the genetic screen , trafficking of the TRPL-eGFP reporter protein was visualized in mosaic eyes by the fluorescence of the deep pseudopupil and the ttd14 mutant was identified by its defective TRPL-eGFP internalization to the cell body after 16 hours orange light illumination . A detailed description of the screen , including crossing schemes , can be found in Ref . [39] . A more detailed analysis using age-matched flies revealed a complex phenotype including defective light-induced TRPL internalization to the storage compartment in young flies , TRPL depletion in the rhabdomere after long-term dark adaptation , and defective redistribution of TRPL to the rhabdomere upon light adaption and subsequent dark adaptation ( Fig 1A ) . To identify the gene affected in the ttd14 mutant , we made use of the lethal phenotype of ttd14 , assuming that lethality in larvae and impaired TRPL trafficking in mosaic eye clones is caused by the same mutation . We performed a mapping approach of the mutated chromosome arm 2R with deficiency strains . A lethal mutation could be mapped between 55C8–55C9 , a region of 30 kilobases containing six candidate genes ( Fig 1B ) . Among these candidate genes , a lethal P-Element mutation in the orphan gene CG30118 ( CG30118KG03769 , see Fig 1C ) failed to complement the lethality of the ttd14 mutant demonstrating that ttd14 is a mutant allele of the CG30118 gene . As shown in Fig 1C , the ttd14 gene encodes three predicted transcripts ( ttd14-A , ttd14-B , and ttd14-C ) . Sequencing of the coding sequence of the ttd14 gene derived from cDNA of wild type fly heads confirmed expression of ttd14-A and -B but not ttd14-C in Drosophila heads . Sequencing of genomic DNA obtained from a heterozygous ttd14 mutant revealed that the mutation present in the ttd14 allele is a C → T transition in the second exon altering the codon for proline75 to a codon for leucine ( P75L ) ( Fig 2C ) . This mutation affects all ttd14 transcripts as P75 is encoded by a common exon . In order to demonstrate that the lethal phenotype and the TRPL transport defect are caused by the same mutation in ttd14 , we generated myc-tagged and untagged rescue constructs driving the expression of ttd14-A or ttd14-B in R1-6 photoreceptor cells under the control of the Rh1 promoter . Upon expression of the constructs in ttd14P75L homozygous mutant eye clones , both ttd14 isoforms rescued the TRPL trafficking defects ( S1A Fig ) . In addition , water immersion microscopy analysis of homozygous mutant eye clones harboring the lethal P-element insertion ttd14KG03769 in the ttd14 gene revealed the same TRPL trafficking defect as was observed in the ttd14P75L mutant ( S1B Fig ) . These results demonstrate that the lethal ttd14P75L mutation causes the TRPL trafficking defect in homozygous mutant eye clones . The protein encoded by the ttd14 gene will hereafter be referred to as TTD14 . No functional data about TTD14 are available so far . Protein domain prediction revealed a P-loop-containing nucleoside triphosphate hydrolase domain ( P-loop ) and a CYTH-like domain ( Fig 2A ) . The predicted P-loop of TTD14 contains a bona fide GxxxxGKT Walker A motif ( amino acids 73–80 ) and a possible hhhhDxG Walker B motif at position 152–158 ( canonical Walker B motif: hhhhDxxG , where h is a hydrophobic and x is any amino acid; [40–42] ) . The mutated amino acid P75 is located in the Walker A motif and is highly conserved in the TTD14 homologs of other invertebrate species . CYTH domains were identified in bacterial adenylyl cyclases and mammalian thiamine triphosphatases [43] . Although highly conserved orthologs of TTD14 are present in other invertebrates such as bees or C . elegans ( Fig 2B ) , BLAST sequence similarity searches did not reveal full-length homologs in vertebrates with significant sequence similarity to TTD14 . Hydrophobicity analysis of TTD14 suggested that the protein is a soluble or a peripheral membrane protein since no sufficiently hydrophobic regions for putative transmembrane domains were predicted ( Fig 3A ) . In order to determine the subcellular localization of TTD14 experimentally , we performed biochemical fractionation experiments and immunocytochemistry . We generated a polyclonal antibody against a recombinantly-expressed full length TTD14-A protein . To test the specificity of the generated antibody , an immunoblot experiment was carried out using protein extracts of dissected Drosophila eyes of wild type flies , of flies overexpressing TTD14-A in photoreceptor cells under the control of the Rh1 promoter , and of flies having homozygous mutant ttd14 eye clones ( comprising 80–90% of the eye ) ( Fig 3B ) . The anti-TTD14 antibody detected two protein bands with an apparent molecular weight of 55 kDa and 130 kDa , respectively , in wild type eyes . These bands were reduced or undetectable in ttd14P75L and ttd14KG03769 mosaic eyes , respectively , and greatly enhanced in eyes overexpressing TTD14-A . Although it cannot be excluded that residual TTD14 protein detected in eyes with ttd14P75L mutant eye clones results from the eye parts that are heterozygous for ttd14P75L , the loss of TTD14 in these eyes seems to be less severe than in ttd14KG03769 mosaic eyes . Therefore , the P75L point mutation may less severely affect the stability of the TTD14 protein but rather impair protein function . Since the calculated molecular weight is 54 , 3 kDa for TTD14-A and 53 , 5 kDa for TTD14-B , we assume that the 55 kDa and 130 kDa bands detected by the antibody represent monomers and dimers of TTD14-A and -B isoforms . For fractionation experiments , Drosophila heads were homogenized in Tris/NaCl-buffer and soluble and membrane proteins were separated by ultracentrifugation ( Fig 3C ) . Using immunoblot analysis , the TTD14 protein was detected exclusively in the soluble fraction . Although the antibody specifically detected TTD14 on immunoblots , it did not yield specific signals when used in immunocytochemistry . Therefore , we used flies expressing a myc-tagged TTD14 ( TTD14-myc ) in photoreceptor cells under the control of the Rh1 promoter and anti-myc antibodies for immunocytochemistry on cross sections through eyes of 24 hours dark- or 16 hours orange light-adapted flies . These experiments revealed a rather uniform signal in the cell body but no signal in the rhabdomeres ( Fig 3D ) . No obvious differences in localization of TTD14-myc between photoreceptor cells of light- or dark-adapted flies were observed . Co-localization of TRPL and TTD14-myc on cross sections through eyes showed that there is indeed a considerable overlap in TRPL and TTD14-myc staining in eyes of light-adapted flies but not in eyes of dark-adapted flies where TRPL is localized in the rhabdomeres ( Fig 3D ) . It has to be noted , however , that in eyes of light-adapted flies , TTD14-myc staining does not completely overlap with TRPL staining , suggesting that the TTD14 protein is not specifically enriched in membrane compartments occupied by TRPL . In order to test the predicted nucleotide binding activity of the TTD14 protein , we performed an in vitro nucleotide binding assay . Purified recombinant full-length TTD14-A protein was incubated with agarose beads coupled to ATP , GTP , or no nucleotide ( control ) . After removal of unbound protein , TTD14 was eluted from the beads with either 25 mM ATP or 25 mM GTP . TTD14 protein was recovered from GTP beads but not from ATP or control beads indicating that TTD14 is a GTP-binding protein ( Fig 3E ) . In order to test an effect of the P75L mutation present in the ttd14P75L allele on GTP-binding , we generated a plasmid for recombinant expression of TTD14 protein harboring the P75L amino acid substitution . This amino acid substitution did not affect protein stability as the mutated protein could be purified in an amount comparable to the wild type TTD14 protein ( Fig 3E ) . However , the P75L mutation abolished GTP binding of TTD14 in the in vitro nucleotide binding assay ( Fig 3E ) indicating that the predicted Walker A motif ( amino acids 73–80 ) is likely to be involved in GTP binding . In addition , the mutant phenotype of the ttd14P75L allele might be attributed to the loss of GTP binding activity , suggesting that GTP binding is essential for the biological function of the TTD14 protein . As differentially-distributed phosphoinositides play a crucial role in membrane trafficking by recruiting trafficking proteins to the membrane [44] , we tested a possible interaction of TTD14 with membrane lipids . In a lipid binding assay we employed nitrocellulose strips spotted with 100 pmol of various phospholipids ( PIP-Strips ) . The PIP-Strips were incubated with purified recombinant full length TTD14-A protein and bound protein was detected with the anti-TTD14 antibody . As a result , among the 15 lipids tested , TTD14 bound to 3-phosphoinositide ( PtdIns ( 3 ) P ) and to phosphatidic acid ( PA ) ( Fig 3F ) . PtdIns ( 3 ) P is predominantly localized at the cytosolic side of membranes of early endosomes [44] , suggesting that TTD14 interacts with early endosome membranes . Collectively , our results reveal that TTD14 is a soluble , most likely cytosolic protein expressed in the Drosophila eye that binds GTP and interacts with PtdIns ( 3 ) P and PA . For a detailed analysis of the TRPL trafficking defect in ttd14P75L , flies expressing TRPL-eGFP in R1-6 photoreceptor cells were subjected to water immersion microscopy of intact eyes and the TRPL-eGFP fluorescence in rhabdomeres was determined as in [35] ( Fig 4A ) . As the generated Rh1>ttd14-A and Rh1>ttd14-B rescue constructs are associated with a white+-marker and result in an orange eye color that interferes with the quantification of water immersion images , a yellow+-marked Rh1>ttd14-A-myc construct bearing a C-terminal myc-tag was expressed in white-eyed flies and used for this quantitative analysis . Qualitatively , rescue of the ttd14P75L phenotype by untagged ttd14 constructs is shown in S1A Fig . In addition , the phenotype in homozygous ttd14KG03769 mutant eye clones , that have red eye color , is depicted in S1B Fig and compared to red-eyed wild type flies . In wild type flies ( Fig 4B green bars ) , long-term dark incubation for up to 7 days only slightly reduced the rhabdomeral TRPL-eGFP fluorescence . In contrast , after 16 hours illumination with orange light , rhabdomeral fluorescence was reduced by 78% in 1 day old flies , indicating that most of TRPL-eGFP had translocated to the cell body . Light-triggered TRPL translocation was less efficient in flies that were previously kept in darkness for 3 days or 7 days but still resulted in a reduction of the rhabdomeral TRPL-eGFP fluorescence by more than 55% . Subsequent dark adaptation for 24 hours fully restored the original rhabdomeral TRPL-eGFP fluorescence irrespective of the duration of the initial dark incubation . In ttd14ttd14 mutant eye clones ( Fig 4B , red bars ) of 1 day old flies , TRPL-eGFP was properly located in the rhabdomere in the dark , but hardly translocated to the cell body upon illumination with orange light for 16 hours . With increasing time of dark incubation , TRPL-eGFP fluorescence progressively disappeared from the rhabdomere resulting in a rhabdomeral TRPL-eGFP fluorescence of only 40% in 7 day old flies . Orange light illumination in these flies did not further reduce the rhabdomeral TRPL-eGFP content . Of primary significance here , subsequent dark adaptation for 24 hours did not affect TRPL-eGFP distribution in 3 day or 7 day old flies , indicating that recycling of TRPL-eGFP to the rhabdomere was severely impaired . This latter phenotype is less prominent in young flies , as the initial rhabdomeral TRPL content before dark adaptation is already elevated . In conclusion , mutation of the ttd14 gene did not affect the initial localization of TRPL-eGFP in the rhabdomere but resulted in reduced transport of TRPL-eGFP from the rhabdomere to the cell body in young flies . Upon prolonged incubation of flies in the dark , net transport of TRPL-eGFP from the cell body to the rhabdomere was reduced as most of TRPL-eGFP was located in the cell body and failed to recycle to the rhabdomere irrespective of the light condition . All aspects of the mutant phenotype described above were rescued by expression of ttd14 wild type constructs ( Fig 4B , blue bars and S1A Fig ) in R1-6 photoreceptor cells under control of the Rh1 promoter showing that TTD14 function is required in photoreceptor cells . In order to reveal the subcellular localization of TRPL with higher resolution , we performed immunocytochemistry of 7 day old wild type , ttd14P75L , and rescue flies ( Fig 4C ) . Like in the water immersion experiments , the flies were first kept in constant darkness , then illuminated with orange light for 16 hours and then again kept in darkness for 24 hours . As observed previously [34 , 37] , wild type flies revealed TRPL labeling in the rhabdomeres when kept in darkness and a relatively uniform labeling of the cell bodies ( except for nuclei ) when kept in light . The same labeling pattern was observed in ttd14 mutant eye clones of flies expressing the rescue construct Rh1>ttd14-myc . Without the rescue construct , ttd14 mutant eye clones revealed a TRPL labeling pattern in the cell body in all light and dark conditions tested that was indistinguishable from that of wild type flies kept in the light . We previously reported a localization defect of a mutated TRPL , in which eight C-terminal phosphorylation sites were abolished [45] . However , the mislocalization of phosphorylation-deficient TRPL resulted in labeling of distinct spots in the cell body quite different from the uniform TRPL labeling pattern observed in the ttd14 mutant ( S2 Fig and see Ref . 45 ) . In addition to the localization defect , removal of TRPL phosphorylation sites affected TRPL stability in the dark and resulted in progressive TRPL degradation [45] . To assay TRPL stability in the ttd14P75L mutant , we carried out immunoblot analyses with protein extracts obtained from 1 day and 7 day old wild type flies and from the ttd14P75L mutant subjected to different light conditions ( Fig 4D and 4E ) . A lower amount of TRPL in 1 day old flies , which were assayed immediately ( that is before further incubation in light or darkness , Fig 4D , lanes 1 and 7 ) , was observed in both wild type and mutant , suggesting that the TRPL content in freshly eclosed flies is lower than in older flies . No indication of TRPL degradation in the ttd14P75L mutant flies was observed . Indeed , except for flies freshly eclosed in the dark ( Fig 4E , first and 7th column ) , ttd14P75L mutant flies exhibited significantly ( p < 0 . 05 ) higher TRPL protein levels as compared to the corresponding condition in wild type flies . Thus , TRPL in the ttd14P75L mutant is not targeted to the lysosomal degradation pathway . It rather seems that the ttd14 mutation affects the recycling of TRPL which accumulates in the internal storage compartment . While TRPL becomes internalized from the rhabdomere in the light and is recycled back to the rhabdomere in the dark , two other important Drosophila photoreceptor membrane proteins , Rh1 and TRP , do not seem to undergo such a regulated change in their subcellular localization . Rh1 is constantly renewed in illuminated photoreceptor cells as the internalized Rh1 becomes partially degraded in the lysosome and rhabdomeral Rh1 is replenished by newly synthesized protein [20] . In addition , Rh1 is also partially recycled via the retromer complex [23] . However , both mechanisms do not result in a major change of the subcellular localization , as most of the Rh1 protein remains localized in the rhabdomere . Albeit less well studied , there is no evidence for a light-dependent translocation of the TRP protein . Indeed , a number of mutations in chaperones and transport proteins have been described that affect the anterograde transport of both Rh1 and TRP , but not TRPL suggesting a common transport pathway for these two photoreceptor proteins and a different pathway for TRPL ( see introduction ) . Assuming that loss of functional TTD14 does not induce a general cytological defect but a specific defect in the internalization and recycling of membrane proteins such as TRPL , one might expect that the ttd14 mutation does not affect trafficking of Rh1 and TRP . To test this assumption , we carried out water immersion microscopy with flies expressing eGFP-tagged Rh1 and TRP in R1-6 photoreceptor cells and also performed immunocytochemical experiments ( Fig 5 ) . While we had observed that TRPL-eGFP disappears from the rhabdomeres of ttd14 mutants upon dark incubation for 7 days ( see Fig 4 ) , no age-related changes in the rhabdomeral content of Rh1-eGFP or TRP-eGFP were observed in water immersion microscopy for up to 28 days in darkness . The fluorescence pattern of Rh1-eGFP or TRP-eGFP expressed in ttd14P75L mutant eye clones was the same as in wild type ( Fig 5A ) . Likewise , immunocytochemistry of cross sections through ommatidia of 7 day old flies kept in the dark , revealed no signs of mislocalization of Rh1-eGFP or native TRP that were both confined to the rhabdomeres in wild type flies as well as in ttd14P75L mutant eye clones ( Fig 5B ) . In addition , we assayed the Rh1 and TRP protein content of 1 day and 7 day old ttd14P75L mutant flies , which were kept in darkness and then subjected to orange light illumination for 16 hours , by immunoblot analysis and observed the same protein content as in wild type flies ( S3 Fig , Fig 4 ) . Taken together , these results strongly suggest that the transport of Rh1 or TRP to the rhabdomere is not affected in the ttd14P75L mutant . Internalization of Rh1 can be assessed by immunoblot analysis when flies are illuminated with bright white light for at least 16 hours . In wild type flies , this illumination results in a reduced Rh1 level due to lysosomal degradation and the original Rh1 level is recovered after a subsequent incubation in darkness for 6 hours [23] . In mutants with defective Rh1 internalization , reduced lysosomal degradation would be expected . In contrast , in mutants affecting components required for Rh1 recycling like the retromer complex , enhanced Rh1 degradation results in a stronger reduction of the Rh1 level as compared to the wild type [23] . In illuminated ttd14P75L mutant flies , the Rh1 level was reduced to a similar degree as in wild type flies ( Fig 5C and 5D ) arguing against a major role of the TTD14 protein in Rh1 trafficking . Finally , in order to show that the ttd14 mutant does not affect the function of Rh1 and TRP , we performed electroretinogram ( ERG ) recordings of wild type and ttd14P75L mutant flies kept in darkness for 7 d . At that time , TRPL was present in the rhabdomeres of wild type flies but not in the rhabdomeres of ttd14P75L mutant flies ( Fig 4A–4C ) . A loss of rhabdomeral TRPL is not expected to have a major impact on the ERG , as the loss of TRPL in the trpl302 null mutant , has an effect on light adaptation , but no impact on the shape of ERG recordings [46] . In contrast , impaired Rh1 or TRP function causes characteristic changes in ERG recordings ( Fig 6A ) . A severe reduction in the amount of Rh1 , which can be achieved by feeding flies with a vitamin A-deprived diet ( see below ) , resulted in a loss of the prolonged depolarization afterpotential ( PDA ) after bright blue light illumination while a complete loss of Rh1 , like in the ninaE17 mutant , was readily indicated by a dramatic reduction of the ERG amplitude ( Fig 6A ) . Loss of TRP function , like in the trpP343 mutant , can be detected by the characteristic transient response to a light stimulus ( Fig 6A ) . Using a stimulus protocol containing both orange and blue light illumination , we did not observe any obvious differences in ERG recordings from ttd14P75L mutant eye clones as compared to wild type ( Fig 6A ) . We conclude that besides TRPL , neither Rh1 nor TRP nor any other major component of the phototransduction cascade is severely affected in ttd14P75L mutant flies . In order to asses long-term effects of the ttd14P75L mutation on photoreceptor function , we carried out ERG recordings of wild type flies , ttd14P75L mutant flies , and flies expressing the rescue construct Rh1>ttd14-A-myc in a ttd14P75L mutant background ( Rescue ) for up to 21 d . In a 12 hours light / 12 hours dark cycle , ttd14P75L mutant flies exhibited a decline in ERG amplitude after 7 days and an almost abolished photoresponse after 14 days ( Fig 6B and 6C ) . After 21 days in constant darkness , ttd14P75L mutant flies also displayed a reduction in the ERG amplitude , albeit to a much lower extent than in a 12 hours light / 12 hours dark cycle ( Fig 6B and 6C ) . In mutants of the retromer complex , photoreceptor degeneration that results in a loss of photoreceptor function is based on the mislocalization of Rh1 and can be attenuated by a reduction of the Rh1 level [23] . The Rh1 level can effectively be reduced by raising the flies on a vitamin A-deprived diet as the opsin protein becomes degraded in the absence of its chromophore ( Fig 6D ) . In contrast to mutants of the retromer complex , diet-induced reduction of the Rh1 level had no significant effect on the ERG amplitude in response to an orange light stimulus in wild type or ttd14P75L mutant flies and did not rescue the declined ERG observed in ttd14P75L mutant flies kept in a 12 hours light / 12 hours dark cycle for 21 days ( Fig 6E ) . In line with our results showing that the ttd14P75L mutation does not affect Rh1 localization ( see Fig 5 ) , this finding suggests that aberrant Rh1 localization is not causal for the loss of photoreceptor performance in this mutant . In order to assess if the decline of the ERG amplitude was associated with morphological alterations in the rhabdomeres , we assessed rhabdomeral structure in wild type and ttd14P75L mutant eye clones for up to 21 days in a 12 hours light / 12 hours dark cycle by transmission electron microscopy and by monitoring TRP-eGFP fluorescence ( Fig 7 ) . While no obvious changes could be detected in wild type eyes , electron microscopy revealed severe degeneration of photoreceptor cells in ttd14P75L mutant flies kept in a 12 hours light / 12 hours dark cycle for 21 days . Most rhabdomeres of photoreceptor cells R1-6 and also the rhabdomere of R7 were absent in these flies . Degeneration was much less pronounced when the ttd14P75L mutant was kept in the dark for 21 days . In these flies the R7 cell was affected frequently while almost all rhabdomeres of R1-6 cells remained intact . These findings show that degeneration of photoreceptor cells in the ttd14P75L mutant is enhanced by light and affects inner and outer photoreceptor cells . Of note , no signs of degeneration of inner or outer photoreceptor cells were detected in ttd14P75L mutant flies kept in the dark for 7 days followed by 16 h orange light , a condition in which TRPL failed to recycle back to the rhabdomere . This finding suggests that the TRPL trafficking defect in the ttd14P75L mutant is not a result of photoreceptor cell degeneration . As observed before Vitamin A deprivation resulted in diminished rhabdomere size [23] . However , degeneration was also observed in vitamin A-deprived ttd14P75L mutant flies showing that a reduction of the rhodopsin content cannot rescue the degeneration phenotype . Using water immersion microscopy with ttd14P75L mutant eye clones that express TRP-eGFP as a rhabdomeral marker we analyzed the time course for the loss of rhabdomeres both under regular and vitamin A-deprived conditions ( Fig 7B and S4 Fig ) . In flies kept in a light-dark cycle first signs of degeneration were detected after seven days while almost all rhabdomeres were lost after 21 days ( Fig 7B ) . Vitamin A-deprivation had a small but not statistically significant effect on the degeneration time course and slightly slowed down the speed of degeneration in the ttd14P75L mutant . Taken together , loss of functional TTD14 in the ttd14P75L mutant resulted in late onset light-dependent , but Rh1-independent retinal degeneration .
The mutant ttd14P75L was initially identified by the internalization defect of TRPL-eGFP after illumination . However , TRPL internalization is not completely abolished in ttd14P75L mutant flies suggesting that the TTD14 protein is not crucial for TRPL internalization but rather acts as a modulator that facilitates TRPL internalization . Alternatively , TTD14 might play an indirect role in TRPL internalization and promote TRPL internalization by enabling recycling of a rhabdomeral protein that is required for TRPL internalization . The most prominent phenotype of the ttd14P75L mutant is the failure of TRPL trafficking from the storage compartment back to the rhabdomere during dark adaptation . This phenotype is not evident in young flies , in which TRPL is localized in the rhabdomeres , suggesting that delivery of newly synthesized TRPL to the rhabdomere via the secretory pathway is not affected by the ttd14P75L mutation . However , when TRPL is redistributed from the rhabdomere to the storage compartment in illuminated 3 day or 7 day old ttd14P75L mutant flies , subsequent dark adaptation for 24 hours does not result in redistribution of TRPL to the rhabdomere ( see Fig 4 ) . This finding indicates that internalized TRPL fails to become recycled to the plasma membrane in ttd14P75L . Continuous dark adaptation of the ttd14P75L mutant for 7 days also results in localization of TRPL in the cell body . This finding indicates that there is a basal level of TRPL internalization in the dark . In wild type flies , TRPL that becomes internalized in the dark is readily recycled back to the rhabdomere resulting in little if any TRPL in the cell body in the dark . In ttd14P75L , where TRPL recycling is blocked , prolonged dark adaptation depletes TRPL from the rhabdomere and results in TRPL accumulation in the cell body . We had previously observed that mutation of C-terminal phosphorylation sites of TRPL also results in depletion of mutated TRPL from the rhabdomere when the flies are kept in darkness for five days [45] . This finding again argues for a basal rate of TRPL internalization in the dark . The phosphorylation-deficient TRPL , however , does not accumulate in the cell body but becomes degraded . Therefore , this mutation might shift the balance between recycling and lysosomal degradation of TRPL towards degradation rather than hindering TRPL entry into the recycling pathway . The proposed cellular trafficking pathways of TRPL in light and darkness and the steps that are presumably disturbed in the ttd14 mutant are illustrated in Fig 8 . Our biochemical analysis revealed that TTD14 is a soluble protein that binds GTP and the phospholipids PtdIns ( 3 ) P and phosphatidic acid ( see Fig 3 ) . Protein domain searches revealed a P-loop nucleotide binding domain comprising amino acids 65–167 of TTD14 . This domain contains a bona fide Walker A and a possible Walker B motif and thus represents a likely site for binding of GTP or ATP . When compared with vertebrate proteins the domain exhibited 37% amino acid identity with the respective domain of mitochondrial GTPase Era ( S1 Table ) . Binding assays with recombinant TTD14 revealed binding to GTP but not to ATP . The P75L mutation of ttd14P75L affects a conserved proline in the Walker A motif and abolished binding of TTD14 to GTP . This finding indicates that the P-loop domain indeed is the GTP binding site . It also suggests that GTP binding is important for TTD14 function as mutation of the site results in the observed ttd14 phenotype . A role of GTP binding proteins in the context of protein trafficking is well established . Important examples are the families of small GTPases , including Rho proteins that regulate actin organization , Rab proteins that regulate docking and fusion of vesicles at different organelles of the endocytic and secretory pathways , and Arf proteins that are involved in protein transport from Golgi to endoplasmic reticulum or at the trans-Golgi network [50] . Other examples for GTP binding proteins in this context are the microtubule forming subunits α and β tubulin and dynamin , which is required to pinch off clathrin-coated vesicles from membranes [51 , 52] . Except for dynamin , GTP hydrolysis by these proteins is not used to generate force but rather to induce conformational changes in the GTP binding proteins that regulate protein function . Besides to GTP , TTD14 also bound to the phospholipids PtdIns ( 3 ) P and phosphatidic acid ( PA ) . Phosphoinositides that differ in the phosphorylation state of the inositol ring are distributed differentially in cellular compartments [44] . Accordingly , trafficking proteins that bind specific phosphoinositides are recruited to distinct membrane compartments [44] . PtdIns ( 3 ) P is located mainly in the early endosome [44] . Therefore , binding to PtdIns ( 3 ) P may recruit TTD14 to the early endosome in order to perform its function in TRPL recycling . PA is an intermediate in the biosynthesis of phosphoinositides and other phospholipids . In Drosophila photoreceptor cells , PA is generated during phototransduction that generates diacylglycerol ( DAG ) by phospholipase Cβ-mediated hydrolysis of phosphatidylinositol 4 , 5-bisphosphate . DAG in turn is translocated to the submicrovillar cisternae located at the base of the rhabdomere where it is phosphorylated to PA by the rdgA-encoded DAG kinase as a first step in the regeneration of rhabdomeral phosphatidylinositol 4 , 5-bisphosphate [53 , 54] . Therefore , upon illumination , TTD14 could be recruited to the base of the rhabdomere via binding to PA , where it could assist light-triggered internalization of TRPL . However , an obvious light-dependent redistribution of myc-tagged TTD14 was not observed in immunocytochemical experiments ( see Fig 3D ) . It has also been suggested that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis in Drosophila photoreceptors as elevated PA levels in Drosophila mutants caused defects in rhabdomere biogenesis [55] . However , defective biogenesis of rhabdomeres during development was not observed in ttd14 mutants and TRPL was properly transported to the rhabdomeres in young flies , arguing against a role of TTD14 in apical membrane trafficking events required for rhabdomere biogenesis . As protein domain prediction did not reveal any lipid binding domain in the TTD14 protein , we performed a BLAST search with low stringency which revealed regions within TTD14 that showed amino acid sequence identities to vertebrate proteins between 30% and 37% . Homologous regions in vertebrate proteins that appeared in more than one species in the BLAST screen and contained specific protein domains within the homologous region included the Phox homologous- ( PX ) domain within vertebrate sorting nexin-8 homologs . This domain displayed sequence similarity with amino acids 254–320 of TTD14 ( S1 Table ) . PX-domains were first identified in NADPH oxidase subunits , sorting nexins , and PtdIns ( 3 ) P-kinases [56] . These domains bind to phosphoinositides and sorting nexins are typically recruited to the early endosome membrane by binding to PtdIns ( 3 ) P via their PX-domain [57 , 58] . Besides in the PX-domain there is little amino acid sequence conservation among the various sorting nexins and the PX-domain is located at varying positions . Sorting nexins are involved in the recycling of internalized proteins . They can be part of retromer complexes that sort internalized proteins in early or late endosomes away from the lysosomal degradation pathway and enable their trafficking back to the trans-Golgi network or to the plasma membrane [24 , 59] . This function would be in accordance with a function of TTD14 in TRPL recycling . Yet , TTD14 is not an ortholog of a known sorting nexin as there is no significant sequence similarity over the whole protein length to any of the described 33 vertebrate sorting nexins . Given the weak sequence conservation between different classes of sorting nexins and the functional analogy of TTD14 with sorting nexins , it is tempting to speculate that TTD14 represents a new class of sorting nexin , composed of a GTP-binding domain and a putative PX-domain that is present in Drosophila and other invertebrates but not in vertebrates . The ttd14P75L mutant displays a light-dependent late onset photoreceptor degeneration as ERG amplitudes diminished and rhabdomere structure became distorted when flies were kept in a 12 hours light / 12 hours dark cycle for more than 7 days ( see Figs 6B , 6C and 7 and S4 Fig ) . While defects in the phototransduction cascade that result in constitutively open TRP channels , for example in rdgA or trpP365 mutants , lead to fast photoreceptor degeneration within days after eclosion [54 , 60 , 61] light-dependent late onset photoreceptor degeneration of Drosophila photoreceptor cells can result from defective Rh1 transport [2 , 62] . Accordingly , mutations in proteins required to transport Rh1 to the rhabdomere like XPORT , Rab11 or Crag cause photoreceptor degeneration [9 , 18 , 63] . Importantly , mutations in Vps26 or Vps35 , components of the retromer complex , also cause light-dependent late onset photoreceptor degeneration [23] . This has been attributed to a failure in Rh1 recycling , which then accumulates in late endosomes or lysosomes and exerts a toxic effect on photoreceptor cells possibly due to an overload of the endolysosomal system [23] . Since ttd14 mutants display a defect in TRPL recycling we wondered if they also have a defect in Rh1 recycling that would then cause photoreceptor degeneration . In contrast to TRPL , in flies dark-adapted for 28 d , Rh1 ( and also TRP ) were properly located in the rhabdomere as revealed by water immersion microscopy ( Fig 5A ) . In addition , in the ttd14P75L mutant , over night illumination of flies with white light under conditions , which markedly reduced Rh1 levels in the Vps261 mutant [23] , resulted in the same Rh1 levels as in wild type . Finally , while a reduction of the Rh1 content by genetic means or by vitamin A deprivation rescued photoreceptor cells from degeneration in the Vps261 mutant [23] , Vitamin A deprivation did not rescue degeneration in the ttd14P75L mutant kept under a 12 hours light /12 hours dark cycle ( see Figs 6E and 7 and S4 Fig ) . Thus , the defective ttd14 allele does not seem to affect Rh1 recycling or trafficking of Rh1 to the rhabdomere , and photoreceptor degeneration caused by ttd14P75L cannot be attributed to defective Rh1 trafficking . These findings indicate that TTD14 is specifically required for TRPL recycling but not Rh1 recycling , either because TRPL utilizes a different recycling pathway than Rh1 or because TTD14 specifically recruits TRPL to a common recycling pathway . What then might be the reason for photoreceptor degeneration in ttd14P75L flies ? It can be excluded that the lack of TRPL in the rhabdomeres , observed in older ttd14P75L flies irrespective of the light condition , underlies photoreceptor degeneration as photoreceptors of the trpl302 null mutant do not degenerate [46] . Rather than by Rh1 accumulation , degeneration of photoreceptor cells in ttd14P75L could be caused by accumulation of TRPL in the endocytic pathway . However , the amount of TRPL in photoreceptor cells is much smaller than the amount of Rh1 , and dark-kept ttd14P75L flies , in which TRPL was found to be accumulated in the cell body , showed only little photoreceptor degeneration . Since a homozygous ttd14P75L mutant is lethal during the larval stage while all known trpl mutants are viable , it is likely that other membrane proteins require TTD14 for their recycling . Therefore , other proteins requiring TTD14 for recycling might be involved in photoreceptor degeneration . These proteins remain to be identified .
The following strains of Drosophila were used: yw , w1118 ( here referred to as wild type ) , Oregon R ( wild type with red eyes ) , ninaE17 [64] , trp343 [65] , yw;;P[Rh1>TRPL-eGFP , y+] [35] , yw;P[Rh1>Rh1-eGFP , w+] [66] , yw;;P[Rh1>TRP-eGFP , y+] [67] , yw , ey>flp; FRT42D , ttd14P75L/CyO , yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO [39] , yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO; P[Rh1>TRPL-eGFP , y+] , yw , P[Rh1>ttd14-A-myc , y+] , yw;;att86Fb[Rh1>ttd14-A , w+] , yw;;att86Fb[Rh1>ttd14-B , w+] , yw , ey>flp; FRT42D , ttd14KG03769[w+]/CyO , yw , ey>flp; P[Rh1>Rh1-eGFP , w+] , FRT42D , ttd14P75L/CyO , yw; FRT42D , ttd14P75L/CyO , P[ActGFP , w+]JMR1 , yw; FRT42D , ttd14KG03769[w+]/CyO , P[ActGFP , w+]JMR1 . Flies were raised on standard cornmeal food at 25°C unless indicated otherwise . Flies were either kept in the dark or in a 12 hours light / 12 hours dark cycle using white light illumination ( 1300 lux ) . For vitamin A deprivation , flies were raised and kept on food containing 10% dry yeast , 10% sucrose , 2% agar , and 0 , 02% cholesterol . For the analysis of TRPL ion channel translocation , flies were kept in the dark for the indicated period and were then illuminated with orange light ( acrylic glass cut off filter transmitting light >560 nm , ~200 lux ) for 16 hours . Light-raised flies were dissected under white light whereas dark-raised flies were dissected under dim red light ( Schott RG 630 , cold light source KL1500 , Schott ) . For the analysis of the amount of Rh1 protein flies were illuminated with white light ( 1800 lux ) . To map the mutation in ttd14 , yw , ey>flp , P[Rh1>TRPL-eGFP , y+]; FRT42D , ttd14P75L/CyO mutant flies were crossed to the Bloomington 2R Deficiency Kit and the offspring was screened for lethality and TRPL translocation . In addition , the deficiency stocks Df ( 2R ) ED3610 ( 54F1-55C8 ) , Df ( 2R ) BSC483 ( 55A1-55B7 ) , Df ( 2R ) 334 ( 55B2-55C4 ) , Df ( 2R ) 3636 ( 55B8-55E3 ) , Df ( 2R ) Excel7153 ( 55B9-55C1 ) , Df ( 2R ) BSC337 ( 55B11-55C9 ) , Df ( 2R ) ED3683 ( 55C2-56C4 ) , Df ( 2R ) BSC335 ( 55C6-55F1 ) , Df ( 2R ) BSC399 ( 55D1-55E10 ) , Df ( 2R ) BSC339 ( 55E2-55F6 ) , and Df ( 2R ) Excel7158 ( 55E9-55F6 ) were used to refine the region of the lethal ttd14P75L mutation . For the analysis of TRPL-eGFP localization in the eye of ttd14P75L mutant Drosophila , mosaic eyes were generated by crossing yw , ey>flp; FRT42D , ttd14P75L/CyO females with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO; P[Rh1>TRPL-eGFP , y+] males . Female offspring with mosaic eyes ( yw/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ; P[Rh1>TRPL-eGFP , y+]/+ ) was analyzed for the localization of TRPL-eGFP using the deep pseudopupil and water immersion microscopy . For wild type flies , yw females were crossed with yw;;P[Rh1>TRPL-eGFP , y+] males . Female offspring of the genotype yw;;P[Rh1>TRPL-eGFP , y+]/+ was analyzed . To analyze the rescue construct ( Rh1>ttd14-A-myc ) in ttd14P75L mutant mosaic clones , we crossed females expressing the rescue construct on the X-chromosome ( yw , P[Rh1>ttd14-A-myc , y+] ) with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO; P[Rh1>TRPL-eGFP , y+] males . F-1 males without the CyO balancer ( yw , P[Rh1>ttd14-A-myc , y+]; FRT42D , w+ , 2R11 . 5 ( lth ) /+; P[Rh1>TRPL-eGFP , y+]/+ ) were then crossed with yw , ey>flp; FRT42D , ttd14P75L/CyO females . In the F-2 generation , females carrying mosaic eyes were selected for the presence of the TRPL-eGFP reporter by fluorescence microscopy using a Leica MZ16 F stereomicroscope prior to water immersion microscopy . Analyzed flies had the following genotype: yw , P[Rh1>ttd14-A-myc , y+]/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ; P[Rh1>TRPL-eGFP , y+]/+ . For the analysis of the rescue by the non-tagged Rh1>ttd14-A and Rh1>ttd14-B construct , the transgenic males yw;;att86Fb[Rh1>ttd14-A , w+] and yw;;att86Fb[Rh1>ttd14-B , w+] were crossed to yw , ey>flp; FRT42D , ttd14P75L/CyO females . The heterozygous rescue construct can be traced by the orange eye color . F-1 males with orange colored eyes and without the CyO balancer ( yw , ey>flp; FRT42D , ttd14P75L/+; att86Fb[Rh1>ttd14-A or B , w+]/+ ) were then crossed with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO; P[Rh1>TRPL-eGFP , y+] females . In the F-2 generation , females carrying mosaic eyes composed of orange and red clones ( yw/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ; P[Rh1>TRPL-eGFP , y+]/att86Fb [Rh1>ttd14-A or B , w+] ) were analyzed . For the analysis of the TRPL localization in the ttd14KG03769[w+] allele , this mutant allele was recombined with the FRT42D locus . Next , yw , ey>flp; FRT42D , ttd14KG03769[w+]/CyO males were crossed with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO; P[Rh1>TRPL-eGFP , y+] females . In the F-1 generation , females lacking the CyO balancer ( yw/yw , ey>flp; FRT42D , ttd14KG03769[w+]/FRT42D , w+ , 2R11 . 5 ( lth ) ; P[Rh1>TRPL-eGFP , y+]/+ ) were analyzed . For the wild type control yw;;P[Rh1>TRPL-eGFP , y+] flies were crossed with Oregon R flies and F-1 females ( yw/+;; P[Rh1>TRPL-eGFP , y+]/+ ) were analyzed . For the analysis of TRP-eGFP localization , flies carrying the TRP-eGFP reporter ( yw;;P[Rh1>TRP-eGFP , y+] ) were crossed with yw , ey>flp; FRT42D , ttd14P75L/CyO females . F-1 males lacking the CyO balancer ( yw , ey>flp; FRT42D , ttd14P75L/+; P[Rh1>TRP-eGFP , y+]/+ ) were then crossed to yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO females . In the F-2 generation female flies with mosaic eyes were selected for the presence of the TRP-eGFP reporter using a Leica MZ16 F stereomicroscope prior to water immersion microscopy . Analyzed flies had the following genotype: yw/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ; P[Rh1>TRP-eGFP , y+]/+ . For the wild type control , yw females were crossed to yw;;P[Rh1>TRP-eGFP , y+] males and F-1 females ( yw;;P[Rh1>TRP-eGFP , y+]/+ ) were subjected to water immersion microscopy . For the analysis of Rh1-eGFP localization in water immersion microscopy and immunocytochemistry , the P[Rh1>Rh1-eGFP , w+] construct was recombined to the FRT42D , ttd14P75L chromosome resulting in the fly stock yw , ey>flp; P[Rh1>Rh1-eGFP , w+] , FRT42D , ttd14P75L/CyO . Flies from this stock were crossed with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO flies and F-1 females with mosaic eyes were analyzed ( yw/yw , ey>flp; P[Rh1>Rh1-eGFP , w+] , FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ) . The w+ marker of the heterozygous Rh1>Rh1-eGFP construct results in a faint orange eye color while flies homozygous for the Rh1>Rh1-eGFP construct exhibit dark orange colored eyes . As the F-1 females with mosaic eye clones display a faint orange eye color , we assume that the Rh1>Rh1-eGFP construct is present with one copy in this genetic background . We conclude that the Rh1>Rh1-eGFP construct is localized on the left arm of the second chromosome which is not affected by FRT-mediated mitotic recombination . Therefore , for the wild type control , yw females were crossed to yw;P[Rh1>Rh1-eGFP , w+] males and F-1 females ( yw;P[Rh1>Rh1-eGFP , w+]/+ ) were subjected to water immersion microscopy . For immunoblot , immunocytochemistry and ERG experiments , yw , ey>flp; FRT42D , ttd14P75L/CyO flies were crossed with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO flies and female F-1 flies with mosaic eyes ( yw/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ) were used for analysis . w1118 was used as wild type control . To analyze the rescue construct ( Rh1>ttd14-A-myc ) in ttd14P75Lmutant mosaic clones , we crossed females expressing the rescue construct on the X-chromosome ( yw , P[Rh1>ttd14-A-myc , y+] ) with yw; FRT42D , w+ , 2R11 . 5 ( lth ) /CyO males . F-1 males without the CyO balancer ( yw , P[Rh1>ttd14-A-myc , y+]; FRT42D , w+ , 2R11 . 5 ( lth ) /+ ) were then crossed with yw , ey>flp; FRT42D , ttd14P75L/CyO females . In the F-2 generation , females carrying mosaic eyes ( yw , P[Rh1>ttd14-A-myc , y+]/yw , ey>flp; FRT42D , ttd14P75L/FRT42D , w+ , 2R11 . 5 ( lth ) ) were analyzed . For the analysis of the subcellular localization of the TTD14 protein by immunocytochemistry yw , P[Rh1>ttd14-A-myc , y+] flies were crossed with yw;;P[Rh1>TRPL-eGFP , y+] flies and female F-1 flies , carrying both reporter constructs ( yw , P[Rh1>ttd14-A-myc , y+]/+;;P[Rh1>TRPL-eGFP , y+]/+ ) were analyzed . To address the larval lethality of mutant alleles of the ttd14 gene , the mutant alleles were crossed with a GFP-labeled CyO-Balancer ( yw; FRT42D , ttd14P75L/CyO , P[ActGFP , w+]JMR and yw; FRT42D , ttd14KG03769[w+]/CyO , P[ActGFP , w+]JMR1 respectively ) and development of non-fluorescent homozygous mutant F-1 larvae was traced . For the generation of the Rh1>ttd14-A and Rh1>ttd14-B rescue constructs , the entire coding sequence , but not the 5´ and 3´ untranslated region of ttd14-A and ttd14-B was PCR amplified from cDNA derived from fly heads . Restriction sites were introduced in the primer pair 5´-TTCCCGAATTCGAAGACATG-3 ( EcoRI ) and 5´-GAGTCAACATAATCGATAGCCA-3´ ( ClaI ) for the amplification of ttd14-A and 5´-TTCCCGAATTCGAAGACATG-3´ ( EcoRI ) and 5´-GTCCCTGAATCGATTTTGCACAC-3´ ( ClaI ) for the amplification of ttd14-B . PCR fragments were cloned into a modified pBluescript II SK vector ( Stratagene ) between the Rh1 minimal promoter ( base pairs -833 to +67 ) and the last 0 . 6 kb of the Rh1 3´ untranslated region using the EcoRI and ClaI restriction sites . After a KpnI site in the Rh1 promoter was eliminated with a site directed mutagenesis Kit ( Agilent ) using the mutagenesis primer 5´-CAGAATCCAGGAACCCTGAGTACCGGATCC-3´ , the Rh1>ttd14-A ( B ) construct was excised from the pBluescript vector using a NotI/KpnI digest and cloned into the pattB vector [68] . The final pattB Rh1>ttd14-A and -B clones were verified by DNA sequencing ( Qiagen ) . Transgenic flies were generated using site specific recombination by injecting the pattB Rh1>ttd14-A/B vector ( 200 ng/μl ) into y1 M{vas-int . Dm}ZH-2A w*; M{3xP3-RFP . attP}ZH-86Fb embryos . For the generation of the Rh1>ttd14-A-myc rescue construct , a PCR amplified ttd14-A construct encoding amino acids 1 to 472 of TTD14-A and a C-terminal myc-tag obtained from the vector pSF-CMV-COOH-EKT-CMyc2 ( Oxford Genetics ) was cloned in the pENTR 1A vector ( Invitrogen ) . Using the Gateway system ( Invitrogen ) , the ttd14-A-myc sequence was recombined with a modified pYC4 vector containing a DEST cassette between the Rh1 minimal promoter ( base pairs -833 to +67 ) and the last 0 . 6 kb of the 3´untranslated region of Rh1 [67] . The final pYC4 Rh1>ttd14-A-myc clone was verified by DNA sequencing ( GATC Biotech ) . The construct was injected into yw embryos . Full length ttd14-A was PCR amplified from cDNA derived from fly heads and cloned in the pQE30 vector ( Qiagen ) , which encodes a N-terminal His-tag , using BamHI and SalI sites . For generating a construct encoding the mutated TTD14[P75L] protein , the respective codon was mutated using the Quick Change Lightning Site-Directed Mutagenesis Kit ( Agilent ) as described in the instruction manual . The resulting pQE30 ttd14-A and pQE30 ttd14-A[P75L] clones were verified by DNA sequencing ( Qiagen ) and transformed in E . coli M15 cells ( Qiagen ) . For the purification of native TTD14 or TTD14[P75L] protein , expression of the recombinant protein was induced by 1 mM IPTG for 2 hours at 30°C . The pellet of 1 liter bacteria culture was lysed in 20 ml lysis buffer ( 50 mM Tris , pH 8 . 0 , 300 mM NaCl , 10 mM Imidazol , 1% Triton X-100 , 2 mM DTT , 50 μM APMSF ) using a French press ( OneShot; Constant Systems ) and incubated with 500 μl Ni-NTA agarose ( Qiagen ) for 60 min at 4°C . After three wash steps in 20 ml wash buffer each ( 50 mM Tris/HCl , pH 8 . 0 , 300 mM NaCl , 50 mM Imidazol , 50 μM APMSF ) , the TTD14 protein was eluted from the Ni-NTA agarose with 400 μl elution buffer ( 50 mM Tris , pH 8 . 0 , 300 mM NaCl , 250 mM Imidazol , 50 μM APMSF ) . For the purification of TTD14 protein under denaturating conditions , expression of the recombinant protein was induced by 1 mM IPTG for 2 hours at 37°C . The pellet of a 300 ml bacteria culture was homogenized in 10 ml lysis buffer ( 8 M Urea , 100 mM Na2HPO4 , /NaH2PO4 , pH 8 . 0 , 10 mM Tris/HCl pH 8 . 0 , 20 mM β-mercaptoethanol , 10 mM Imidazol ) and incubated with 2 ml of Ni-NTA agarose ( Qiagen ) for 30 min at room temperature . After three wash steps in 20 ml wash buffer each ( 8 M Urea , 100 mM Na2HPO4 , /NaH2PO4 , pH 6 . 3 , 10 mM Tris/HCl pH 6 . 3 , 10 mM Imidazol ) , the TTD14 protein was eluted with 1 ml elution buffer ( 50 mM Tris/HCl , pH 7 . 5 , 300 mM NaCl , 250 mM Imidazol ) . 1 μg of TTD14 protein purified under denaturating conditions was used to immunize two rabbits for 61 days ( Pineda Germany ) . IgG were purified from the serum using a HiTrap Protein A HP-column ( GE Healthcare ) according to the manufacturer’s instructions . PIP-Strips ( Echelon Research Laboratories ) which contain various phospholipids at distinct spots ( 100 pmol ) were blocked in TBS-T ( 10 mM Tris/HCl pH 8 . 0; 150 mM NaCl , 0 . 1% Tween-20 ) containing 3% bovine serum albumin ( BSA ) . The membrane was incubated with recombinant TTD14 protein purified under native conditions ( 0 . 4 μg/ml ) in TBS-T plus 3% BSA for 90 min at 4°C . After three wash steps for 5 min each in TBS-T , the membrane was incubated with the α-TTD14 antibody in TBS-T plus 3% BSA for 90 min at 4°C , washed again three times for 5 min each in TBS-T and finally incubated with horseradish peroxidase-coupled anti-rabbit IgG ( 1:10 , 000 Sigma ) in TBS-T plus 3% BSA for 90 min at 4°C . Lipid-bound protein was detected by enhanced chemiluminescence ( 0 . 091 M Tris-HCl pH 8 . 6; 0 . 0227% ( w/v ) luminol; 0 . 01% ( w/v ) para-hydroxycoumarin acid 0 . 01% H2O2 ) using a ChemiDocXRS+ Imaging system ( Bio-Rad ) . 20 μg of recombinant wild type TTD14 or TTD14[P75L] protein was diluted to 225 μl with binding buffer ( 20 mM Tris-HCl pH 7 . 0; 150 mM NaCl; 5 mM MgCl2; 0 . 1% ( v/v ) Triton X-100 ) and incubated with agitation ( 1000 rpm ) at 4°C for 1 hour with 25 μl of either control agarose ( Pierce ) , ATP-agarose ( Sigma Aldrich ) or GTP-agarose ( Sigma Aldrich ) pre-equilibrated in binding buffer . Beads were centrifuged at 4°C at 13 , 000 g for 2 min and washed three times with 1 ml ice-chilled binding buffer . Proteins were eluted by adding 50 μl of binding buffer containing either 25 mM ATP or 25 mM GTP for 5 min on ice . After centrifugation ( 2 min at 13 , 000g at 4°C ) 15 μl of the resulting supernatant was subjected to immunoblot analysis . For immunoblot analyses of TRPL , TRP , Rh1 , and Tubulin in wild type and ttd14P75L mutant flies ( Figs 4D , 5C and 6D and S3 Fig ) , fly heads were homogenized in SDS extraction buffer ( 4% SDS , 1 mM EDTA , 75 mM Tris/HCl , pH 6 . 8 ) using 4 μl of extraction buffer per head and incubated for 1 hour at room temperature . After 10 min centrifugation at 16 , 000 g to remove debris the supernatant was subjected to SDS-PAGE . For immunoblot analysis of TTD14 protein in wild type and ttd14 mutant flies ( Fig 3B ) , eye cups were dissected and homogenized in Tris-buffer ( 50 mM Tris/HCl pH 8 . 0; 150 mM NaCl; 50 μM APMSF; 1 μl per eye ) and extraction was carried out for 30 min on ice . The supernatant obtained after 10 min centrifugation at 16 , 000 g was subjected to SDS-PAGE . For immunoblot analysis of membrane and soluble proteins ( Fig 3C ) , fly heads were homogenized in Tris-buffer ( 50 mM Tris/HCl pH 8 , 0; 150 mM NaCl; 50 μM APMSF; 2 μl per head ) and extraction was carried out for 30 min at room temperature . A 3 min centrifugation step at 2 , 500 g was applied to remove cuticular particles of the fly head . The supernatant was subjected to ultracentrifugation ( 10 min at 100 , 000 g , 4°C ) and the resulting supernatant was loaded as soluble fraction on a SDS-gel . The membrane pellet was washed three times in Tris-buffer and solubilized in SDS extraction buffer ( 4% SDS , 1 mM EDTA , 75 mM Tris/HCl , pH 6 . 8; 1 μl per head ) for 20 min at room temperature and loaded as membrane fraction on a SDS-gel . SDS-PAGE was performed according to Laemmli [69] using 10% or 12% polyacrylamide gels . Separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes ( Bio-Rad ) . The membrane was then blocked for 20 min in TBS-T with 5% skim milk ( 10 mM Tris/HCl , pH 7 . 5 , 150 mM NaCl , 0 . 1% Tween 20 , 5% skim milk ) . α-TRPL [34] , α-TRP ( Mab83F6; Developmental Studies Hybridoma Bank , University of Iowa ) , α-Rh1 ( 4C5; Developmental Studies Hybridoma Bank ) , α-tubulin ( E7; Developmental Studies Hybridoma Bank , University of Iowa ) and α-TTD14 antibodies were used for immunological detection in TBS-T with 5% skim milk over night at 4°C . Signals were detected by enhanced chemiluminescence ( ( 0 . 091 M Tris-HCl pH 8 . 6; 0 . 0227% ( w/v ) luminol; 0 . 01% ( w/v ) para-hydroxycoumarin acid; 0 . 01% H2O2 ) using the ChemiDocXRS+ Imaging system ( Bio-Rad ) . Quantification of immunoblot signals was performed with Image Lab 4 . 0 ( Bio-Rad ) by determining the integrated density of each protein band . The Rh1 signals were normalized by the TRP signals of the same sample . Fluorescence in the deep pseudopupil was observed in CO2-anaesthetized flies using a Leica MZ16 F stereomicroscope with 63x magnification and the GFP3 filter set . Images were captured with a Leica DFC420 C camera . Water immersion microscopy of eGFP-tagged proteins in intact eyes was performed as previously described [35] . Living flies were anaesthetized on ice , spiked on an insect needle , mounted with plasticine on an object slide and covered with ice-chilled distilled water . The eGFP fluorescence was observed with an AxioImager . Z1m microscope ( Zeiss , Germany; objective: Achroplan 20X/0 . 5 water immersion ) . Images were captured with the AxioCamMrM ( Zeiss ) camera and the Axio-Vision 4 . 6/4 . 8 or Zen 2012 ( Zeiss ) software . For images of flies with white and orange colored eyes , exposure time was determined individually for every single eye to be closely below overexposure . For the analysis of the red colored wild type and ttd14KG03769 eyes , an exposure time resulting in images closely below overexposure in 1 day old dark-adapted wild type flies was determined and applied for all other experimental conditions . For quantitative analyses of the relative TRPL-eGFP fluorescence in the rhabdomeres , fluorescence images obtained with the water immersion technique were analyzed with ImageJ 1 . 42j software ( National Institute of Health , USA ) . The relative amount of TRPL-eGFP present in the rhabdomeres ( R ) was calculated using the formula R = ( Ir–Ib ) /[ ( Ir–Ib ) + ( Ic–Ib ) ] , where Ir , Ib , and Ic are the fluorescence intensities in the rhabdomeres , in the background , and in the cell body , respectively . Background intensities were determined in the center of the ommatidium where the rhabdomere of the R7/R8 cells is located . For each eye , three ommatidia were analyzed and five individual flies were analyzed per data point . The data were normalized to the value obtained for 1 day old dark raised wild type flies that was set to 100% . For determining the time course of rhabdomere degeneration flies expressing TRP-eGFP in R1-6 photoreceptors were inspected for the presence of R1-6 rhabdomeres using water immersion microscopy . Clearly visible rhabdomeres were scored 2 , weakly visible rhabdomeres were scored 1 and absent rhabdomeres were scored 0 . Three ommatidia per eye were scored , resulting in a degeneration score of 36 for fully intact eyes or in a score of 0 for fully degenerated eyes . The maximal degeneration score of 36 was set to 100% . Five individual flies were analyzed per data point . For immunocytochemical analyses , Drosophila eyes were fixed in 2% paraformaldehyde ( PFA ) in PBS ( 175 mM NaCl , 8 mM Na2HPO4 , and 1 . 8 mM NaH2PO4 , pH 7 . 2 ) for 1 hour at room temperature , and then washed twice in 0 . 1 M phosphate buffer ( 0 . 1 M Na2HPO4 and 0 . 1 M NaH2PO4 , pH 7 . 2 ) . Subsequently , three wash steps in 10% sucrose and two wash steps in 25% sucrose were performed for 15 min each . Eyes were finally infiltrated with 50% sucrose overnight at 4°C , cryofixed in melting pentane , and sectioned at 10 μm thickness in a Leica CM3050S cryostat ( Leica , Germany ) at −25°C . Cryosections were first fixed in 2% PFA in PBS for 10 min and then washed twice in PBS . After blocking of sections in PBS-T ( 1% BSA , 0 . 3% Triton X-100 in PBS ) for 2 hours at room temperature , sections were incubated with α-TRPL [35] , α-TRP ( Mab83F6; Developmental Studies Hybridoma Bank of the University of Iowa ) and α-myc ( Sigma ) in PBS-T overnight at 4°C . The sections were subsequently washed three times in PBS and were then incubated either with α-mouse-Alexa Fluor 488 and α-rabbit-Alexa Fluor 680 or with α-mouse-Alexa Fluor 680 and α-rabbit-Alexa Fluor 488 ( Life Technololgies ) in 0 . 5% fish gelatine and 0 . 1% ovalbumin in PBS for at least 4 hours at room temperature . Phalloidin-Alexa Fluor 546 ( Life Technologies ) was added to stain F-actin in rhabdomeres . The eGFP-tagged Rh1 protein was visualized by its own fluorescence . The sections were finally mounted in Mowiol 4 . 88 ( Polyscience ) and examined with an AxioImager . Z1m microscope ( objective: EC Plan-Neofluar 40×/1 . 3 Oil , Zeiss , Germany ) using the ApoTome module ( Zeiss , Germany ) at room temperature . Images were captured with the AxioCamMrM ( Zeiss ) camera and the Axio-Vision 4 . 6/4 . 8 or Zen 2012 ( Zeiss ) software . For electroretinogram recordings , flies were immobilized in a pipette tip and mounted with a mixture of colophonium and bee’s wax ( 1:3 ) . Electroretinogram recordings were performed at room temperature after 3 minutes of dark adaptation prior to the first orange light-stimulus . Light-stimuli of 5 s duration were delivered by an orange light-emitting diode ( Roithner , Austria ) and a blue light-emitting diode ( Roithner , Austria ) in a setup of two collimating lenses ( Linos , Germany ) within the light path . The light intensity at the position of the fly eye was 2 . 15 mW/cm2 for orange light and 1 . 3 mW/cm2 for blue light . A DPA-2FS amplifier ( NPI electronic , Germany ) with a low pass filter ( 700 Hz ) was used for signal amplification . Analog-to-digital conversion was accomplished with a BNC-2090A rack-mounted terminal block ( National Instruments , Germany ) and a PCI-6221 PC card ( National Instruments , Germany ) . Data recording was achieved by the Whole Cell Analysis Program software WinWCP 4 . 7 . 6 . ( University of Strathclyde ) . The recording electrode glass capillary was filled with Davenport solution ( 100 mM NaCl , 2 mM KCl , 1 mM CaCl2 , 1 . 8 mM NaHCO3 , pH 7 . 2 ) . Fly heads were separated from the body , dissected into two halves and incubated in fixative solution ( 4% paraformaldehyde , 2 . 5% glutaraldehyde in 1 x PBS , pH 7 . 4 ) for 1 h at room temperature . The semi-heads were washed 3 times with 0 . 1 M sodium cacodylate buffer ( pH 7 . 4 ) for 10 min and postfixed in 2% OsO4 in 0 . 1 M cacodylate buffer ( pH 7 . 4 ) for 1 h . After 3 washes in 0 . 1 M sodium cacodylate buffer for 10 min the semi-heads were dehydrated through a graded series of ethanol from 30% to 100% . The dehydrated semi-heads were incubated in 100% propylene oxide twice for 10 min each , and then transferred to 50% propylene oxide: 50% Renlam M-1 resin ( Serva Electrophoresis , Heidelberg , Germany ) and incubated overnight . After that the semi-heads were incubated in 100% Renlam® M-1 resin overnight , embedded in 100% Renlam® M-1 resin and polymerized at 60°C for two days . Ultrathin sections ( 60–70 nm ) were obtained using a Reichert Ultracut E microtome ( Leica ) . Sections were counterstained with heavy metal staining ( 2% uranyl acetate in 50% ethanol; aq . 2% lead citrate ) . Ultrathin sections were analyzed in a Tecnai 12 BioTwin transmission electron microscope ( FEI , Eindhoven , The Netherlands . Images were obtained with a charge-coupled device SIS Megaview3 SCCD camera ( Surface Imaging Systems , Herzogenrath , Germany ) . Image contrast was adjusted with Adobe Photoshop CS using different tools .
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Protein trafficking in neurons occurs throughout the lifetime of a cell and includes the internalization and redistribution of plasma membrane proteins . Regulated protein trafficking controls the equipment of the plasma membrane with receptors and ion channels and thereby attenuates or enhances neuronal function . Defects in recycling of plasma membrane proteins can cause detrimental neurodegenerative diseases such as Alzheimer’s disease , Parkinson’s disease and Down´s syndrome . In Drosophila photoreceptors , the TRPL ion channel , together with the TRP channel , mediates vision and light-dependently shuttles between an endomembrane storage compartment and the apical plasma membrane . Here , we report the identification of a mutation in the ttd14 gene that inhibits TRPL-trafficking in both directions and also results in photoreceptor degeneration . The TTD14 protein contains a region with weak homology to a PX-domain , which is also found in proteins that sort cargo in the endosome and enable protein recycling . We characterize TTD14 as a new regulator of photoreceptor maintenance and ion channel trafficking that binds to GTP and PtdIns ( 3 ) P , a phospholipid enriched in early endosomes .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
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The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells
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Plasmodium vivax transmission in Thailand has been substantially reduced over the past 10 years , yet it remains highly endemic along international borders . Understanding the genetic relationship of residual parasite populations can help track the origins of the parasites that are reintroduced into malaria-free regions within the country . A total of 127 P . vivax isolates were genotyped from two western provinces ( Tak and Kanchanaburi ) and one eastern province ( Ubon Ratchathani ) of Thailand using 10 microsatellite markers . Genetic diversity was high , but recent clonal expansion was detected in all three provinces . Substantial population structure and genetic differentiation of parasites among provinces suggest limited gene flow among these sites . There was no haplotype sharing among the three sites , and a reduced panel of four microsatellite markers was sufficient to assign the parasites to their provincial origins . Significant parasite genetic differentiation between provinces shows successful interruption of parasite spread within Thailand , but high diversity along international borders implies a substantial parasite population size in these regions . The provincial origin of P . vivax cases can be reliably determined by genotyping four microsatellite markers , which should be useful for monitoring parasite reintroduction after malaria elimination .
Although the global incidence of malaria has been greatly reduced in recent years , Plasmodium vivax remains the most geographically widespread human malaria parasite [1] . In Thailand , intensified malaria control has resulted in ~50% reduction of clinical cases over the past 10 years . In parallel , the distribution of malaria cases in Thailand has become more heterogeneous . The central region is virtually malaria free , whereas malaria remains endemic along the western border with Myanmar , the northeastern border with Laos , the eastern border with Cambodia , and to a lesser extent , the southern border with Malaysia [2] . As a result , malaria along these four international borders now accounts for 95% ( 56 , 17 , 13 and 9% ) of the total malaria cases in Thailand [3] . An increasing proportion of the remaining infections is caused by P . vivax , which is now the source of 63% of all clinical cases [3] . In 2015 , P . vivax annual parasite incidence was 0 . 37 per 1000 . Given the successful interruption of transmission in other parts of the country , information on diversity and genetic relationship of parasites from the remaining transmission hotspots can help target control programs to track the sources of remaining infections and to direct control programs towards remaining transmission hotspots . This is particularly relevant for P . vivax , since the dormant liver hypnozoites can relapse months or even years after the initial infection in seemingly parasite-free migrants . Genotyping of microsatellite ( MS ) markers [4 , 5] , polymorphic antigens [6] , mitochondrial DNA [7 , 8] , and genome-wide single nucleotide polymorphisms ( SNPs ) [9] has revealed high levels of diversity among P . vivax populations on both global and local scales . Whole-genome sequencing of >400 isolates has confirmed high P . vivax diversity and population structure following continental origins of the isolates [10] , as well as a clear separation of parasite populations between western Thailand and Cambodia [11] . Earlier studies on P . vivax diversity in Thailand in the 1990s and early 2000s , when transmission levels were significantly higher , found high diversity of several antigens and population genetic differentiation between regions [12 , 13] . More recently , genotyping of different polymorphic markers further confirmed the high diversity in most regions [14–16] . However , a recent study with the msp3β gene revealed contrasting genetic structure of the P . vivax populations between the southern and northwestern border regions , with the northwestern parasite population showing very high genetic diversity as compared to southern population with the same msp3β allele [17] . To further characterize the population genetic structure and relatedness of P . vivax populations in Thailand after the scaling-up of malaria control in recent years , we have genotyped P . vivax parasite samples from three residual malaria transmission foci along the western and eastern borders .
A total of 127 P . vivax isolates from two western border provinces and one eastern border province of Thailand ( Fig 1 ) were genotyped using 10 MS markers [18] . Complete genotyping data for all 10 loci was obtained for 112 isolates ( 87 . 5% ) . Three samples with fewer than five MS markers genotyped were removed from analysis . The predominant allele of each locus in each sample was used for population genetic analysis . MS2 was the most diverse marker with >17 alleles in each parasite population , whereas MS1 was the least diverse with only 6–7 alleles ( S1 Fig and S1 Table ) . For all markers except MS5 , at least one allele was shared among the three populations; the most extensive allele sharing was found with MS1 ( 3 alleles ) and MS6 ( 4 alleles ) . Parasite diversity was high; when all sites were combined , the mean expected heterozygosity ( HE ) was 0 . 851 , and it was not significantly different among the three sites ( 0 . 837 , 0 . 850 , and 0 . 867 for Kanchanaburi , Ubon Ratchathani and Tak , respectively ) ( Table 1 ) . Twenty four ( 19 . 3% ) parasite isolates contained multiclonal infections ( more than one peak for at least one MS marker ) , which resulted in a mean multiplicity of infection ( MOI ) of 1 . 297 ( Table 1 ) . Tak had a higher proportion of multiclonal infections ( 23 . 70% ) than Kanchanaburi ( 14 . 60% ) and Ubon Ratchathani ( 19 . 70% ) ; the differences were significant among all sites with pairwise comparisons ( p < 0 . 05 , Pearson Chi-square Test ) ( Table 1 ) . Despite the hypoendemic nature of malaria in Thailand , the haplotype diversity was extremely high . A total of 116 haplotypes were observed and no haplotypes were shared among the three sites . In Tak , identical haplotypes 25 , 79 , 92 , and 109 were shared by two , three , and four parasites , respectively , whereas in Kanchanaburi , two parasite isolates carried the same haplotype ( S2 Table ) . Significant genetic differentiation was observed among the three parasite populations ( Fst = 0 . 113–0 . 126 , p < 0 . 05 , Table 2 ) . STRUCTURE analysis showed a clear distribution pattern of parasite haplotypes . When K = 3 , isolates clustered according to their provincial origin ( Fig 2A ) . Principal coordinate analysis ( PCoA ) confirmed the genetic separation of the three parasite populations . In line with the STRUCTURE analysis , PC1 separated Tak from Kanchanaburi and Ubon Ratchathani , while PC2 separated Tak from Kanchanburi ( Fig 3 ) . PC1 and PC2 explained 6 . 34% and 4 . 91% of the variance in the data , respectively . Nevertheless , parasites collected from each province had a few haplotypes that shared their phylogenetic relationship . Weak but significant correlation was found between genetic and geographic distances ( Mantel rank test , r2 = 0 . 1993 , p < 0 . 05 ) ( S2 Fig ) . The separation of these three parasite populations was further demonstrated by the minimum spanning tree analysis , which revealed a similar pattern of relatedness of parasite isolates by provinces ( Fig 4 ) . Only a few haplotypes clustered with parasites from other provinces . Effective population size ( NE ) was large in all provinces ( Table 3 ) . The overall NE was estimated to be 8704 [95% CI: 3741–19828] using the infinite allele model ( IAM ) , while it was 3–4 fold larger based on the stepwise mutational model ( SMM ) . Significant linkage disequilibrium ( LD ) , suggesting of inbreeding , was observed in Tak ( ISA = 0 . 0679 , p < 0 . 00001 ) and Kanchanaburi ( ISA = 0 . 1245 , p < 0 . 00001 ) , whereas LD did not reach significance in Ubon Ratchathani ( ISA = 0 . 0109 , p = 0 . 0769 ) . To detect whether there were significant changes in the parasite population sizes , we performed BOTTLENECK analysis using SMM ( Table 4 ) . The models detected significant deficiency in HE from the mutation-drift equilibrium , indicating events of population size reduction with possible clonal expansion in all areas . We sought to identify a minimum set of markers suited to differentiate parasite populations based on their provinces of origin , and for differentiating parasites within each province . Stepwise removal of MS markers based on their HE ranking , starting with the least diverse , showed that four highly diverse MS markers ( MS2 , MS6 , MS12 and MS20 ) were enough to distinguish more than 96% of all haplotypes ( 112/116 haplotypes ) in the study populations ( Fig 5 and S3 Table ) , and >94% in each province . Due to subtle differences in the diversity of the MS markers in different provinces , the optimal set of haplotypes for each province differed slightly ( S3 Table ) . A similar cluster pattern was obtained when performing STRUCURE analysis using these four MS markers as compared with that using all 10 MS markers ( Fig 2B ) .
This study presents an updated analysis on P . vivax diversity and population structure along the Thai-Myanmar and the Thai-Cambodian borders with samples collected after malaria control had been intensified and transmission reduced . Our results clearly show that despite reduction of malaria prevalence in recent years , P . vivax diversity ( HE ) remained high . There were still ~20% of the isolates containing mixed-strain infections , and MOI was only slightly lower than in studies conducted 15–20 years ago in Thailand [5 , 12] . Similarly high P . vivax diversity despite low prevalence has been reported from other countries [19–21] . Compared to a previous study [4] which used a nearly identical set of microsatellite markers to study isolates from around the world , our observed HE ( 0 . 84–0 . 87 ) in Thailand are similar to those in Cambodia ( 0 . 87 ) and Vietnam ( 0 . 84 ) and clearly higher than those in hypoendemic regions including Peru ( 0 . 71 ) , Brazil ( 0 . 74 ) , and central Asia ( 0 . 75 ) . Thus HE generally tracks transmission intensity [4 , 22] . Interestingly , despite the higher transmission intensity in the southwest Pacific , HE in Solomon Islands ( 0 . 81 ) and Papua New Guinea ( 0 . 80–0 . 82 ) are similar to those in Thailand , Cambodia , and Vietnam , if not lower . This may reflect the more restricted gene flow imposed by the island geography of these areas compared to the mainland Southeast Asia . The maintenance of high genetic diversity in low-transmission areas of Southeast Asia may be due to a large proportion of asymptomatic and submicroscopic infections [23 , 24] . In addition , relapses from hypnozoites may also be frequent [25] . Both processes favor the maintenance of genetic diversity . The highly diverse P . vivax populations from the three Thai provinces are in sharp contrast to data from southern Thailand , where sequencing of the Pvmsp3β gene revealed the same genotype in all of 28 isolates [17] . Even though microsatellites have a higher mutation rate than antigens , the results based on Pvmsp3β suggest that P . vivax population in southern Thailand is clonal or near-clonal . Though truly clonal expansion–i . e . many isolates sharing the same haplotype–has been reported only sporadically from other regions , e . g . , Uzbekistan and Ethiopia [4 , 26] , it may be a good indicator of that the parasite population size has been reduced to very low levels . On the other hand , our results suggested that parasite population sizes in our study sites were still large and this may represent the general situation in the Greater Mekong Subregion ( GMS ) . Therefore , more intensified control efforts are needed to bring down the relatively large parasite populations during the malaria elimination phase in this region . As control efforts intensify with the aim of malaria elimination in the GMS by 2030 , the remaining parasite transmission foci are expected to be localized along international borders and separated by areas with extremely low or no malaria transmission . Such geographical separation eventually will result in parasite population division . The Mantel test detected a weak correlation between the genetic distance and the geographic distance among our sites ( S2 Fig ) . However , given the small number of populations tested , this correlation may be coincidental . It is interesting to note that the east-west differentiation between Kanchanaburi and Ubon Ratchathani is less strong than between the Kanchanaburi and Tak , as revealed by the structure analysis at K = 2 . This could be due to the mountainous terrains on the western border , which limit the gene flow between the two western sites . Another factor that may have an impact on parasite population division between different sites is malaria vectors . P . falciparum has been shown to be adapted to local vectors , e . g . infectivity of Asian isolates is lower in African vectors than in local vectors [27] . Furthermore , P . vivax populations in Mexico have different infectivity in two local Anopheles species , and parasite population structure was associated with vector distribution [28 , 29] . Analogously , we can speculate that the highly divergent vector systems in the GMS may be partially responsible for the parasite genetic structures . Different distribution patterns of malaria vectors between western and eastern regions of Thailand were reported [30] . Major vectors in western Thailand are An . dirus , An . minimus and An . maculatus complexes , whereas An . barbirostris is distributed in Ubon Ratchathani . The latter species has been shown to be a vector for P . vivax [31 , 32] and may explain differentiation of parasites in Ubon Ratchathani from the western provinces . Likewise , An . harisoni is restricted to Kanchanaburi [33] and may contribute to parasite differentiation in this area . It remains to be determined whether these vector species have differential capacity for different P . vivax genotypes . Once malaria transmission becomes very low , the identification of the source of residual infections and potential reintroduction becomes increasingly important [34] . The use of appropriate molecular markers that can clearly separate parasite populations will allow us to identify the sources of parasite introduction . This is especially relevant if parasites are introduced to regions where malaria has been eliminated . As the parasite population evolves over time , the choice of suitable markers will need to be updated and tailored to the questions at hand . For Thailand , genotyping the parasites can be particularly useful if there are malaria resurgence in malaria-free regions of the country . On the other hand , distinguishing local transmission from cross-border reintroduction in areas along the national border may still be challenging . In these areas , local parasite populations likely do not display a clear genetic structure due to heavy cross-border human traffic . Further studies using parasites from both sides of the border will be required . Our findings that a small panel of MS markers can allocate haplotypes to their provincial origin provide a proof of principle as well as important baseline information for the malaria control programs . Future detection of local cases in areas that are assumed to be malaria-free would indicate that parasite reservoirs are overlooked and that intensified control activities should be implemented . In contrast , detection of cases acquired from border regions would call for heightened efforts for malaria control among migrants .
Written informed consent was obtained from all blood donors or their legal guardians for participants under the age of 18 . This study was approved by the Institutional Review Boards of Mahidol University and the Pennsylvania State University . Malaria transmission in Thailand is perennial following the patterns of rainfall , and P . vivax transmission typically has two peaks: one in July–September and one in November [12] . Parasite samples were collected from Tak and Kanchanaburi provinces in western Thailand bordering Myanmar and Ubon Ratchathani Province in eastern Thailand bordering Cambodia and Laos ( Fig 1 ) . The western provinces are the traditionally high malaria transmission areas . In 2016 , however , Ubon Ratchathani reported the highest number of malaria cases in Thailand , whereas Tak and Kanchanaburi were the second and fourth highest , respectively [3] . Malaria in Thailand has decreased significantly in recent years due to intensified national control . According to the annual malaria report ( http://203 . 157 . 41 . 215/malariar10/index_newversion . php ) by the Thailand Malaria Elimination Program , Ministry of Public Health , the number of malaria cases in Kanchanaburi has continually declined since 2012 , with the annual number of 4122 in 2012 , 2623 in 2013 , 1739 in 2014 , 1081 in 2015 , to 539 cases in 2016 . A similar decline was observed in Tak , with the annual case numbers of 13706 , 1279 , 6306 , 3259 and 1364 from 2012–2016 . In contrast , Ubon Ratchathani saw a rise in the malaria cases from 1119 cases in 2012 , to 1248 cases in 2013 , and to 8834 in 2014 after which a decline was seen to 3436 in 2015 and 788 cases in 2016 . Blood samples were collected from symptomatic patients attending local malaria clinics . Diagnosis was based on microscopy of Giemsa-stained thin and thick smears and ~100 μl of finger-prick blood from P . vivax patients were spotted on filter paper , dried and stored in individually zipped plastic bags before DNA extraction . A total of 127 P . vivax samples were collected , including 38 collected in 2013–2014 from patients attending the malaria clinics in Sai Yok and Srisawat districts , Kanchanaburi , 54 samples in 2013–2015 in Tha Song Yang District , Tak Province , and 35 samples in 2015–2016 in Bun Tharik and Na Chaluai districts , Ubon Ratchathani . The distance between the sites in the two western provinces is approximately 694 km , and they are 776 and 1101 km away from Ubon Ratchathani , respectively ( Fig 1 ) . Ten MS markers ( MS1 , MS2 , MS5 , MS6 , MS7 , MS9 , MS10 , MS12 , MS15 and MS20 ) previously used to differentiate P . vivax populations [18] were selected for genotyping ( S4 Table ) . A nested PCR protocol was applied , with a 10-plex primary PCR followed by individual semi-nested PCRs for each marker , using a 40-fold dilution of the primary PCR product as the template [35] . The size of the PCR product was assessed by capillary electrophoresis in a 3730 XL ABI Sequencer ( Applied Biosystems , Macrogen , South Korea ) . Electropherograms were analyzed using Peak Scanner v . 2 ( Macrogen , Seoul , South Korea ) . Only peaks with a height of ≥200 relative fluorescence unit were selected , and in case of several peaks only those with an intensity of at least one-third of the dominant peak . Isolates containing more than one peak for any marker were considered to be multiple clone infections . MOI of a given isolate was measured as the highest number of observed alleles at any of the 10 loci , or at any of the two most diverse loci . Alleles were binned using TANDEM software [36] . The mean number of alleles , allelic richness and expected heterozygosity ( HE ) were calculated using FSTAT v . 2 . 9 . 3 [37] . Pairwise genetic differentiation ( Weir & Cockerham Fst values ) was calculated using FSTAT v . 2 . 9 . 3 . Fst varies from 0 ( no genetic differentiation among populations ) to 1 ( no shared alleles among populations ) . LD was calculated using the program LIAN 3 . 7 [38] with 50 , 000 iterations for burn-in and then 100 , 000 Markov Chain Monte Carlo ( MCMC ) iterations . Samples with missing data were excluded for this analysis . To evaluate the optimal panel of MS markers for differentiating between different clones in Thai parasite isolates , MS markers were removed in a stepwise manner [4] . The number of haplotypes after removing one marker at a time was counted using GenAlEx 6 . 5 [39] and expressed as the percentage of haplotypes identified compared to the full panel of 10 markers . The correlation between geographic and genetic distances was done by using Mantel rank test in GenAlEx 6 . 5 . STRUCTURE 2 . 3 . 2 software was used to assess clustering of haplotypes [40] . Twenty iterations were run for each cluster ( K = 1–12 ) with a burn-in of 50 , 000 steps and then 500 , 000 MCMC steps using the admixture model . The cluster number K = 3 is considered optimal based on its consistency with other clustering methods including the principal component analysis and the phylogenetic tree analysis . PCoA was conducted by using the GenAlEx 6 . 5 . The goeBURST algorithm within PHYLOViZ 2 . 0 was used to generate a unique minimum spanning tree [41] . Any haplotype with missing data was excluded from the tree . The unique tree was ruled by the n Locus Variants level ( nLV ) method , where n is the number of MS markers . All 116 complete multilocus haplotypes of the total 127 isolates were included in the tree . The effective population size was calculated using two models , SMM and IAM , following the formula Neμ = 1/8{[1/1-HE_mean]2–1 and Neμ = HE_mean/4 ( 1-HE_mean ) , respectively . HE_mean is the average of the expected heterozygosity across all loci , while μ is the mutation rate per generation . Mutation rate values were derived from P . falciparum: 1 . 59 × 10−4 ( 95% confident interval 3 . 7 × 10−4–6 . 98 × 10−5 ) [42] . Assuming the SMM [43] , we assessed whether our results deviate from the mutation-drift equilibrium [44] . Alternative models exist , including the IAM and the Two-phase Model [45 , 46] . We chose the SMM because it is considered most appropriate for the interpretation of simple repeat units of microsatellite data [43] .
|
This study presents an updated view of the P . vivax populations along the Thai-Myanmar and the Thai-Cambodian borders . Genotyping of parasite samples collected after intensified malaria control demonstrated that despite the decline in overall transmission intensity , the genetic diversity of the P . vivax parasites remained high . Parasite populations from three border provinces showed clear genetic separation . This indicates successful interruption of parasite gene flow within Thailand , but suggests frequent parasite migration across international borders . From the analysis of 10 microsatellite markers , we further refined a set of four that are sufficient for distinguishing the provincial origins of these parasites , which should allow tracking of parasite introduction among these provinces .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
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] |
2017
|
Substantial population structure of Plasmodium vivax in Thailand facilitates identification of the sources of residual transmission
|
Bacteriophages ( or phages ) dominate the biosphere both numerically and in terms of genetic diversity . In particular , genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages , favoring a high evolution rate . Molecular mechanisms of this pervasive mosaicism are mostly unknown . One hypothesis is that phage encoded recombinases are key players in these horizontal transfers , thanks to their high efficiency and low fidelity . Here , we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism . The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants . In the second assay , recombination was monitored between sequences on phage λ , and allowed to compare the performance of three different Rad52-like recombinases on the same substrate . We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E . coli virulent and temperate phages or prophages . We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence . We show that phage encoded Rad52-like recombinases act independently of RecA , and that they are relatively more efficient when the exchanged fragments are divergent . We also show that accessory phage genes orf and rap contribute to mosaicism . A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments . We found no evidence of exchanges between virulent and temperate phages of E . coli . Altogether , our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages , accelerating their evolution . This mechanism may prove to be more general , as other mobile genetic elements such as ICE encode Rad52-like functions , and play an important role in bacterial evolution itself .
Bacteriophages , or phages , the viruses that attack bacteria , have gained a renewed interest in the last decade with the emergence of antibiotic resistant bacteria . Despite their early discovery [1] , [2] and the in-depth molecular and genetic characterization of few model phages , overall phage biology is still globally poorly understood comparatively to their ecological importance . Indeed , phages and related elements dominate the biosphere both numerically and in terms of genetic diversity . Despite more than 105 genes already described in phage genomes , recent studies suggest that the majority of phage genes remains to be discovered [3] . The great genetic diversity of these viruses is due to their very ancient origin , their large population size and their high evolvability . Understanding evolvability of bacterial viruses will likely become a major issue with the prospective massive use of phages as alternatives to antibiotics . Notably , the mutation rate of viruses is much higher than that of cellular organisms [4] , [5] . Horizontal gene transfer also plays a major role in virus evolution by creating new combinations of genetic material through the pairing and shuffling of related DNA sequences [6]–[8] . Initial observations of hybridizing segments between phage genomes by electron microscopy , and more recent genomic analyses , have revealed the pervasive mosaicism of temperate phage genomes [9]–[11] . Mosaicism refers to the patchwork character of phage genomes , which can be considered as unique combinations of exchangeable genomic segments [11]–[15] . Temperate phages , as opposed to lytic phages , have the ability to enter a prophage dormant state upon infection , in which they stably replicate with the bacterial genome . Nearly all bacterial genomes contain multiple active or defective prophages , the latter being unable to produce phage particles . In Escherichia coli , prophage genes can constitute up to 14% of the genome [16] , and represent 41% of a 20 species pangenome [16]–[18] . Intergenomic rearrangements are thus facilitated for temperate phages by frequent encounters of different viruses inside the same bacterial host , for example between an invasive virus and a resident prophage . While genome shuffling appears as a key driver of phage evolution , a quantitative description of mosaicism and analysis of its underlying molecular mechanisms are lacking . In particular , it is still debated whether phage genetic mosaicism is the product of recombination at sites of limited homology between genomes [19] , [20] , or the result of random , cut and paste , illegitimate recombination [11] , [21] . In the latter hypothesis , the conservation of synteny would result from the counterselection of deleterious non-ordered gene combinations . Functions involved in homologous recombination ( HR ) have been extensively studied in E . coli and phage λ [22] , [23] . In E . coli , the RecA recombinase is essential for catalyzing DNA exchanges between homologous molecules . However , the rate of successful RecA-dependent exchanges rapidly decreases with increasing sequence divergence [24] , [25] , suggesting that homologous exchanges should not happen between very divergent phage genomes . Phage genomes also encode recombinases catalyzing HR reactions , that have been classified into 3 super-families known as Rad51- , Rad52- and Gp2 . 5-like [26] . These recombinases are also found sporadically on non-bacterial viruses: archaeal proviruses encode Rad52-like genes [27] and Mimi and Herpes viruses encode a recombinase sharing homology with Gp2 . 5 [28] . Among the phage recombinases , the Rad52-like family is the largest and most diversified , and is itself subdivided into the Redβ , Erf and Sak groups . Among them , the λ recombinase Redβ is known to be efficient in the recombination of diverged sequences [19] , [29] , leading us to suggest that phage recombinases could be key actors in genomic shuffling of related phages . The λ HR system , known as Red , is expressed during phage lytic development . Red consists of two functions encoded by the redβ and redα genes . The main activity of Redβ is to mediate single-strand DNA annealing between a Redβ-bound single-stranded region and a complementary sequence . λ Redα is a double-strand-specific exonuclease that generates single-strand DNA for Redβ annealing . Redβ can also promote recombination by a RecA-like strand-invasion mechanism , especially on short DNA sequences [30] . Two other genes in the λ nin genomic region , orf ( former name ninB ) and rap ( former name ninG ) , were shown to facilitate RecA-dependent gene exchanges in vivo between strictly identical sequences [31] . The orf gene product is a mediator protein that participates in the loading of the bacterial RecA recombinase on SSB-coated DNA in the absence of the three bacterial proteins RecFOR [32]–[36] . The rap gene codes for a Holliday junction resolvase [35] . Orf and Rap have numerous homologs among temperate phages , and form ∼500 members families in Pfam . Interestingly , their distribution pattern among phage genomes is contrasted: among the 465 completely sequenced phage genomes collected in the ACLAME database , 191 encode a recombinase [26] . The presence of Orf is tightly associated with Recombinase+ genomes , as among the 55 phages encoding Orf , 49 also encode a recombinase , of the Rad52- or Rad51-like family . On the contrary , among the 180 genomes encoding Rap , only 100 are Recombinase+ , as if Rap and phage recombinase occurrences were independent ( MAP et al . , to be published elsewhere ) . These distributions are suggestive of a more important role of Orf on phage recombinase activity . However , whether Orf or Rap stimulates Redβ mediated recombination is unknown at present . Here , we study quantitatively the generation of mosaics between functional ( i . e . , infectious ) temperate phages and defective prophages , and identify the genetic determinants of these exchanges . These assays reveal the preponderant role of Rad52-like phage recombination genes in exchanges involving short and diverged sequences . Moreover , a global analysis of mosaics between active temperate phages and defective prophages further reveals that these exchanges are commonplace in phage genome evolution .
The aim of this first assay was to determine the extent and the mechanisms of genomic exchanges between an invasive infectious phage and defective prophages residing in the host chromosome . Homologous genomic regions between the MG1655 E . coli strain and λ phage were identified by a Blast search using relaxed parameters ( same parameters as for the ANI analysis , see Fig . S6 legend ) . Twelve regions sharing over 70% mean identity on a stretch of at least 100 bp were found , all inside defective prophages . 3 such regions , differing in size and in the extent of identity , were selected for the experiments: i ) a region in Dlp12 defective prophage , sharing 98% mean identity over 3353 bp with λ , ii ) a region in Qin defective prophage sharing 96% mean identity over 657 bp , and iii ) a region in Rac defective prophage sharing 88% mean identity over 703 bp , followed by another homology region nearby of 95% mean identity ( Fig . 1 A , B & C ) . The length distribution of segments of perfect identity ( segment without mismatch ) is accordingly very different in the 3 regions ( Fig . S1 ) . Notably , the longest stretches of perfect identity are 298 , 115 and 49 bp for Dlp12 , Qin and Rac respectively . An antibiotic resistance gene , conferring resistance to either chloramphenicol ( cat ) or kanamycin ( kanR ) , was introduced in the middle of each selected region ( Fig . 1 and Materials and Methods ) . As a control , we used a strain with the cat gene into ilvD , which has no homology with λ and whose deletion does not impact phage growth . Defective prophages can reportedly excise and even replicate in different strain backgrounds under certain conditions , notably during phage infection [31] , [37] , [38] . We thus verified by PCR that the 3 studied defective prophages do not excise during infection by λ . Moreover , as Rac has an active but normally repressed replication origin , we checked by semi-quantitative PCR whether its replication was induced upon λ infection . We found no evidence of such a phenomenon ( Fig . S3 ) . Recombination between λ and the marked defective prophages can result in the integration of cat or kanR antibiotic resistance gene into λ ( Fig . 1E ) . As this does not produce a directly detectable phage phenotype , E . coli cells were further lysogenized with the resulting phage , and the proportion of cells lysogenized by WT or antibiotic-resistant recombinant phages was determined by plating on selective antibiotic media ( Fig . 1D and Materials and Methods ) . To be able to detect all lysogenized bacteria , we used a λ strain marked with the phleomycine resistance gene ble ( Urλble strain , Table S1 and Materials and Methods ) . This recombination assay enables the estimation of the horizontal gene transfer rate into the λ genome when it infects its host . λ recombinants with Rac and Qin were observed at frequencies around 2×10−6 , and were a hundred fold more numerous with Dlp12 ( Fig . 1E ) . Interestingly , the frequencies of recombinants were similar for Rac and Qin despite different extent of identity on comparable lengths ( Fig . 1 and S1 ) . In contrast , the recombinant frequency with the control chromosomal gene ilvD , presenting no homology with λ genome , was below our detection threshold of 5×10−9 . PCR analysis on 20 λ recombinants within each of the 3 loci showed that all had incorporated the resistance gene at the expected position for an homologous exchange . 12 to 19 were sequenced to confirm that homologous recombination occurred in the targeted region and to identify the exact junctions ( Fig . 2 ) . Among recombinants with Rac , the majority resulted from recombination events within the adjacent 88% identity regions , but one third had recombined on the right in the nearby 95% homology region in tfa . HR with Rac and Qin leads to the inactivation of λ side tail fibers genes ( stf and tfa ) , which reportedly improves phage growth compared to the parent in a soft-agar overlay [39] . To evaluate if this increases the recombinant frequency by favoring their growth , we performed the same recombination assay with the sequenced λPaPa strain of λ , mutated in its stf gene [40] . The frequency of λPaPa recombinants with Rac was not significantly different from that with Urλ ( p = 0 . 18 , Student T-test ) , ruling out the possibility that the advantage of side tail fiber inactivation distorts our conclusions . This can be explained by the low recombination rate: most recombinants statistically arise only during the last ( and third ) cycle of phage growth , as phage engaging in the third lytic cycle are 10 , 000 more numerous than those involved in the first one . Sequencing of 12 λ-Rac recombinants showed that they all correspond to different hybrids ( Fig . 2A ) , confirming the absence of amplification and also indicating the absence of a recombination hotspot . The high recombination frequency with Dlp12 is expected for two reasons . First , Dlp12 shares the largest region of homology with λ ( 3353 bp ) , and secondly , the region of homology contains the λ cos site ( Fig . 1A ) . Double-strand DNA breaks at cos sites , created for genome encapsidation , stimulate recombination on its left side , the right side bound by the terminase being protected [41] . Double-strand breaks in both λ and Dlp12 might thus stimulate the recombination in this region . Sequencing of 19 λ-Dlp12 recombinants indeed revealed a high proportion of junctions in the immediate proximity to cos ( 10 out of 19 clones , Fig . 2B ) . 3 out of 19 junctions were nevertheless found on the right side of cos , indicating that double-strand breaks at cos improve but are not necessary for recombination events . Interestingly , all tested λ-Dlp12 recombinant phages were active , including those that had replaced the essential λ genes R and Rz ( lysis ) or nu1 ( terminase ) by those of Dlp12 , indicating that these Dlp12 genes are functional in λ . We have shown that genetic exchanges with λ are driven by the presence of homologous regions . We next questioned the respective roles on these exchanges of the bacterial and phage HR genes , recA and redβ , and also of two other λ recombination genes , orf and rap . As redβ and redα λ mutants have a reduced burst size ( Fig . S2 ) , conditions of phage growth on the marked strains were adapted to ensure that the same number of phage generations was realized ( see Materials and Methods ) . The frequency of recombination with the large , quasi-identical segment of Dlp12 ( 3 . 3 kb , 98% identity ) was not affected by a single recA deletion , and only 3-fold by a redβ deletion ( Fig . 3A , blue bars ) . However , when both recombinase genes were deleted , exchanges were completely abolished . This shows that both RecA and Redβ produce recombinants independently of one another , which is indicative of redundant pathways . Within Qin and especially Rac regions , showing more divergence with λ , single recA deletion had similarly no effect on recombination frequencies . The single redβ deletion however had a pronounced effect on the recombination frequencies ( 6- and 13- fold respectively , Fig . 3A , orange and green bars ) , indicating that most recombination events are formed by Redβ when homology is reduced , reflecting a lowest activity of RecA on these substrates . The residual recombinants formed in the absence of Redβ almost completely disappeared with the double deletion of recA and redβ . Only 2 recombinant clones were obtained in the recA redβ double mutant , with Rac , and were found by PCR to result from homologous recombination . They might have originated from the expression of the recET recombination genes of Rac . Normally the recET genes are completely repressed in E . coli MG1655 , even upon λ infection [42] , and thus cannot promote recombination , but rare sbcA mutations [43] or incorporation in λ ( the so-called λ-rev genotype , [44] ) can activate them . The activities of recombinases are stimulated by numerous host or phage-encoded cofactors , which either prepare the substrate for recombination or act at latter stages to resolve the DNA heteroduplex structure . The main partner of Redβ is the double-strand-specific exonuclease Redα that transforms double-strand DNA into single-strand DNA , the substrate for Redβ . Redα is dispensable for Redβ recombination if the DNA substrate is initially in a single-strand form [45] . Here , deletion of redα gene had the same effect as the redβ deletion , and basically disabled Red-mediated recombination in Dlp12 and Rac regions ( Fig . 3B&C ) . This finding reveals a need for single-strand DNA formation for Red-mediated recombination in our assay . We then tested the effects of other recombinase helper proteins on recombinant frequencies . λ encodes two such proteins , Orf and Rap , which belong to protein families highly prevalent in phage genomes ( respectively 55 and 180 homologues in 465 phage genomes , our unpublished work ) . Orf and Rap participate in RecA-dependent recombination by supplying a function equivalent to bacterial encoded recombination cofactors , respectively RecFOR and Ruv resolvase [36] , [46] . Here we asked whether they could also participate in Red recombination , and whether their role was more pronounced on diverged sequences than on easy to recombine long sequences . Single deletion of orf or rap resulted in a not significant 3-fold reduction of λ recombination with Dlp12 ( Fig . 3B , first set of data ) . However , in a recA strain , in which all gene exchanges are Red-mediated , recombination was reduced by 10-fold in the Δorf mutant , and by significant 3-fold in the Δrap mutant . This result reveals that Orf and to some extent Rap participate in Red-mediated recombination . Finally , in the genetic context where RecA mediates all gene exchanges , the orf deletion had no effect , while the rap deletion decreased recombinant frequency by 300-fold , as expected from an earlier report [47] . The same genetic analysis in the Rac region assay revealed the same dependencies ( Fig . 3C ) . In conclusion , Orf and to a lesser extent Rap facilitate homologous recombination with λ when Redβ is the only recombinase , with no indication of an increased role when DNA sequences are more divergent . In order to determine the generality of our observations with λ , we performed similar recombination assays with the lambdoid phage Φ80 . Its genome homology with λ is mostly clustered in the capsid region , while the rest of the genome is highly divergent with only few segments of homology [48] . Φ80 encodes a putative recombinase from the Rad52 family , hereafter named RecTΦ80 , that shares 32% identity at the amino acid level with Redβ . As previously described with λ , we detected by Blast search 9 regions of homology between Φ80 and E . coli MG1655 genomes , and selected 3 of them for the recombination assay: i ) the 3′ part of the bacterial core gene yecD , of unknown function , presenting 96% mean identity over 300 bp , ii ) in the nohD region of the defective prophage Dlp12 , sharing 79% identity over 980 bp with the terminase genes nu1 and A , and iii ) within the nin region of Dlp12 , sharing 67% identity over 500 bp ( Fig . 4 A , B , C ) . For each region , the longest stretch of perfect identity is 116 , 41 and 11 bp for yecD , nohD and nin , respectively ( Fig . S1B ) . Homology regions were labeled with the cat gene , and Φ80 genome was marked with the ble gene . Φ80 phage was then propagated on the modified E . coli strains and the number of recombinant phages was scored ( Fig . 4D ) . Recombinants were produced at a frequency of 6×10−7 per phage with the yecD locus and 8×10−7 with the nohD locus of Dlp12 ( Fig . 4D ) . In the last case , recombination disrupts the essential terminase genes nu1 and A , but this does not prevent encapsidation of recombinant Φ80 genomes , due to the terminases encoded by the other copies of Φ80 . However , the recombinants formed will not give lytic progeny upon infection of a new host , ensuring that the recombinants counted in the assay are exclusively those formed during the last lytic cycle . As discussed above , the presence of the cos site within the homology region probably increases the production of recombinants . Within the last region , sharing only 67% identity over 500 bp with λ , the recombination frequency was below 1×10−8 , our detection threshold ( ybcN locus , Fig . 3C ) . PCR analysis revealed that the 12 recombinants scored had incorporated the resistance gene at the locus expected for homologous exchange . As with λ , we then determined the respective role of homologous recombination enzymes RecA and RecTΦ80 in these genetic exchanges . recA deletion slightly diminishes recombinant frequency by 2-fold on both loci , whereas recTΦ80 deletion has a pronounced effect on the more diverged sequences ( Fig . 3C ) . These results show that homology-dependent mosaic formation driven by phage encoded recombinases is not restricted to λ and may be a general event among temperate phages encoding their own homologous recombination functions . Exchanges occurred even when the resulting recombinant phages were no longer viable as a lytic phage , and appeared to be only limited by the degree and length of sequence homology . The capacities of Rad52-like phage encoded recombinases and cofactors were further explored with an intra-bacteriophage recombination assay , which enables the comparison of recombinase activities on the same DNA substrate . We monitored homologous recombination within the λ genome , between two inverted 800 bp sequences introduced on each side of the PL promoter ( described in Fig . 5A and [19] ) . Briefly , inversion of the PL promoter leads to a detectable phenotypic switch , as it prevents transcription of the red and gam genes , enabling growth on a P2-lysogen , contrary to the non-inverted phage . Vice versa , inverted PL Red− Gam− phage cannot grow on a recA strain , in contrast to the non-inverted wild-type phage . Two different recombination cassettes were used , the inverted sequences being either strictly identical , or 78% identical . The switch was measured in the two directions , and in all cases , the recombination assay was performed by growing phages on a restrictive host for the multiplication of the recombinants , ensuring that the recombinants are produced only during the last generation . In this study , λ redβ and redα genes were replaced by other pairs of recombinase genes and their respective associated exonuclease . The first pair is recT and recE ( fragment coding for the Cter part of the protein ) , from the defective prophage Rac . The second pair is the predicted Erf family recombinase gene erf , with its associated predicted exonuclease gene exo , from D3 phage infecting Pseudomonas aeruginosa [49] . Resulting phages were grown to confluence on a bacterial lawn , and the frequency of recombinants was measured by differential platings . On both the 100% and 78% identical substrates , efficiency of recombination mediated by the RecT/RecERac and Erf/ExoD3 pairs was similar to that mediated by λ Red proteins ( Fig . 5B ) . First , this demonstrates that the D3 erf gene product is indeed a recombinase , and as efficient as Redβ and RecTRac . For all 3 recombinase/exonuclease pairs , recombination between identical or diverged sequences was respectively 20- and 100-fold higher than in the RecA pathway . This result extends our previous observation with λ and Φ80 , and suggests that during the lytic cycle , the high efficiency and low fidelity of phage-dependent recombination might be a general phenomenon that promotes horizontal gene transfer into phage genomes . We thus attempted to quantify the traces that such exchanges might have left on phage genomes on an evolutionary time scale by analyzing a collection of E . coli phages ( Table S4 ) . In particular , we examined whether traces of HR events could be detected at the boundaries of recently exchanged DNA fragments . In a previous study of mosaics formed between temperate lambdoid phages [19] , we showed that half of the mosaics , defined by Blast hits with more than 90% identity , were flanked by at least one region sufficiently similar to be an indication of a region of homology preexistent to the recent exchange identified by the Blast hit . These above background homology regions were hereafter named “HR traces” . This study was extended and refined here , so as to include more genomes from temperate ( 24 genomes ) , defective ( 34 genomes ) or virulent ( 26 genomes ) E . coli phages . Results are summarized in Table 1 . We detected a large number of recently exchanged genomic segments among temperate and defective phages ( between 0 . 8 and 2 . 4 mosaics per genome pair ) . No mosaic was found between virulent and temperate or defective phages . We also found a few mosaics between virulent phages ( 0 . 06 per genome pair , Table 1 ) . Interestingly , among temperate and defective phages , the density of mosaics , i . e . the numbers of mosaics per 10 kb , was similar ( Fig . 6 , lower part of the diagonal ) , suggesting that exchanges do occur at similar frequencies among defective and functional temperate phages . Among temperate phages , two sub-groups not sharing mosaics were found: the larger one contained lambdoid phages ( i . e . λ-like , ε15-like and P22-like ) , the smaller group contained P2-like , P1 and Mu-like phages . Among pairs involving defective phages , exchanges between these two groups are found , probably because non functional gene combinations are not counterselected in defective prophages . Interestingly , the size distribution of mosaics indicates that small gene fragments are much more often exchanged than complete functional modules ( Fig . S5 ) . We next investigated whether the mosaics had HR traces , i . e . flanking regions that have a lower mean identity than the mosaic but a higher than expected identity ( see Figure 7 and Text S1 for the principle of analysis ) . Briefly , HR traces were detected by complete realignment of the mosaic flanking regions , and identification of above-background levels of nucleotide identity . We found regions suggestive of HR for 32% of the mosaics occurring among temperate phages , 35% of those formed between defective and temperate phages , and 31% for the inter-defective mosaics ( Fig . 6 , upper side of the diagonal , and Table 1 ) . In most cases , only one , rather than two HR traces were found flanking the mosaics . This may be explained by other mechanisms than HR to generate exchanges but also by successive rounds of exchanges , involving different phage pairs , as illustrated in Fig . S7 .
We demonstrate that phage encoded Rad52-like recombinases play a primordial role in recombining regions sharing only short stretches of homology . Interestingly , on these substrates , all four Rad52-like recombinases that we tested ( Redβ , RecTRac , ErfD3 and RecTΦ80 ) had a higher activity than RecA during λ replication . These results strongly support the view that phage Rad52-like recombinases , predominant among E . coli lambdoid phages [54] , play a crucial role in genomic shuffling . Interestingly , a recent bioinformatic study showed that the level of mosaicism is higher for phages encoding a recombinase than for others [54] , supporting this hypothesis . Whether the same holds true for the two other large families of recombinases encountered in phages ( Gp2 . 5 and Rad51 , [26] ) remains to be investigated experimentally . Staphylococcus aureus temperate phages exhibit a large level of mosaicism [19] , and encode indifferently all three types of recombinases , which suggests that the two other families might also have the same property of relaxed fidelity . Further work aiming at comparing these recombinases side by side will allow addressing this point in detail . The high efficiencies of Rad52-like recombinases on diverged sequences could be due to their higher concentration or activity , compared to RecA , during phage infection . However , on highly homologous sequences , RecA or phage recombination pathways result in a similar yield of recombinants ( Fig . 3A and 4D ) . The 10–20 fold higher efficiency of Redβ or RecTΦ80 compared to RecA pathway is unveiled only on more divergent sequences . It could result from activity on shorter perfect sequence identity segments to recombine as compared to RecA . The Minimal Efficient Pairing Segment ( MEPS ) , the minimal size of exact pairing required for efficient exchange in vivo , has indeed been found to be 31–34 bp for RecA [24] and only 23–27 for Redβ [24] , [55] . Our in vivo assays involve sequences that do not have regularly spaced mismatches , so that slight changes in the number of segments above MEPS size may have drastic effects . Interestingly , Li and collaborators have recently reported that recombineering with regularly spaced , diverged oligonucleotides is effective up to 1 mismatch every 5 nucleotide , which corresponds to a mean divergence of 17% [29] , and fits nicely our observations . The low sensibility to divergence of phage recombinases could also result from different sensitivities to methyl-directed mismatch repair ( MMR ) , comprising MutSLH proteins . Indeed , the high fidelity of homologous recombination is caused not only by the intrinsic properties of the enzymes , but also by MMR inhibition of exchange if mismatches are present in recombination intermediates [56] . Classically , defects in MMR provoke a 100-fold increase of HR , either in RecA [57] or Redβ pathways [58] . However HR is less affected by the MMR during the λ lytic cycle: RecA-dependent recombination is enhanced by only 2 to 8 fold in a mutS background , depending on the level of divergence , and Redβ-dependent recombination is not affected at all [19] . Whether mismatch repair is titrated or inhibited during the phage lytic cycle is an open question that deserves further inquiry . In λ , red mutants have a 5-fold reduced burst size ( Fig . S2 ) , which at present remains unexplained ( see [59] for a review discussing this point ) . Our observation that RecA substitutes completely for Red for HR on identical sequences seems to exclude that the loss of viability of red mutants is due to a strict HR defect . RecBCD pathway is inactive in λ infected cells as λ lacks Chi sites and moreover express a RecBCD inhibitor . Further , we could not detect any synergy of the red and recA mutations on plaque sizes ( Fig . S4 ) . Whether Red impacts replication or any other stage of the phage cycle remains to be investigated , as well as why Φ80 recombinase does not impact Φ80 plaque size . Biochemical evidence suggests that Sak and Sak3 , two Rad52-like recombinases of lactococcal phages , act as cofactors of RecA [60] , [61] . Some in vivo work also pointed to such a role for Redβ on non-replicating λ genomes [62] . In the present work , the phage and host recombinases appear rather to work independently from each other , and redundantly on highly similar sequences . The RecA cofactor function of phage-encoded Rad52-like proteins may therefore be a minor activity in vivo . This situation is contrasted with the yeast-encoded Rad52 protein , which is essentially known for its Rad51 cofactor activity . However , Rad52 also performs some repair reactions in a Rad51 independent way [63] . Whether these Rad52 activities are tolerant to diverged DNA is unknown at present . Interestingly , another kind of mobile genetic elements that present genomic mosaicism , the integrative conjugative elements of the SXT/R391 family , has been reported to encode a recombinase of the Rad52 family ( named s065 ) [64] . It was found to act in the formation of hybrid ICEs , independently of RecA . The assay involved 95–97% identical sequences , and RecA was the dominant pathway . Whether s065 becomes dominant when recombination involves more diverged sequences , remains to be investigated . Interestingly , both Orf and Rap proteins have numerous homologs among temperate phages , and the presence of Orf is strongly associated with the presence of a recombinase . Notably , Φ80 possesses homologs of both orf ( gp53 ) and rap . Rap ( recombination adept with plasmid ) increases RecA-dependent recombination between λ and a plasmid sharing perfect identity by a 100-fold [47] . In line with this result , we found that in the RecA pathway , rap deletion results in a 500-fold decrease in recombination with Dlp12 . With the Rac substrate , where the activity of the RecA pathway is minor , rap deletion results in a further 10-fold decrease , again underlining the importance of rap in this pathway . For Redβ-dependent recombination , the decreases due to rap deletion were only 3 and 6-fold for Dlp12 and Rac substrates , respectively . This is probably related to the fact that the Rap substrates , Holliday junctions , are not necessarily formed by Redβ Indeed , Redβ principally recombines by a strand assimilation mechanism , which consists in the single-strand annealing between Redβ-bound single-strand DNA and the lagging strand template of a replication fork , a reaction that does not need a resolution step [65]–[68] . On the contrary , RecA catalyses mainly strand invasion reactions , which generate Holliday junctions [69] , [70] . The Rap protein acts essentially independently from Redβ , a conclusion which agrees nicely with the independent genomic distribution of these two genes . Orf is involved in displacing of SSB from single-stranded DNA , which facilitates RecA binding . We found that orf deletion decreases Redβ dependent recombination by 10-fold , and does not affect the RecA pathway . This is in line with the strong association observed between orf and recombinase genes in phage genomes . This Orf effect might reflect the proportion of cases where Redβ enters in competition with SSB . As Redβ loads onto single-strand DNA immediately after Redα , the two proteins being supposedly in interaction , the Orf activity to remove SSB should not be essential , as SSB might not be present on most of the substrates recombined by Redβ . The presence of all these recombination genes in phage genomes influences their long term evolution . Indeed , here we add evidences that HR leaves frequent traces among phage genomes . We found recently formed mosaics - defined as sequences sharing more than 90% identity - between temperate and defective genomes , but also a few ones among virulent phages . Interestingly , recent gene exchanges were identified only inside the lambda-like group or the P1-P2-Mu group , but not between each group . Traces of homology flanking the mosaic were detected in ∼30% of the mosaics between temperate phages and/or defective prophages . Whether the remaining 70% mosaics were formed by HR but have shorter or masked traces ( Fig . S7 ) , or were created by non-homologous recombination remains an open question . Remarkably , no recent mosaics were found between temperate and virulent phages infecting E . coli . In fact , a well documented case of ancient horizontal gene transfer among a large group of unrelated temperate and virulent phages exists , and concerns the side tail fiber ( stf ) genes [71]–[73] . However , this case is very specific , as these genetic exchanges are most probably driven by stf associated phage invertases that catalyze site-specific recombination [74] . The very low potentiality of virulent phages to acquire genes from temperate phages , which often carry virulence genes , is reassuring for the development of phage therapy as a means to combat bacterial infections . It should be noted however that some virulent phages are in fact former temperate phages that lost their lysogeny module . The case is well documented among dairy phages [75] . Such “virulent” phages , that should rather be named “ex-temperate” , do exchange sequences with temperate and defective prophages [75] and should therefore be avoided for phage therapy , as already indicated [76] . To ascertain the choice of virulent phages for therapeutic use in the future , it may be relevant to conduct an analysis similar to the one presented here , by looking for mosaics ( i . e . Blast matches above 90% identity ) between the selected ( and sequenced ) phage , and all possible sequenced phages and prophages infecting the targeted bacterial species . The relaxed exchange of genetic information among phages might result from different selective pressures acting on viruses as compared to bacteria . The level of HR is indeed the result of contradictory demands . Recombination between homologous sequences must be an efficient process to repair rapidly DNA lesions . On the other hand , recombination between different homologous loci , by creating genomic rearrangement , can destroy chromosomal integrity and functional gene associations and must be avoided . This problem is especially pronounced in eukaryotes , which contain repeated sequences with slight polymorphism , that are prone to recombine together , but bacterial chromosomes also possess repeated elements and related genes . Phage genomes , however , do not possess repeated sequences usually , and can thus better tolerate recombination between diverged sequences without risking chromosomal rearrangements . This might explain the phage tolerance to low fidelity recombination , which then accidentally contribute to rapid phage evolution . Alternatively , as recombination of diverged sequences is sometime advantageous for phages , it is tempting to speculate that it is under positive selection . First , high levels of shuffling inside genes can generate new functions ( reviewed in [77] ) . We found that small sequences of 100 bp within genes are frequently exchanged . This mechanism to generate new genes could explain the much larger viral gene repertoire as compared to bacterial gene repertoire . Secondly , creation of new gene combinations is likely valuable in numerous functions to allow greater phage propagation , e . g . , escape from CRISPR-mediated bacterial immunity systems [78] or expansion of bacterial host range [72] , [79] . Temperate phage genes are alternatively submitted to very different selective pressures depending on the nature of their replicating cycle . Once integrated , prophage DNA becomes subject to the selective forces working on the bacterial chromosome . This explains that temperate phages constitute a reservoir of genes that improve bacterial phenotypes by lysogenisation [76] , [80] . Likewise , defective prophages , that were long considered to be mere genetic junk , are now known to confer a broad range of beneficial phenotypes to the bacterial hosts , with respect to virulence , stress resistance , or even mutation rate [80]–[83] . The present study illustrates that inversely , prophage remnants can be a reservoir of functional lytic cycle genes for temperate phages . Dlp12 lysis module , helpful in certain strains of E . coli for biofilm development [43] , is also active in λ for lysis . The constant exchange of genes between prophage remnants entrapped in host chromosome and active phages , via homologous recombination , blurs even more the distinction between evolutionary pressures acting on temperate phages and their bacterial host , tightly links their evolution , and indirectly accelerates bacterial evolution itself .
All phage and bacterial strains are listed in Table S1 . Unless specified , all gene replacements , deletions or insertions were done by recombineering as already described [84] , with primers specified in Table S2 . The MG1655 stfR::cat ( in Rac prophage ) and tfaQ::cat ( in Qin prophage ) were constructed by inserting the cat cassette from the pKD3 plasmid into the respective genes with the stfR::cat and tfaQ::cat oligonucleotide pairs listed in Table S2 , respectively . The MG1655 ybcV::Kan ( in Dlp12 prophage ) was constructed by P1 transduction from the Keio collection strain ECK0550 [85] . The Urλble phage was constructed from Urλ isolated from the E . coli K12 ancestral strain . The phleomycine/bleomycine resistance gene ble , cloned under the Bacillus subtilis promoter psacB , taken from plasmid pUCphleo ( gift from E . Dervyn ) , was introduced between the tfa and ea47 genes of λ , and oriented as tfa . The presence of ble does not modify phage growth ( Fig . S2 ) , nor the frequency of lysogenization . Complete sequencing of Urλble showed that no important mutation other than the presence of the ble gene and the expected frameshift in the stf gene [40] differentiate our strain from the sequenced λPaPa strain ( Table S2 ) . Deletions of redβ , redα , orf and rap genes were done by recombineering in E . coli K12 Urλble . In the case of the deletion of redβ , a RBS was introduced to maintain the expression of redα , as the two genes are overlapping and redα RBS is inside redβ . Phage Φ80 was isolated in our laboratory from a strain contamination . Phage Φ80ble was constructed by introducing the psacB-ble construct after gp63 , and in the same orientation . The λNec9 and 10 were constructed by replacing redα and redβ by the Rac-encoded recE and recT genes , in λNec4 and λNec6 , respectively ( phage strains listed in Table S1 ) . To do this , the fragment corresponding to the C-terminal ( from a . a . 588 ) of RecE and the full recT gene was added into pKD4 at the BmgBI site , to give pJA100 . The PCR fragment generated from pJA100 , using the pair of oligonucleotides pKD4 ( Table S3 ) was then introduced between the gam and orf60a genes of λNec4 and λNec6 by recombineering [84] . To construct λNec11 and λNec12 , redα and redβ were replaced by erf and exo genes from D3 ( a P . aeruginosa phage ) in λNec4 and λNec6 , respectively . First , these two genes were cloned into pKD4 ( pJA82 , constructed with erf/exo oligonucleotides described in Table S3 ) at the BmgBI site , then the PCR fragment generated from this plasmid ( using the oligonucleotides pKD4 described in Table S3 ) was introduced between the gam and orf60a genes of λNec4 and λNec6 , following the same steps as for the recET constructions . Final constructs were verified by sequencing . Burst size was determined on E . coli MG1655 bacteria growing exponentially in LB broth supplemented with maltose ( 0 . 2% w/v ) and magnesium ( MgSO4 at a concentration of 10 mM ) . When OD600 reached 0 . 2 , 10 ml of the culture were concentrated 10 times , and the phage added at a multiplicity of infection of 0 . 002 . The mix was incubated for 7 minutes at 37°C and then diluted 100-fold and 10 , 000-fold in LB at 37°C . The number of non-adsorbed phage at the start of the phage growth was evaluated by plaque assay after bacterial centrifugation . Samples of the two dilution mixes were taken repeatedly throughout time and assayed immediately for plaque-forming units . The burst size is the factor between final phage number and initial phage number , subtracting the number of non-adsorbed phage at time of dilution . Plaque size was determined after overnight growth on a layer of E . coli MG1655 bacteria embedded into top agarose supplemented with maltose and magnesium ( 2 g/L agarose , 10 g/L tryptone , 5 g/L yeast extract , 5 g/L NaCl , 10 mM MgSO4 , 2 g/L maltose ) . Plates imaging and analysis was realized with the Colony Doc-it imaging station ( UVP , Upland , Canada ) with the same settings for all plates . The phage to be tested was multiplied on the appropriate E . coli strains on plates . Briefly , 104 ( for Red+ λ strains and Φ80 strains ) or 106 ( for Δredβ λ strains ) PFU were mixed to 100 µl of overnight bacterial culture grown on LB maltose . After 5 minutes of incubation , 4 ml of top agar containing MgSO4 10 mM were added and then poured on a fresh LBA plate containing 0 . 2% of glucose . After 6 to 8 hours of incubation at 37°C , when lysis was confluent , 5 ml of water were poured on the top of the plates and incubated at 4°C for two hours . The phage supernatant was then recovered and filtrated at 0 . 2 µm . Stocks were around 1010 PFU/ml for Red positive strains and 109 PFU/ml for Δredβ or Δexo strains . A theoretical calculation based on the measured burst sizes in liquid medium allowed to estimate that under such conditions Redβ+ and Redβ− phages performed similarly 2 . 8 generations during the recombination assay . Φ80 experiments were performed similarly but with few differences: no maltose was added to the medium , and the same amount of RecT+ and RecT− phages ( 104 ) were used for inoculation as no difference in plaque sizes was observed between the two genotypes . 1 ml of E . coli MG1655 culture growing in LB+0 . 2% maltose+ MgSO4 10 mM was added to the phage stock in which recombinants were produced ( m . o . i = 1 ) when the OD600 of the culture reached 1 . The mix was then incubated for 1 . 5 hours at 37°C in a closed 2 ml tube . The total number of lysogenized bacteria was measured by plating the bacteria at the appropriate dilution on phleomycine ( 5 mg/L ) plates . Recombinant phage concentration was estimated by plating on either chloramphenicol ( 20 mg/L ) or kanamycine ( 50 ml/L ) plates . In these conditions , 5 to 10% of the bacteria were lysogenized , as indicated by the proportion of bacteria that acquired phleomycine resistance . Recombinant frequencies are the ratio of chloramphenicol or kanamycine resistant bacteria on phleomycine resistant bacteria . Figures given are an average of at least three independent recombination assays , followed for each of them by at least 3 read outs by lysogenisation . PCR amplification of the λ genomic region susceptible of containing the resistance gene acquired from the host chromosome , if recombination was guided by homology , were realized on more than 20 colonies per genotype ( oligonucleotide sequences are given in Table S3 ) . For each colony tested , a fragment at the expected size was obtained . About 35% of the colonies gave an additional band corresponding to the native size of the λ region tested . A PCR using divergent oligonucleotides at int and ea59 ( that give a product if λ is excised or integrated in multiple copies ) allowed confirming that these colonies were polylysogens . As the same proportion of polylysogens was found for each mutant , this phenomenon was neglected for the calculation of the frequency of recombinants . 2×106 λNec phages ( 100 µL ) were incubated 5 min with 2×107 ( 500 µL ) of exponentially growing C600 recA cells . Then 5 mL of top agar containing 10 mM MgSO4 was poured onto the mix and plated on LB plates , which were incubated 6 h at 37°C , until confluent lysis . 5 mL of a 10 mM MgSO4 solution were poured on the lysed plates , and the whole top agar layer was recovered , vortexed , and centrifuged for 10 min at 4°C . The supernatant , containing the phage , was then recovered and filtered at 0 . 2 µm . To estimate the amount of recombinants produced during lytic growth on plates , the stock was titrated on C600 recA strain to count parental phage , and on a C600 P2 lysogen strain , where only recombinant phage can grow , to count the recombinants . The frequency of recombination was then calculated by the number of recombinant PFU divided by the number of PFU corresponding to parental phages , counted on the recA strain . A set of 50 non-redundant genomes ( less than 92% overall identity or coverage <80% ) of phages infecting E . coli , and having either temperate ( n = 24 ) or virulent ( n = 26 ) lifestyles were chosen ( see Table S4 ) . The 34 non-redundant defective prophages were those of strains MG1655 and 0157:H7 Sakai [37] as well as the prophages identified in IAI39 E . coli strain ( all contained Insertion Sequences ( IS ) in genes important for the phage life cycle , and were therefore classified as defective ) . The analysis was focused on recent exchanges only ( mosaics with more than 90% identity ) , because it aimed at detecting traces of recombination in mosaic flanking sequences . Among the selected phages , some pairs were too closely related to contribute to the analysis: when more than 50% of the smaller genome of the pair shared more than 70% average nucleotide identity with its partner , the pair was excluded ( black squares in Fig . 6 , the strategy used to examine phage relatedness is shown Fig . S7 ) . To detect recent exchanges , a Blastn was run on each genome pair , and all hits of a minimal size of 100 bp and sharing more than 90% identity were selected . Among these hits , some corresponded to IS sequences: they contributed for 9% of the total hits found among temperate phages , but as much as 33% among the defective phage pairs . The rest of the hits were named mosaics . Under the hypothesis of mosaics formed by homologous recombination between diverged sequences , the scenario depicted in Fig . 7 is supposed to take place . Two ancestral phages A and B sharing in average 60% identity except for some more conserved regions at 80% identity , recombine across these 80% identical sequences ( light grey ) . As a consequence , the new piece of DNA in the recombined phage C , when compared to its parent B providing the mosaic , exhibits a 100% identity region ( dark grey ) , flanked by the two 80% identical sequences , above the background level of 60% identity shared by the two genomes at the time of exchange , and somewhat less at the time where genomes are analyzed . Following this scheme , to detect traces of homologous recombination at the vicinity of the mosaics , pairs of 2 kb-long sequences flanking the mosaics were realigned by dynamic programming , and the identity level of successive 50 bp-long windows along the alignment was measured . The 50 bp length was chosen as a close value to the minimum required for Redβ recombination ( 30 bp ) . The probability to encounter such HR traces at random ( last line of Table 1 ) was calculated as described in Text S1 .
|
Temperate bacteriophages ( or phages ) are bacterial viruses that , unlike virulent phages , have the ability to enter a prophage dormant state upon infection , in which they stably replicate with the bacterial genome . A majority of bacterial genomes contain multiple active or defective prophages , and numerous bacterial phenotypes are modified by these prophages , such as increased virulence . These mobile genetic elements are subject to high levels of genetic exchanges , through which new genes are constantly imported into bacterial genomes . Phage-encoded homologous recombination enzymes , or recombinases , are potentially key actors in phage genome shuffling . In this work , we show that gene acquisition in temperate phages is strongly dependent on the presence of sequence homology , but is highly tolerant to divergence . We report that gene exchanges are mainly catalyzed by recombinases found on temperate phages , and show that four different Rad52-like recombinases have a relaxed fidelity in vivo , compared to RecA . This high capacity of exchange speeds up evolution of phages , and indirectly also the evolution of bacteria .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"model",
"organisms",
"genomics",
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"microbiology",
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2014
|
Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases
|
We have characterized a novel pleiotropic role for CymR , the master regulator of cysteine metabolism . We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus . Genes involved in detoxification processes , including oxidative stress response and metal ion homeostasis , were differentially expressed in a ΔcymR mutant . Deletion of cymR resulted in increased sensitivity to hydrogen peroxide- , disulfide- , tellurite- and copper-induced stresses . Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the ΔcymR mutant , with an increase in the intracellular cysteine pool and hydrogen sulfide formation . Since resistance to oxidative stress within the host organism is important for pathogen survival , we investigated the role of CymR during the infectious process . Our results indicate that the deletion of cymR promotes survival of S . aureus inside macrophages , whereas virulence of the ΔcymR mutant is highly impaired in mice . These data indicate that CymR plays a major role in virulence and adaptation of S . aureus for survival within the host .
Cysteine , an important sulfur-containing amino acid , plays a major role in cellular physiology . Cysteine residues are required for the biogenesis of [Fe-S] clusters , are found in the catalytic sites of several enzymes and assist in protein folding and assembly through disulfide bond formation [1] , [2] . In several pathogenic bacteria , links between bacterial virulence and cysteine metabolism have been described . In toxinogenic clostridia and Bordetella pertussis , toxin synthesis is repressed in the presence of cysteine [3]–[5] . Sulfur metabolism genes are also induced upon interaction of Neisseria meningitidis and Mycobacterium tuberculosis with human cells [6] , [7] and decreased virulence of mutants inactivated in various steps of sulfur metabolism has been reported in several microorganisms [6] , [8] , [9] . Cysteine-containing molecules such as thioredoxin and glutathione play an important role in protecting cells against oxidative stress [10] , [11] . In Gram-positive bacteria , mycothiol , coenzyme A and bacillithiol are thought to function as antioxidant thiols [12]–[14] . Several studies have shown that cysteine itself plays a role in bacterial sensitivity to oxidative stress [15]–[21] . More generally , recent data report the existence of links between cysteine metabolism and the response to various stressors such as hydrogen peroxide , superoxide , diamide , nitric oxide , thiol-reactive electrophiles and metal ions [18] , [20] , [22]–[24] . Due to the reactivity of the SH group of cysteine and to its toxicity , cysteine metabolism is tightly controlled in bacteria . The CymR repressor , belonging to the poorly characterized Rrf2 family of regulators , has recently been identified as the master regulator of cysteine metabolism in Bacillus subtilis and Staphylococcus aureus [15] , [25] . CymR forms a regulatory complex with the key cysteine biosynthesis enzyme , CysK ( O-acetyl-serine ( OAS ) thiol-lyase ) , to repress genes involved in cysteine formation pathways [26] . We have recently compared the expression profiles of the S . aureus ΔcymR mutant and the parental SH1000 strain grown in the presence of cystine to characterize global changes in gene expression . The presence of cystine corresponds to conditions where the CymR repressor is active and binds to its direct targets [25] , [26] . This transcriptome analysis identified sulfur metabolism genes including direct CymR targets and cell envelope associated genes as differentially expressed in the ΔcymR mutant . Moreover , we have shown the involvement of the S . aureus CymR regulator in utilization of sulfur sources of human origin and its requirement for efficient biofilm formation [25] . This suggested a potential role for this metabolic regulator in adaptation and survival within the host . S . aureus is an important human opportunistic pathogen responsible for a broad spectrum of diseases ranging from food poisoning and minor skin lesions to life-threatening postsurgical infections in humans [27] . This bacterium is a major cause of nosocomial infections of increasing importance due to the spread of antibiotic resistance , particularly methicillin-resistant strains [28] . Oxidative stress is one of the challenges S . aureus faces during host infection . Following ingestion by phagocytic cells such as neutrophils and macrophages , bacteria are exposed to an oxidative burst [29] . Reactive oxygen species ( ROS ) , such as superoxide anion ( O2− ) , hydrogen peroxide ( H2O2 ) , and hydroxyl radicals ( ⋅OH ) , may also be generated as by-products of endogenous metabolism . Their actions lead to damage of DNA , proteins and lipids [30] . Several other stressors such as diamide , resulting in disulfide stress , and metal ions or metal-containing compounds , including copper and tellurite , can also induce oxidative stress [31] . Staphylococci are highly resistant to potassium tellurite ( K2TeO3 ) , a selective agent often used for their isolation [18] , [32] . Copper is an essential trace element that is toxic to cells at high concentrations and its homeostasis is maintained by copper uptake and efflux systems [33] . Detoxification enzymes that allow transformation of ROS mediate oxidative stress resistance . In particular , in S . aureus , there are two cytoplasmic manganese superoxide dismutases , SodA and SodM that catalyse superoxide radical dismutation . Hydrogen peroxide resulting from this reaction is then eliminated by the action of catalase ( KatA ) . Oxidative stress response and metal ion homeostasis are tightly controlled via a complex regulatory network involving the PerR , Fur , Zur , and MntR repressors [34]–[37] . The PerR regulator of peroxide response controls the expression of genes encoding antioxidants and iron storage proteins and predominantly protects S . aureus cells against H2O2-induced oxidative stress [34] . Fur , the ferric uptake regulator , represses iron uptake genes and positively controls catalase expression , helping to prevent formation of the toxic hydroxyl radical via the Fenton reaction [35] . In addition , the sodA and sodM superoxide dismutases genes are directly regulated by SarA , a global virulence regulator [38] , [39] . In this study , we show that the pleiotropic CymR repressor plays an important role in the response to various stresses and in virulence of S . aureus . Comparative transcriptome analysis showed the significant upregulation of genes involved in detoxification processes , including oxidative stress response and metal ion homeostasis in a ΔcymR mutant . Increased sensitivity of the ΔcymR mutant to H2O2 , disulfide , tellurite and copper stresses might be explained by profound metabolic changes in this mutant . We observed increased survival of the ΔcymR mutant inside macrophages but drastically decreased virulence in mice . This indicates for the first time in S . aureus the existence of a direct link between the control of cysteine metabolism and adaptation to the host .
We have revisited our previously reported expression profiling of ΔcymR mutant and parental SH1000 strains focusing on the role of CymR in the S . aureus stress response . A more detailed analysis of these transcriptome data , carried out by hierarchical clustering ( see Materials and Methods ) , not only showed derepression of directly CymR-dependent sulfur metabolic genes in the ΔcymR mutant [25] , but also revealed increased expression of genes involved in detoxification processes such as oxidative stress response and metal ion homeostasis . These include genes belonging to the PerR regulon such as ahpFC , trxB , ftnA , dps , perR and fur [34] as well as the sodA and sodM genes encoding superoxide dismutases [39] ( Table 1 ) . Notably , the copAP operon encoding a copper efflux system [33] is also strongly up-regulated in the ΔcymR mutant . Quantitative RT-PCR analysis for selected genes was in accordance with transcriptome data and the ratios obtained were generally greater than those resulting from the expression profiling . In particular , we observed about a ten-fold derepression of the ahpF and copA genes in the ΔcymR mutant ( Table 1 ) . In addition , several genes differentially expressed under H2O2 , nitrosative , disulfide or paraquat stress conditions also showed altered expression in the ΔcymR mutant as compared to the parental strain ( Tables 1 and S1 ) . The CymR regulator functions as a transcriptional repressor . Similar levels of derepression in the ΔcymR mutant were observed for stress-related genes and for previously identified direct targets of CymR [25] . To determine whether CymR also directly controls stress-related genes , we performed electrophoresis mobility shift assays ( EMSAs ) with promoter regions of the copA , ahpC , sodA , ftnA and dps genes using crude extracts of a S . aureus ΔcymR mutant overexpressing or not cymR . We have previously used this approach successfully to demonstrate specific direct interactions of CymR with several promoter regions [25] . No specific DNA-protein complexes for the promoters of stress-related genes were formed under these conditions , indicating that these genes are likely to be controlled indirectly by CymR ( data not shown ) . This is in agreement with the absence of a CymR binding motif in the promoter regions of stress-related genes [25] . CymR also controlled the synthesis of the two oxidative stress regulators , Fur and PerR , and several genes derepressed in the ΔcymR mutant belong to the PerR regulon ( ahpFC , trxB , dps , ftnA ) . However , CymR does not appear to bind directly to the promoter regions of either fur or perR in EMSAs ( data not shown ) . To determine the relative roles of CymR , PerR and Fur on the expression of stress-related genes , we carried out quantitative RT-PCR analysis of gene expression in various mutants inactivated for the cymR , perR and/or fur genes . While some genes of the PerR regulon , such as dps and ahpF , are strongly repressed by PerR and negatively affected to a lesser extent by Fur and CymR , we did not observe any synergistic effect of the combined mutations . On the contrary , these experiments revealed an antagonistic effect of the ΔcymR and ΔperR mutations on the expression of the dps and ahpF genes ( Table S2 ) . These genes , belonging to the PerR regulon , were strongly derepressed in a perR mutant as compared to the SH1000 strain . However , in a perR cymR mutant , the derepression of these genes was lower , and similar to that observed in the ΔcymR mutant suggesting antagonistic effects of these two mutations . Inactivation of CymR had similar effects in different mutant backgrounds on the expression of other genes including sodA , sodM and copA . Thus , the CymR effect on stress response does not appear to be mainly mediated by the known oxidative stress regulators , PerR and Fur , even though these regulatory systems may interfere with each other . To compare the changes induced by peroxide stress in the ΔcymR mutant and the parental SH1000 strain , we analyzed the expression of several stress-related genes after an H2O2 challenge ( Table S2 ) . As expected , we observed a strong induction of ahpF and dps in strain SH1000 . However , the induction of ahpF expression by H2O2 was lost in a ΔcymR mutant while the extent of dps induction was reduced in this mutant as compared to SH1000 . This illustrated that the cymR deletion resulted in an altered stress response at the molecular level . S . aureus can survive a wide range of stresses during its life cycle [29] . As mentioned above , a set of genes involved in stress response was differentially expressed in the ΔcymR mutant as compared with the parental strain . The role of CymR in responses to various stress stimuli was tested using either disk diffusion assays or survival analyses . In disk diffusion assays , the ΔcymR mutant was significantly more sensitive than SH1000 to 1 M diamide , a specific thiol oxidant that causes disulfide stress ( Fig . 1A ) . The SH1000 strain showed high resistance to 200 mM K2TeO3 in disk diffusion assays without a detectable growth inhibition area around the 6 mm-disk . By contrast , the ΔcymR mutant was extremely sensitive to tellurite stress with a growth inhibition area of 28 mm under the same conditions ( Fig . 1B ) . Bacterial detoxification of tellurite leads to formation of insoluble tellurium ( Te° ) , appearing as black deposits in the growth plates . We also observed increased sensitivity of the ΔcymR mutant to copper stress in disk diffusion assays carried out with 200 mM CuSO4 ( Fig . 1C ) . Sensitivity of the ΔcymR mutant to other metal ions ( FeCl3 , Pb ( CH3COO ) 2 , MnSO4 , CoCl2 , ZnSO4 , HgSO4 and NiSO4 ) was similar to that of the parental strain ( data not shown ) . In all cases , increased stress sensitivity of the ΔcymR mutant could be complemented by the introduction of plasmid pDIA5780 carrying the cymR gene ( Fig . 1 ) . With respect to oxidative stress , no significant differences in sensitivity were observed between the ΔcymR mutant and the parental strain in disk diffusion assays in the presence of paraquat ( 2 M methyl viologen ) ( data not shown ) . Viability of the ΔcymR mutant and the SH1000 strain grown in TSB medium with cystine was also tested 1 h after addition of 20 mM H2O2 . A 1000-fold reduction in survival was observed for the ΔcymR mutant as compared to the parental strain and viability was restored in a ΔcymR mutant complemented by pDIA5780 ( Fig . 2 ) . We further tested the oxidative stress response in mutants inactivated for CymR , PerR and/or Fur . We observed a 10-fold decreased viability in a ΔperR ΔcymR mutant as compared to the ΔcymR mutant and extremely low survival capacities for the ΔcymR ΔperR Δfur mutant as compared to the ΔperR Δfur mutant ( data not shown ) . Taken together , our results indicate that CymR plays a major role in staphylococcal stress response , independently of other known regulators . Increased oxidative stress sensitivity of the ΔcymR mutant could be due to elevated intracellular cysteine pools , driving production of hydroxyl radicals via the Fenton reaction and leading to cellular damage [20] . We tested the effect of cystine on stress sensitivity in a ΔcymR background . We observed decreased sensitivity to tellurite and copper stress of the ΔcymR mutant in the presence of cystine in several genetic backgrounds including perR and/or fur mutants ( Fig . S1 and S2 ) . By contrast , the ΔcymR mutant showed increased sensitivity to both diamide and H2O2 stress in the presence of cystine ( data not shown ) . Similarly , the decrease in H2O2 and diamide stress resistance in the ΔcymR mutant due to perR inactivation was more pronounced in the presence of cystine ( data not shown ) . In agreement with altered oxidative stress response , the presence of cystine affected the expression of genes associated with stress response in the ΔcymR mutant as shown by quantitative RT-PCR analysis ( Table 1 ) . These genes were differentially expressed in a ΔcymR mutant only in the presence of cystine . The altered stress response linked to cymR inactivation can be explained by an imbalance in thiol redox status . The derepression of genes involved in cystine uptake and cysteine biosynthesis from sulfide and homocysteine may result in cysteine accumulation in the ΔcymR mutant . Analysis of the intracellular pools of several metabolites using HPLC revealed a strong up to 68-fold increase of the intracellular cysteine concentration in the ΔcymR mutant in comparison with the parental strain during growth in TSB medium with cystine ( Fig . 3A and Table S3 ) . We also observed a 2-fold increase in cystine and cystathionine content and a 6-fold increase in homocysteine content . This analysis also revealed a 36-fold increase in the cysteine to cystine ratio in the ΔcymR mutant as compared to the SH1000 strain , reflecting the imbalance in thiol redox status of the cell in the absence of CymR . The estimated glutamate concentration decreased 4-fold while the concentration of other amino acids was unchanged in the ΔcymR mutant as compared to the SH1000 strain . As cysteine is probably toxic for the cell at high concentrations , it may then be rapidly transformed into hydrogen sulfide , pyruvate and ammonia by cysteine desulfhydrases . The MccB , MetC and CysK enzymes have cysteine desulfhydrase activities in B . subtilis [40] and orthologous proteins are present in S . aureus . We then compared production of hydrogen sulfide , the main product of cysteine catabolism , in the ΔcymR mutant and SH1000 strains grown in the presence of cystine . An important increase in H2S production was observed in the ΔcymR mutant in a qualitative lead-acetate-paper assay and a 40-fold increase was further confirmed by an H2S quantification assay ( Fig . 3B ) . The introduction of a plasmid carrying the intact cymR gene into the ΔcymR mutant led to a level of H2S production similar to that observed in the parental SH1000 strain ( Fig . 3B ) . In the absence of cystine , H2S production was undetectable in the ΔcymR mutant and SH1000 strains ( data not shown ) . We also measured the pH of the TSB medium after 16 h culture in the presence of cystine . A significant acidification of the medium was observed with the ΔcymR mutant as compared with the parental strain ( Fig . 3C ) . This may be associated with pyruvate production from cysteine and/or with a decreased capacity to catabolize organic acids . These changes in the pH of the growth medium of the ΔcymR mutant could be reversed by the introduction of a plasmid carrying the cymR gene . Since the CymR regulator was shown to be involved in stress adaptation , we tested its role in the S . aureus survival inside macrophages . Professional phagocytes are the first line of defense encountered by pathogens during the infection process . Since it has been shown that S . aureus is particularly efficient in persisting within professional phagocytes [41] , [42] , the survival of the parental SH1000 strain and the ΔcymR derivative inside RAW 264 . 7 murine macrophages was investigated over a 3-day period ( Fig . 4A ) . We also examined the survival of a sodAsodM mutant as a positive control of macrophage stress generation . As shown in Fig . 4B , clearance of the sodAsodM mutant was much faster than that of the wild type strain , directly correlating its increased stress sensitivity with lowered survival within RAW 264 . 7 murine macrophages . We measured the internalization rates of the parental and ΔcymR mutant strains , which were identical ( about 90% of entry for a multiplicity of infection ( m . o . i ) = 5 ) . Viable bacterial counts inside macrophages over time allowed us to demonstrate that the ΔcymR mutant is more resistant to macrophage stress than the parental strain ( Fig . 4A ) . Professional phagocytes are part of the host anti-microbial defense and the ΔcymR mutation seems to favor intracellular survival of S . aureus . We therefore tested whether this selective advantage could have an effect on global S . aureus virulence , using a murine intraperitoneal infection model with BALB/c mice since this lineage has been shown to be susceptible to S . aureus infection [43] . We infected mice intraperitoneally with 3 . 108 colony-forming units ( CFU ) of either the SH1000 strain or the ΔcymR mutant . As shown in Fig . 5A , while mice infected by the parental strain were all dead ( 7 mice/7 ) 18 h after inoculation ( black curve ) , those infected with the ΔcymR mutant displayed a significant extension of time-to-death and 3 mice ( out of 7 ) were still alive 6 days post-infection ( grey curve ) . As a control , we tested a sodA sodM mutant previously described as impaired in its capacity to develop abscesses in a mouse subcutaneous infection model [39] . We observed that decreased mouse mortality linked to the bacterial sodA sodM inactivation was equivalent to that caused by cymR inactivation ( Fig . 5A , dotted lines ) . In order to follow bacterial dissemination within the animal , we also infected mice with a sub-lethal dose ( 5 . 107 CFU ) of either the SH1000 strain or the ΔcymR mutant . We followed bacteraemia at 1 and 3 days post-infection and quantified the renal load 7 days post-infection . The bacterial load drastically decreased in the cymR mutant ( Fig . 5B ) . In the blood , one -day post-infection , there was at least 1-log-unit decrease for the ΔcymR mutant compared to the wild type strain ( Fig . 5B , left panel ) . The difference between the two strains increased 3 days post-infection with more than 3-log-unit less bacteria in the blood in a ΔcymR background . The bacterial load in the kidneys also showed a colonization defect of the ΔcymR mutant since 7 days post-infection there was more than 1-log-unit less CFU with the ΔcymR strain than with SH1000 ( Fig . 5B , right panel ) . Thus , although the ΔcymR mutation appears to be beneficial with regard to survival within professional phagocytes , it largely decreases global S . aureus virulence . In order to determine whether the production of major virulence factors is affected in the ΔcymR mutant , we performed hemolytic activity assays on blood agar plates . These experiments clearly show that the ΔcymR mutant is impaired in its capacity to produce δ-hemolysin as compared to the parental SH1000 strain ( Fig . 5C , left panel ) . On sheep blood agar plates , we can distinguish between δ- and β-hemolysin [44] . As shown on Fig . 5C ( right panel ) we have confirmed that the δ-hemolysin production was strongly reduced in the cymR mutant strain with respect to the parental strain , whereas β-hemolytic activity was not affected by the mutation . α-hemolytic activity was not tested since it is inhibited by β-hemolysin [45] . This significantly altered δ-hemolysin production likely contributes to the virulence defect observed in the absence of CymR .
In S . aureus , diamide and H2O2-induced stresses result in induction of several direct CymR target genes including mccAB , cysM , tcyABC and metNPQ , indicating an increased requirement for cysteine under these conditions [46] , [47] . Conversely , here we show an upregulation of part of the peroxide stress PerR regulon , of superoxide stress ( sodA and sodM ) and copper efflux system ( copAP ) genes together with other stress-related genes in a S . aureus mutant lacking the master regulator of cysteine metabolism , CymR . However , the effect of CymR on these genes appears to be indirect . We investigated possible connections between CymR and the PerR and Fur regulators of oxidative stress response . CymR appears to affect stress response independently of these regulators , since the effect of cymR inactivation on stress sensitivity and gene expression is still observed in perR and/or fur mutant backgrounds . We propose that these different stress response systems may recognize a common stress signal that is present in the ΔcymR mutant . This signal could be related to thiol-redox homeostasis imbalance and to increases in intracellular cysteine pools or changes in other cysteine-related compound content including H2S ( Fig . 6 ) . Cysteine is one of the major cellular thiols in S . aureus . Metabolite content estimation revealed a 36-fold increase in the cysteine to cystine ratio in the ΔcymR mutant reflecting the imbalance in thiol redox status in the absence of CymR . It is worth noting that the simultaneous induction of the PerR and CymR regulons as well as metal-ion efflux systems , including CopA , by thiol-reactive electrophiles leading to imbalance of thiol-redox homeostasis has been reported in B . subtilis [24] . In agreement with this metabolic hypothesis , the addition of cystine to the culture medium affected stress-related phenotypes of the ΔcymR mutant . Recent studies suggested the existence of links between cysteine and/or cysteine-containing molecules and oxidative stress defense in several bacterial systems with positive or negative effects of this amino acid . Cysteine protects Lactobacillus reuteri from H2O2 stress while cysteine or thiol-derived compounds such as glutathione are important for defense against damages [10] , [19] . By contrast , in E . coli , a 8-fold increase in intracellular cysteine concentrations promotes oxidative DNA damages by driving the Fenton reaction due to the efficient reduction of Fe3+ by cysteine [20] . We observed a strong 68-fold increase in the intracellular cysteine pool in the ΔcymR mutant grown in the presence of cystine , leading to a 1000-fold increase in sensitivity to H2O2 stress . However , the addition of extracellular or cell-penetrating iron and copper chelators ( dipyridyl , desferal , neocuproine and ferrozine ) had no positive effect on viability of the ΔcymR mutant after an H2O2 challenge ( data not shown ) . This suggests more complex mechanisms of altered stress response in addition to the Fenton reaction-mediated process , as recently proposed for other microorganisms [48] , [49] . High cysteine levels are correlated with the production of H2S by cysteine desulfhydrases ( MccB , MetC , and CysM ) ( Fig . 6 ) . H2S increases the formation of H2O2 and other ROS in several organisms and inhibits human superoxide dismutase activity and S . aureus catalase activity in acid medium [50]–[52] . This could also contribute to the oxidative stress sensitivity of the ΔcymR mutant . The S . aureus ΔcymR mutant exhibited increased susceptibility to disulfide , copper , tellurite , and H2O2-induced oxidative stresses . Diamide , tellurite and copper can each cause both oxidative stress as well as an imbalance in the thiol redox status of the cytoplasm ( Fig . 6 ) . A recent proteomic study that analyzed the diverse S . aureus responses to H2O2 , diamide and paraquat [47] indicates a close relationship between disulfide and H2O2 stress responses , in agreement with the similar behavior of the ΔcymR mutant toward these compounds . Tellurite ( TeO32− ) is toxic for most forms of life , even at very low concentrations . The genetic and biochemical basis underlying bacterial tellurite toxicity is still poorly understood [32] . However , several tellurite resistance determinants have been identified , mainly in E . coli , suggesting mechanisms involving cysteine metabolism and cellular oxidative stress due to its strong oxidizing ability . Cysteine synthases from various bacteria and molecules containing cysteine including glutathione are involved in tellurite resistance via reductive detoxification of this compound [32] . In S . aureus , the cysM mutant defective in cysteine synthase is more sensitive to tellurite , probably due to cysteine depletion [18] . Inactivation of cymR also leads to extreme sensitivity to tellurite , even greater than that of the cysM mutant . However , the addition of cystine to the culture medium resulted in a drastic decrease in tellurite toxicity in both the cymR and cysM mutants ( Fig . S1 and data not shown ) . The accumulation of cysteine and/or H2S ( Fig . 3 ) under these conditions could promote tellurite detoxification leading to the formation of nontoxic tellurium . As observed with tellurite , a ΔcymR mutant is more sensitive to copper stress than the parental SH1000 strain , and this effect is more pronounced in the absence of cystine ( Fig . S1 ) . The copA and copP genes encoding a copper efflux system involved in maintaining copper homeostasis in S . aureus [33] are strongly upregulated in the ΔcymR mutant in the presence of cystine ( Table 1 ) . Further studies will be required to characterize the molecular mechanisms linking CymR to tellurite and copper sensitivity . The intracellular cysteine level is kept within a narrow range to address both the cysteine supply for protein synthesis and the production of other essential molecules and the necessity of maintaining cysteine levels below the toxicity threshold . Elevated cysteine or H2S levels must also be avoided as they may lead to cysteine autooxidation , the production of ROS and protein thiol oxidation [53] , [54] . The CymR regulator in S . aureus plays an essential role in maintaining intracellular cysteine levels . However , H2S together with cysteine may be a signal recognized by several oxidative stress defense systems in S . aureus . During infection , this pathogen must cope with host phagocytic attack , accompanied by the release of a number of ROS including superoxide anion , hydrogen peroxide , hydroxyl radical , peroxynitrite and hypochlorous acid [29] , [55] . In this study , we showed that the ΔcymR mutant has an increased long-term survival rate within macrophages . This result could be related to increased transcription in the ΔcymR mutant of a number of genes known to be differentially expressed under several host-related stress conditions , including H2O2 , nitrite and nitrosative stresses ( Tables 1 and S1 ) . The differences observed in vitro after a H2O2 challenge and in vivo in macrophages may be explained by variations in the level of H2O2 formed as well as a multitude of reactive species produced inside macrophages . Despite the fact that cymR inactivation promotes survival of S . aureus inside the macrophages , virulence of the ΔcymR mutant in mice is drastically impaired as previously observed for an S . aureus strain lacking catalase and beta-toxin [56] . During the infectious process , the CymR regulator influences different virulence pathways . Indeed , we have shown that mice infected with a lethal dose of the SH1000 strain died very rapidly ( less than 18 hours post-inoculation ) , suggesting that toxemia is responsible . Accordingly , we observed that the ΔcymR mutant has impaired hemolytic activity . Reduced hemolysin production may be responsible at least in part for the virulence defect observed in the absence of CymR . In addition , bacteraemia and the bacterial load in kidneys following infection with a sub-lethal dose were significantly decreased in the absence of CymR . Bacterial metabolism has been linked to virulence of Staphylococci by several studies [57] . Some CymR regulon cysteine metabolic genes ( mccA , cysM and tcyAB ) were shown to be differentially expressed upon internalization of S . aureus in human epithelial cells [58] . In the ΔcymR mutant , we also observed differential expression of genes known to be affected upon internalization in human cells ( Table S4 ) . Our results suggest that the link between cysteine metabolism control by CymR , stress response and virulence is likely indirect and may be integrated into the general concept that alterations of the bacterial metabolic status create metabolic signals that may be “sensed” by the regulatory network controlling virulence determinants , as proposed by Somerville and Proctor [57] . One hypothesis may be that the alteration in redox cell status and metal ion homeostasis modulates the activity of virulence and stress-response regulators , including SarA , SarZ and PerR . Indeed , recent results have shown that the central virulence regulator , SarA , is responsive to redox and pH [59] and that SarZ is a redox active regulator [60] , [61] . Thus , cymR inactivation may affect redox-mediated virulence control in S . aureus at several levels of the regulatory network . Indeed , a number of genes differentially expressed in strains deficient for virulence regulators ( such as SarA , AgrA , ArlSR , SaeSR , Rot and MgrA ) showed altered expression in the ΔcymR mutant in comparison with the parental SH1000 strain ( Table S4 ) . The role of CymR in virulence is most likely multifactorial since , as we show here , it controls several steps in the infectious process , including dissemination within the host and colonization of different organs . The ΔcymR mutant is also affected in biofilm formation and in synthesis of exotoxins ( hemolysins ) and cell envelope components , functions that could be important for host colonization [25] . Our data bring important insights into understanding the interactions between sulfur metabolism and virulence of this major pathogen and suggest interesting possibilities for metabolic strategies to attenuate S . aureus infection . Proteins involved in controlling cysteine metabolism may therefore represent potential targets for antibacterial compounds aimed at treating staphylococcal infections .
All the animal experiments described in the present study were conducted at the Institut Pasteur according to the European Union guidelines for the handling of laboratory animals ( http://ec . europa . eu/environment/chemicals/lab_animals/home_en . htm ) and were approved by the Institut Pasteur animal care and use committee . Bacterial strains used in this study are listed in Table 2 . S . aureus was grown in brain heart infusion ( BHI ) ( Oxoid ) or tryptic soy broth/agar ( TSB/TSA ) ( Difco ) [25] . Antibiotics were added at the following concentrations: chloramphenicol , 5 µg ml−1; erythromycin , 1 or 5 µg ml−1 , tetracycline , 5 µg ml−1 and kanamycin , 50 µg ml−1 . S . aureus was transformed by electroporation [62] . The chromosomal perR , fur , sodA and sodM mutations [34] , [35] , [39] ( Table 2 ) were introduced into the SH1000 strain or ΔcymR mutant by Φ11 phage transduction [63] . Disk diffusion assays were performed as follows: 5 ml of TSB or BHI top agar ( 0 . 7% , wt/vol ) was seeded with 100 µl of an exponential-phase S . aureus culture in TSB or BHI medium ( OD600 = 0 . 2 ) and used as an overlay on a TSA or BHI agar plates . When indicated 2 mM cystine was added to the culture medium and to the agar plates . Sterile 6 mm disks were placed on top of the overlay , and 10 µl of either 1 M diamide , 200 mM K2TeO3 , 200 mM CuSO4 , 10 M H2O2 or 2 M paraquat ( methyl viologen ) ( Sigma ) was added to the disk . Diameters of growth inhibition zones were measured after 24 h of incubation at 37°C . Hydrogen peroxide resistance assays were carried out as previously described with some modifications [18] . Cells were grown in TSB medium with or without 2 mM cystine . At exponential phase ( OD600 = 0 . 2 ) , H2O2 was added to a final concentration of 20 mM in TSB medium . After 1 h of incubation , cells were serially diluted in BHI medium and viability was assessed by overnight growth on BHI agar . Previously obtained transcriptome data [25] were analyzed using hierarchical clustering as the less a priori-based method for transcriptome data exploitation . Uncentered Pearson correlation was used for distance calculation , and the average-linkage clustering was performed on logarithmically transformed data for gene expression ratio in SH1000 versus ΔcymR mutant . We used the Michael Eisen Cluster software program , followed by tree diagram visualization with TreeView [64] . This analysis revealed several specific clusters including the group of genes upregulated in the ΔcymR mutant and involved in detoxification processes . Strains were grown in TSB medium with 2 mM cystine to an OD600 of 1 ( with 1/10 medium-to-flask volume ratio at 160 rpm shaking ) . H2S production was revealed using lead-acetate paper ( Macherey-Nagel ) which turned black following incubation for up to 3 h at 37°C . H2S production was quantified by the modified methylene blue reaction as previously described [65] . Intracellular concentrations of amino acids and other ninhydrin-reactive compounds were estimated using high-pressure liquid chromatography ( HPLC ) [26] , [66] . Briefly , cells were suspended in a sulfosalicylic acid buffer ( 3% final concentration ) and disrupted using a FastPrep apparatus ( Bio101 ) . Supernatant samples were analyzed by cation-exchange chromatography , followed by ninhydrin postcolumn derivatization as previously described [66] . Total RNA was isolated from S . aureus strains grown in TSB with or without 2 mM cystine as previously described [15] . For H2O2 stress induction bacteria were incubated with 20 mM H2O2 for 10 minutes followed by RNA extraction . Quantitative real-time PCR analysis was performed as previously described [25] . Oligonucleotides used in this study are listed in Table S5 . DNA fragments containing various promoter regions were amplified by PCR using specific primers and chromosomal DNA of S . aureus strain SH1000 . PCR products were labeled using [γ32P]ATP 5′-end labeled specific primers . Protein-DNA complexes were formed in 10 µl reaction volumes , by incubating labeled DNA fragments with various amounts of crude extracts of the S . aureus ΔcymR mutant carrying either pDIA5780 ( pMK4-cymR ) or pMK4 as previously described [66] . Murine macrophage RAW 264 . 7 cells were used for bacterial survival assays as previously described [67] with some modifications . Briefly , bacteria were grown in TSB until OD600 ∼2 . Cultures were washed in PBS and adjusted to the desired inoculum in RPMI 1640 medium ( Gibco ) , and CFU counts were verified by plating serial dilutions on TSA plates . Macrophages grown to confluence were counted and incubated with bacteria ( m . o . i . ∼5 ) in RPMI 1640 at 37°C with 5% CO2 for 1 h to allow bacterial phagocytosis . They were then washed once with RPMI and incubated in RPMI-10%Fetal Calf Serum-streptomycin ( 100 µg ml−1 ) /penicillin ( 100 U ml−1 ) . At the indicated times , infected macrophages were washed once with RPMI and then lysed by incubation in ice-cold water for 15 min . CFU counts were determined by plating serial dilutions on TSA plates . Female inbred BALB/c mice ( 4 to 5 weeks old ) were obtained from Janvier Laboratories ( Le Genest-St-Isle , France ) . S . aureus strains ( SH1000 and the ΔcymR or the sodA sodM derivatives ) were grown in TSB until OD600 ∼2 , cells were pelleted and resuspended to the appropriate concentration in sterile PBS . Mice were injected by the intraperitoneal route with ∼3 . 108 CFU ( mortality assays ) , or 5 . 107 CFU ( sub-lethal dose ) in 0 . 2 ml PBS . For mortality rate assays , mice were monitored daily for signs of illness and death . At the end of the experiment , surviving mice were humanely sacrificed ( CO2 asphyxiation ) . Results were statistically analyzed by the log-rank test using Prism 5 . 0b software ( GraphPad Software , San Diego , CA ) . For measuring the bacterial load in blood and kidneys , animals were followed during 7 days post-infection . Blood samples were collected from the retro orbital sinus 1 and 3 days post-infection , immediately mixed with heparin and plated on TSA plates . Seven days post-inoculation , mice were sacrificed ( CO2 asphyxiation ) , and the kidneys were removed and homogenized for determination of CFU counts . Hemolysis was detected on Columbia blood agar plates ( BioMérieux ) . Strains were grown overnight in TSB medium and then either spotted ( 20 µl ) on horse blood agar plates or streaked on sheep blood agar plates . The plates were incubated for 24 hours at 37°C , and specific hemolytic activities ( β- and δ-hemolysins ) were identified as previously described [44] .
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Staphylococcus aureus is a very harmful human pathogen that is a major cause of nosocomial infections . Humans have developed sophisticated defense strategies against invading bacteria , including the innate immune response , with the generation of an oxidative burst inside phagocytic cells . Staphylococcal infections are extremely difficult to eradicate due to the remarkable capacity of these bacteria to adapt to different environmental conditions both inside and outside the host organism . Sulfur metabolism is essential for all living organisms and is tightly controlled by regulatory proteins . In this paper , we revealed an important role for CymR , a major regulator of sulfur metabolism , in adaptation of S . aureus to the host environment . Inactivation of the gene encoding this regulator in S . aureus leads to a mutant bacterium with increased vulnerability to stress conditions including oxidative stress encountered inside the host . More importantly , the deletion of the cymR gene strongly affected the interaction of S . aureus with its host , leading to impaired virulence in mice . Our results place CymR among the potential targets for attenuation of S . aureus infections .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"microbiology/microbial",
"physiology",
"and",
"metabolism",
"microbiology/cellular",
"microbiology",
"and",
"pathogenesis"
] |
2010
|
The Pleiotropic CymR Regulator of Staphylococcus aureus Plays an Important Role in Virulence and Stress Response
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Here we set out to standardize long-lasting , visually-attractive devices for Glossina swynnertoni , a vector of both human and animal trypanosomiasis in open savannah in Tanzania and Kenya , and in neighbouring conservation areas used by pastoralists . The goal was to determine the most practical device/material that would induce the strongest landing response in G . swynnertoni for use in area-wide population suppression of this fly with insecticide-impregnated devices . Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of traps and targets of different sizes and colours , with and without chemical baits , at different population densities and under different environmental conditions . Adhesive film was used as a simple enumerator at these remote locations to compare trapping efficiencies of devices . Independent of season or presence of chemical baits , targets in phthalogen blue or turquoise blue cloth with adhesive film were the best devices for capturing G . swynnertoni in all situations , catching up to 19 times more flies than pyramidal traps . Baiting with chemicals did not affect the relative performance of devices . Fly landings were two times higher on 1 m2 blue-black targets as on pyramidal traps when equivalent areas of both were covered with adhesive film . Landings on 1 m2 blue-black targets were compared to those on smaller phthalogen blue 0 . 5 m2 all-blue or blue-black-blue cloth targets , and to landings on all-blue plastic 0 . 32–0 . 47 m2 leg panels painted in phthalogen blue . These smaller targets and leg panels captured equivalent numbers of G . swynnertoni per unit area as bigger targets . Leg panels and 0 . 5 m2 cloth targets show promise as cost effective devices for management of G . swynnertoni as they can be used for both control ( insecticide-impregnated cloth ) and for sampling ( rigid plastic with insect glue or adhesive film ) of populations .
Glossina swynnertoni Austen ( Diptera , Glossinidae ) is restricted to open savannah in north-western Tanzania and south-western Kenya , extending from Tarangire in the south through Manyara to the Serengeti plains , and into the Maasai Mara in the north [1] . Swynnerton [2] found it at 900–1800 m above sea level and considered temperature , humidity , vegetation and the presence of wildlife as the key factors controlling its distribution . It is a vector of both human and animal trypanosomiasis in wildlife reserves and in neighbouring conservation areas used by pastoralists [3]–[6] . The challenge is to minimize disease transmission through effective management of the vector in the presence of abundant wildlife reservoirs , especially in protected areas . G . swynnertoni is in the savannah or morsitans group of tsetse . Considerable progress has been made in developing visually-attractive control devices such as traps [7] and insecticide-impregnated targets [8] , [9] for this group . However , no comparable effort has been made to develop cost-effective devices for G . swynnertoni since initial tests were conducted in Tanzania in 1991–1993 [10] . Presently , local control of this species is being attempted with techniques refined for other species . For example in Kenya , pastoralists deploy insecticide-impregnated targets or apply pyrethroid sprays to livestock [11] in a largely uncoordinated effort at vector control . Savannah tsetse are attracted to artificial objects of modest size [9] that are conspicuous relative to their immediate environment [12] . Traps and targets of phthalogen blue ( peak reflectance at 465 nm ) and/or black cloth of about 1 m in dimension are typically effective for this group of tsetse [13] . G . swynnertoni , like G . morsitans [8] , is nevertheless difficult to catch with simple stationary devices , as movement and other subtle visual cues [14] are likely involved in host-seeking behaviour . Vehicle patrols or “fly-rounds” were previously used for sampling G . swynnertoni [15] , [16] , but recent studies now mostly use blue-black cloth traps designed for other tsetse [6] . There have been few comparative tests of the efficacy of modern tsetse traps or targets relative to other methods for collecting G . swynnertoni outside of the works of Ndegwa & Mihok [17] and Ndegwa et al . [18] . These studies showed that trap designs other than the S3 trap were relatively inefficient compared to a 1 m2 black sticky target . Unlike Glossina pallidipes Austen ( Diptera , Glossinidae ) , baiting traps with attractants such as acetone , 1-octen-3-ol , phenols and/or cow urine does not result in large increases in catch [10] , [19] . Within the Africa-wide WHO-TDR initiative to develop innovative control strategies for tsetse , we set out to standardize long-lasting , visually-attractive devices for G . swynnertoni . The trials were based on existing trap/target/bait technology following a similar experimental approach throughout Africa [20] . Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of pyramidal traps and targets in phthalogen blue and various alternatives at different population densities and under different environmental conditions . A simple enumeration method ( sticky film ) was used at these remote locations to compare trapping efficiencies of devices made of well-characterized colour-fast fabrics ( and a blue-painted plastic ) . The relative performance of devices was also compared with and without chemical baits . Various alternatives were compared to a standard phthalogen blue and black pyramidal trap , which has been the tsetse survey device of choice in Tanzania in recent years [4] . The overall goal was to determine the most practical device/material that would induce the strongest landing response in G . swynnertoni for future use in area-wide population suppression of this fly with insecticide-impregnated devices .
Studies were conducted in open Acacia-Commiphora-Balanites dry savannah woodland in pastoral areas near the Maasai Mara National Reserve in Kenya and deep within the neighbouring Serengeti National Park across the border in Tanzania . Wild hosts remain abundant in the Serengeti but have declined considerably in the Mara in the last few decades [21] . Livestock were present only in the Mara . In the Serengeti , three sets of studies took place at Death Valley 2°19′51″ S , 34°49′60″E at an altitude of 1548 m near Seronera . A first set of experiments was conducted in 2009 in the wet season ( July ) and repeated at the same sites in the dry season ( October ) . In 2010 and 2012 , a second and third series were conducted at the same location , both at the start of the dry season ( September ) . G . pallidipes was also present in scattered evergreen thickets . Findings for this species are reported where captures were adequate for analysis . In the Mara , studies were undertaken at the Olarro hills 1°25′45 . 4″S , 35°35′0 . 9″E at an altitude of 1910 m , and at the Nyonorri hills at 1°27′18 . 9″S , 35°33′43 . 5″ E at an altitude of 1877 m . Unbaited and baited trials were conducted at these locations separately in the nominal wet season ( June ) , and then repeated only at one location ( Nyonorri ) in the subsequent dry season ( November ) of 2009 . The shift in locations was necessitated by the unanticipated use of pyrethroid spray-ons by pastoralists at Olarro . Habitats have been extensively altered by human activities in pastoral areas near the Mara . Hence , G . swynnertoni is mainly found in remnant woodlands along hillsides that receive moisture from the highlands all year-round . A severe drought was underway during these tests; hence wild hosts were present only in the nominal “wet season” . Vegetation cover was particularly sparse during all seasons in the Mara due to the drought . Cover in the Serengeti was not affected as much , and hence wet and dry season contrasts were more typical of natural climatic cycles in Tanzania . In 2009 , three catching devices were tested: standard cloth pyramidal traps [22] , rectangular cloth targets , and smaller all-blue “leg” panels [23] . The dimensions and design of the targets and leg panels were chosen to reflect current practices in East Africa and are summarised in Table 1 . Devices were set in the open , 30 cm off the ground; vegetation was removed from within a few metres of each site . The targets in Kenya were 1 . 5 m2 ( 1 . 5 m wide by 1 m high ) divided vertically into equal rectangles of blue and black cloth [24] . In Tanzania , the targets were the same size but were divided vertically into three equal rectangles of blue-black-blue [25] . Leg panels in Kenya were made of blue cloth with a surface area of 0 . 32 m2 ( 65 cm wide by 46 cm high for the upper “torso” , plus two “legs” 15 cm high by 8 cm wide ) . Two kinds of slightly larger leg panels were tested in Tanzania . One was 0 . 47 m2 blue cloth ( 70 by 64 cm plus 14 by 9 cm legs ) and the other was 0 . 45 m2 blue-painted plastic ( 90 by 45 cm plus 15 by 15 cm legs ) . The plastic was 3–4 mm thick and was painted glossy phthalogen blue . All targets were mounted on supports that allowed for limited rotational movement in the wind . The wet season trial in Tanzania occurred under particularly windy conditions . Two blue fabrics were tested: C180 Azur 623 phthalogen blue 100% cotton ( 180 g/m2 , TDV , Laval , France ) with a reflectance peak at 460 nm as measured with a Datacolor Check Spectrophotometer ( Datacolor AG , Dietlikon , Switzerland ) , referred to hereafter as standard blue cotton , and turquoise blue 65% polyester/35% viscose ( 234 g/m2 , Q10067 Sunflag , Nairobi , Kenya ) with a peak at 480 nm . The phthalogen blue paint on the plastic leg panel had a peak of 460 nm . A 100% polyester black ( 225 g/m2 , Q15093 Sunflag , Nairobi ) was used for all devices in all trials described here . To monitor the number of tsetse landing on cloth targets and leg panels , one-sided adhesive film ( 30 cm wide rolls , Rentokil FE45 , UK ) was stitched with thread to both sides of the trapping devices . However , in 2009 in Tanzania only the lower 60 cm of the targets was covered . Plastic leg panels in Tanzania were coated with a non-setting shiny glue ( Temoocid , Kollant , Italy ) . Transmittance spectra for both adhesives are compared to polybutene in Figure 4 . 4 in IAEA TECDOC 1373 [26] . All of these adhesives are highly transparent in the visible spectrum , but Rentokil film absorbs significantly in the ultraviolet ( <400 nm ) . In 2010 , two supplementary trials were conducted in Tanzania to enumerate flies landing on pyramidal traps compared to 1 m2 square targets , divided vertically into equal parts of blue and black material ( referred to hereafter as the standard target ) . For this , adhesive film was also attached to the blue-black fabric of the pyramidal traps to enumerate flies that land but may not be captured . In an additional test only 1×1 m squares of adhesive film on its own ( i . e . without targets as a backdrop ) were compared to 1 m2 square targets ( with equal parts of blue and black material ) covered with adhesive film to ascertain whether the adhesive film in itself was attractive . A further set of trials was conducted in Tanzania in 2012 to compare six different two-dimensional cloth targets to evaluate the influence of size , shape and colour combination on fly landing rates . The six devices were: two types of 1 m2 square targets , one divided vertically into equal parts of blue and black material , the other divided vertically into three equal parts of blue-black-blue; two types of 0 . 5 m2 targets ( 0 . 9 m×0 . 55 m ) , one divided vertically into equal parts of blue-black-blue and the other all blue , both set up horizontally; and two types of 0 . 25 m2 square targets ( 0 . 5 m×0 . 5 m ) one divided vertically into equal parts of blue-black-blue , the other all blue ( see Table 1 ) . An 1∶4∶8 mixture of 3-n-propylphenol ( P ) , 1-octen-3-ol ( O ) , and p-cresol ( C ) ( Ubichem research LTD , Budapest , Hungary with a global purity of up to 98% ) was used as an attractant for experiments comparing baited devices based on general efficacy for several tsetse species [26] . Sachets made of 500 gauge/0 . 125 mm polyethylene containing 3 g of the mixture were placed below the catching devices , 10 cm above the ground , next to a 250 ml bottle buried in soil up to the shoulders containing acetone ( A ) with a 2 mm aperture in the stopper . This combination of chemicals is termed the POCA bait . In all trials randomization was set up using design . lsd in the package agricolae [27] , R version 2 . 13 . 0 [28] . Data were analysed using a linear model in R version 2 . 13 . 0 [28] , including the following additional packages: MASS [29] and multcomp [30] . Analysis was performed on log ( x+1 ) transformed data including day and position as ordering parameters and Tukey contrasts were calculated to compare treatments . The Wilcoxon paired test was used to compare fly landings on the blue and black portions of targets and to compare catches on the transparent film versus the blue-black target . Unless otherwise specified , results are presented as detransformed means . G . pallidipes is not mentioned where captures were too low for meaningful analysis .
When unbaited , both types of blue-black targets ( Kenyan and Tanzanian ) covered with adhesive film were the best devices for G . swynnertoni . In both countries and irrespective of season or fabric , sticky targets in the unbaited trials captured more G . swynnertoni than pyramidal traps ( P≤0 . 001 , Table 2 and Figure 2 ) . Catches were 2 . 4–6 . 7 times higher in three of the trials , and nearly 20 times higher in one trial ( wet season , Tanzania ) . Targets covered with adhesive film also out-performed the smaller all-blue leg panels ( all types and regardless of adhesive ) , capturing 2 . 2–3 . 7 times more flies in Kenya ( P≤0 . 01 , Table 2 ) and 1 . 5–2 . 8 times more flies in Tanzania ( P<0 . 05 for the plastic leg panel , not significant for the cloth leg panel , Table 2 ) . The leg panels similarly captured more flies than the pyramidal traps in Kenya ( P<0 . 05 , wet and dry season , Table 2 ) and Tanzania ( P≤0 . 001 , wet season , not significant P>0 . 05 in the dry season , Table 2 ) . There was no difference between the performance of any of the same devices made from the different blue materials ( P>0 . 05; Table 2 and Figure 2 ) , and sex ratios were similar on the different devices . The relative rankings of the POCA-baited devices were very similar to those of unbaited devices for G . swynnertoni . As before , the sticky targets greatly outperformed the pyramidal trap , with the largest difference in catch in the wet season in Tanzania ( P≤0 . 001 , Table 2 and Figure 2 ) . Catches were 5 . 5–6 . 7 times higher in three of the trials and up to 12 . 7 times higher in the wet season in Tanzania . In Kenya , the baited target captured 3 . 2–4 . 2 times more flies than the smaller leg panels in both seasons ( P≤0 . 001 , Table 2 ) , and an average of 5 . 6 times more in the dry season in Tanzania . In contrast , in the wet season in Tanzania , the catches of the target were on average 2 . 2 times higher than on leg panels and this was not significantly higher on either the cloth or plastic leg panels ( P>0 . 05 , Table 2 ) . The baited leg panels consistently caught more flies than baited pyramidal traps , but not all contrasts were significant; In Kenya , where just cloth leg panels were tested , only two out of eight comparisons were significant ( P≤0 . 05 , Table 2 , the standard blue leg panel compared with both pyramidal traps in the wet season and both leg panels compared to the turquoise blue pyramidal in the dry season ) . In Tanzania , leg panels in both cloth and plastic caught significantly more flies than pyramidal traps in the wet season ( 4 . 6 times , P≤0 . 001 , Table 2 ) , but there was no difference amongst all four devices in the dry season ( P>0 . 05 , Table 2 ) . As in the unbaited trials , there was no difference between the performance of any of the same devices ( trap , target , leg panel ) made from different blue materials ( P>0 . 05 , Table 2 and Figure 2 ) , and sex ratios were similar on the different devices . Baited devices were tested shortly after unbaited devices for logistical reasons , hence differences in catches across the two sets of experiments of the same devices have not been interpreted . A 2-d blue-black 1 m2 target with attached adhesive film induced more G . swynnertoni to land relative to a 3-d pyramidal trap with its blue-black surfaces covered with the same surface area of film ( Figure 3 ) . Twice the number of flies landed on the target relative to the pyramidal trap ( 110 . 6/55 . 5 , P<0 . 05; Figure 3 ) , and six times more flies landed on the target than were caught in a control trap without film ( 110 . 6/17 . 6 , P<0 . 05; Figure 3 ) . For G . pallidipes , 3 . 3 times more flies landed on the target than the pyramidal trap covered with adhesive film ( 25 . 1/6 . 0 , P<0 . 05; Figure 3 ) , and 1 . 5 times more landed on the target than in the control trap without adhesive film ( 25 . 1/16 . 7 , P>0 . 05; Figure 3 ) . Sex ratios were similar on the three devices for both species . Trap efficiency , i . e . the proportion of flies caught in the trap cage of those that approach at close range , was estimated by dividing the mean daily catch in the cage of the unaltered pyramidal trap by the mean daily catch of the trap with adhesive film on the cloth , i . e . summing flies caught on adhesive film and in the cage . Efficiency for G . swynnertoni was 30% ( 17 . 6/ ( 55 . 5+3 . 6 ) ×100 , Figure 3 ) . Very few flies were caught in the cage of traps with adhesive film ( 6% ) , suggesting that few flies are caught without first landing on the blue-black cloth . Trap efficiency for G . pallidipes could not be estimated as the trap with adhesive film caught fewer flies than the trap without film . The 1 m2 target of adhesive film on its own ( unbaited ) caught very few tsetse of either species compared to the cloth target covered with adhesive film . This target caught 2% of the detransformed mean daily catch of G . swynnertoni on the cloth target ( 2 . 9/119 . 1 , P≤0 . 05 ) , and 6% of the detransformed mean daily catch of G . pallidipes ( 1 . 3/21 . 3 , P≤0 . 05 ) . Note that the sticky surface area of the cloth targets was twice that of the stand-alone adhesive film target . Catches were low in the indicative experiment to test if a standard 1 m2 blue-black target would catch as many flies as a local blue/black 1 . 5 m2 Kenyan target as pastoralists were attempting to reduce tsetse in the study area in Kenya when the experiments were conducted and vegetation was also being heavily-grazed . The 1 . 5 m2 target caught a mean of 5 . 5 flies versus 3 . 0 flies on the standard 1 . 0 m2 target ( fly counts not statistically analysed ) . However , when landing heights were tallied , 101 of 132 G . swynnertoni ( 77% ) landed on the bottom 60 cm of the targets . This information was used to obtain an indicative normalized catch per m2 for the Tanzanian targets in the main experiments of 2009 where only the bottom 60 cm was covered . The detransformed mean catch on adhesive film was multiplied by 1 . 30 ( 1/0 . 77 ) for flies that could have landed on the upper 40 cm of the target without them landing on the lower 60 cm with adhesive ( an assumption , i . e . a maximum estimate ) , divided by 3 m2 . All other detransformed mean catches were normalized only for the size of the trapping device ( Table 1 ) as in every other case the entire device was covered in adhesive film . Based on this logic , detransformed mean catches per m2 of total surface area of Kenyan cloth leg panels averaged 1 . 5 times those of the Kenyan local target ( range 1 . 0–2 . 1 times , Table 3 ) , with similar trends among turquoise and standard phthalogen blue cloth . After adjusting for the partial adhesive coverage of Tanzanian targets , detransformed mean catches on Tanzanian leg panels averaged 1 . 0 times those of the Tanzanian local target ( range 0 . 4–1 . 7 times , Table 3 ) , with similar trends for turquoise and standard blue cloth , or blue-painted plastic . Note that the leg panels in Kenya were smaller than in Tanzania , and the Kenyan target , although of the same size as in Tanzania , had a different configuration of blue - black . Considering the high performance of the leg panels relative to the , on average , 3 . 5 times bigger targets , we conducted an extra experiment to assess the effect of target size , shape and colour on the landing responses of flies . The 1 m2 targets in blue-black ( standard ) and blue-black-blue equal sized vertical stripes ( Tanzanian style ) both caught very similar numbers of both G . swynnertoni and G . pallidipes which suggests that there is no difference between the two designs for inducing landing ( P>0 . 05; Figure 4 ) . The daily landing rate by flies on the blue-black-blue 0 . 5 m2 oblong targets was higher than the all blue targets of similar size for G . swynnertoni ( 48 . 1 and 34 . 2 flies/day , respectively; Figure 4 ) , but this difference is not significant ( P>0 . 05 ) . The landing rate by G . pallidipes on the two 0 . 5 m2 oblong target types was very similar ( P>0 . 05; Figure 4 ) . Likewise , there was little difference between the daily landing rates by flies of either species on the blue-black-blue and all blue smaller 0 . 25 m2 square targets ( P>0 . 05; Figure 4 ) . The linear model indicates that beside the ordering factors of target position and experimental day , size is the only significant parameter retained ( P<0 . 001 ) , i . e . neither colour pattern ( blue-black-blue , blue-black or all blue ) nor shape ( oblong or square ) significantly affects landings by G . swynnertoni or G . pallidipes . Fly landings on the 0 . 5 m2 targets were reduced to 44% of the 1 m2 targets for G . swynnertoni and to 60% for G . pallidipes ( P<0 . 01 for both ) , and to 19% and 14% , respectively , on the 0 . 25 m2 target compared to the 1 m2 targets ( P<0 . 01 for both; Table 4 ) . Reducing target size from 0 . 5 m2 to 0 . 25 m2 caused landings to be reduced to 44% for G . swynnertoni but to only 23% for G . pallidipes ( P<0 . 01 for both; Table 4 ) . All percentages were calculated by de-transforming the coefficients from the linear model and are very similar to the catch indices calculated from the detransformed means in Table 4 . Analysis of fly landings on the blue-black-blue targets alone also retained size as a significant factor ( P<0 . 001 ) . When the daily landing rates are corrected to an equal target size of 1 m2 , landings on the 0 . 5 m2 oblong blue-black-blue targets are nearly the same as on the standard blue-black targets for G . swynnertoni and G . pallidipes ( Table 4 ) . These corrected landing rates also indicate that landings on the best performing 0 . 25 m2 square target are 74% of those on the standard 1 m2 target for G . swynnertoni but only 66% for G . pallidipes ( Table 4 ) . For G . swynnertoni , the ratio of flies landing on the blue and black portions of the bicolour targets was very close to 50∶50 , irrespective of the area of each colour , with females showing a slight preference for the black portion ( 55% ) and males a slight preference for the blue ( 55% ) . In contrast , in G . pallidipes , both sexes showed a strong preference for landing on the blue portion of targets ( 85%; P<0 . 001 ) . In the experiment with adjoining adhesive film targets placed next to the 0 . 5 m2 oblong targets ( Figure 1 ) , only 1% of the total G . swynnertoni catch on the blue-black-blue target was caught by the adjacent transparent target ( 6 of 464 flies ) and the proportion was 6% for the all blue target ( 15 of 256 flies ) . In the case of G . pallidipes , only 2% of the total catch on both coloured target types was made on the adjacent transparent target ( 11 of 457 flies for the blue-black blue target and 5 of 288 flies for the all blue target ) .
In both Tanzania and Kenya , and independent of season or the presence of baits , targets covered with adhesive film were the best trapping devices for G . swynnertoni in all situations , catching over 19 times more tsetse than the pyramidal traps . When not baited , large blue-black targets captured 1 . 5 to 3 . 7 times more tsetse than much smaller leg panels . Blue leg panels made of either phthalogen blue , turquoise cloth or phthalogen blue-painted plastic nevertheless captured more tsetse than pyramidal traps , or at worst , equivalent numbers . Both targets and leg panels were particularly effective relative to pyramidal traps during the wet season in Tanzania . Of all the experiments , the wet season trials in the Serengeti represented the greatest challenge in terms of attracting flies to artificial devices during peak vegetation cover [31] . Small leg panels that deviate from large square or oblong blue-black fabric targets , [32] , [20] , [33] , were tested as an alternative for G . swynnertoni based on their efficacy for sampling G . austeni in Zanzibar [34] . Indeed the performance of leg panels covered with insect glue was remarkably high in capturing G . swynnertoni . Leg panels were 21% of the surface of targets in Kenya and 30% of the surface in Tanzania , but per unit area , captured 1 . 5 times more flies than the targets in Kenya and the equivalent number to targets in Tanzania . These results with leg panels in 2009 were confirmed in Tanzania in 2012 when similarly sized 0 . 5 m2 oblong blue-black-blue cloth targets covered with adhesive tape induced landings per unit area at nearly the same level as on the 1 m2 targets for G . swynnertoni . The potential cost-effectiveness of small targets for different tsetse has been demonstrated only very recently for a few tsetse species [32] , [35] . This success with smaller all-blue leg panels and all-blue or blue-black-blue cloth targets as landing devices for G . swynnertoni stands out relative to poor results for small all-black targets for two other savannah species G . pallidipes and G . morsitans morsitans in Zimbabwe [9] . However , earlier results have shown that all blue and blue-black targets perform better than all-black targets for G . pallidipes [25] . Indeed , the 0 . 5 m2 oblong target in blue-black-blue or all blue cloth induced landings per unit area at nearly the same level as on the 1 m2 targets for G . pallidipes in our trials . This may be related to the predominance of blue in the 0 . 5 m2 targets tested here , but concurs with earlier findings where doubling the target size doubles the catch for G . pallidipes [25] . G . swynnertoni lives in very open and often windy habitats where visual cues ( including colour ) may be more important than host odours; this is also manifested in terms of its well-known attraction to large , moving objects [16] . Regardless of this , small blue leg panels or targets of approximately 0 . 5 m2 in size clearly show promise for trapping G . swynnertoni , particularly as wind damage can be a problem with the larger local targets at many sites . However , 50% of flies captured landed on the black portion of all the targets tested , even those with only one third of the surface area in black . It would therefore be advisable to maintain a black element in visual devices targeting this species , as the presence of adhesive film used in these experiments has been shown to significantly reduce the landing rate on the black section of targets by G . tachinoides and G . palpalis gambiensis [20] . It is therefore likely that the proportion of G . swynnertoni landing on the black would be higher on unmodified targets . Similar catches with cloth and plastic leg panels also indicate that this strategy can be used for both control ( insecticide-impregnated cloth ) and sampling of this species ( rigid plastic with insect glue or adhesive film ) . Considering the fall off in landings per unit area on the very small 0 . 25 m2 targets , it would be inadvisable to employ targets much smaller than 0 . 5 m2 for control programmes . There was no difference between the performance of any of the same devices made from the different blue materials between seasons and locations . The two blue fabrics chosen for these experiments ( phthalogen blue cotton and turquoise blue polyester/viscose ) were manufactured with only minor differences in fabric texture and with slight but clear differences in blue-green colour . These fabrics , and the equivalent blue paint used on plastic leg panels [36] , performed equally well in targets/leg panels and traps under diverse conditions for G . swynnertoni . These results agree with findings for the same fabrics tested in similar devices for several tsetse species in West Africa [20] . Phthalogen blue cotton cloth has been used for about 30 years in tsetse sampling and control , and is the standard against which all other blues should be compared for attractive properties [13] . It has the maximum colour fastness possible for a pure blue fabric due the formation of copper phthalocyanine ( Pigment Blue 15 ) in situ through a unique dyeing process but now remains in limited production in just a few countries . This has resulted in the ad hoc use of several alternative blue fabrics in tsetse control , some of which are less than optimal for attracting tsetse [37] . Hence , it has become important to develop appropriate fabrics that can be produced locally with non-proprietary methods . The turquoise blue fabric , produced in Kenya by Sunflag for these experiments using generic dyes and processes , performed well in our studies . This clearly shows that it is possible to produce a deep turquoise that can be used as a practical alternative to phthalogen blue , as suggested by Mihok et al . , [38] . Their suitability in control devices is currently being investigated in terms of optimising colour-fastness and insecticide-retaining qualities . This study provides a comparison of the efficacy of several target designs relative to the most common simple trap ( pyramidal ) in current use for the savannah tsetse G . swynnertoni . For a standard 1 m2 blue-black target , catches were twice as high as on the equivalent area of a pyramidal trap for G . swynnertoni , and three times higher for G . pallidipes . The adhesive film used to enumerate tsetse here ( and also in Rayaisse et al . , [20] ) was found to be unattractive when used alone . The fabrication of insecticide-impregnated cloth targets has obvious practical advantages over traps for area-wide population suppression programmes . The potential cost effectiveness of using target-type devices for controlling G . swynnertoni is highlighted in this study by the efficacy with which leg panels trap the species . Per unit area , leg panels and 0 . 5 m2 oblong targets were as effective as the two local styles of bigger targets in common use in Kenya and Tanzania . The efficacy of smaller 2-d devices for capturing G . swynnertoni follows a pattern recently demonstrated for a range of riverine spp . [32] , [33] , [35] , [39] . Evidently , simple blue-black-blue or all-blue targets and all-blue leg panels of equivalent size are clearly effective in providing adequate visual stimuli to attract G . swynnertoni to land , the key behaviour that underlies the principle of insecticide-impregnated control devices and they are also less prone to wind damage because of their smaller size . Pyramidal trap entry/retention did not appear to be improved by baiting traps with POCA , i . e . baited targets still caught far more tsetse than baited pyramidal traps . As the baited and unbaited trials were sequential , they could not be compared directly . Nevertheless , our results are consistent with previous failures to substantially improve catches of G . swynnertoni with traditional tsetse baits [17] , [19] . Here , baiting devices with POCA did not affect their performance relative to one another; altogether results were remarkably constant between seasons at the same location and between the Serengeti and Mara . Considering the efficacy of the leg panels and targets , one should consider how much effort to invest in deploying and maintaining chemical baits ( some of which are toxic , e . g . phenols ) when it may be possible to adequately compensate by simply deploying more targets . In particular , the deployment of many small leg panels or targets with long-lasting insecticide impregnation may prove to be a cost-effective strategy . This approach would be particularly appropriate in conservation areas in East Africa , especially if fabrics could be engineered to be biodegradable after their effective lifespan . As expected from many other studies on savannah tsetse , the pyramidal trap was found to be inherently inefficient as a trapping device for G . swynnertoni , i . e . less than two thirds of the flies that landed on attractive surfaces of the trap ended up being captured in the cage of the trap . This trapping rate is very similar in magnitude ( 31% efficiency ) to that already measured for the S2 trap [17] where most G . swynnertoni that landed on the panels of the trap were never captured . In contrast to this , the pyramidal trap proved more efficient for G . pallidipes . There has been an underlying deficiency with traps for savannah species of tsetse . An absolute estimate of trapping efficiency is , however , difficult as there are many untested assumptions concerning fly behaviour and counting accuracy near traps that could affect the outcome . In contrast to this , the low number of flies ( <2% ) landing on transparent 0 . 5 m2 sticky targets compared to the number alighting on adjoining blue-black-blue cloth targets of the same size and shape suggests that very few G . swynnertoni or G . pallidipes circle the oblong 0 . 5 m2 targets before landing . Ndegwa and Mihok [17] used electric nets placed radially from the S2 trap and found that most ( 89% ) G . swynnertoni flying in the vicinity of the trap circled within 0–1 m of it , and by covering the blue trap sides with adhesive fly rolls they found that 84% of approaching flies landed on the trap before entering it . Area-wide tsetse population suppression typically requires the deployment of many thousands of devices; devices need to be effective and inexpensive , and ideally should also be maintenance-free [40] . G . swynnertoni is abundant in areas with difficult logistics; it also presents limited options for dealing with as it occurs in protected areas frequented by large numbers of tourists . Simple targets that attract flies , which then land on insecticide-impregnated surfaces , are most suitable in this context . The most practical device for area-wide suppression of G . swynnertoni populations would be a large blue-black , insecticide-impregnated 1 m2 target . Our results show that there is no significant difference between the blue-black and blue-black-blue 1 m2 targets . A number of smaller targets in blue and black or all-blue leg panels with the same surface area would achieve the same result . Although all-blue leg panels would also provide a satisfactory control device a black element in the target is recommended where G . swynnertoni is the target species . The most cost-effective size of these devices and the associated costs of fabricating , deploying and maintaining large targets versus a higher number of small targets or leg panels still need to be determined in field trials . Our findings indicate that targets smaller than 0 . 5 m2 are not recommended for either G . swynnertoni or G pallidipes . Long-lasting but ultimately biodegradable devices of simple construction could be used to reduce disease transmission in the high-profile wildlife conservation areas of Tanzania and Kenya where G . swynnertoni is the main vector of human and animal trypanosomiasis . Either phthalogen or turquoise blue would be suitable for these visual control devices . Effective control will also require adaptive management [41] whereby tsetse populations are monitored and disease-transmission hot spots are identified for additional intervention . For long-term eradication goals , the detection of very low-density , residual pockets of tsetse is also critical [42] . The best monitoring tool would clearly be a leg panel or cloth target of equivalent size covered with adhesive film . Since this approach is not very economical or practical outside of a research context , all-blue plastic leg panels covered with insect glue can be used as an effective alternative .
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Glossina swynnertoni is restricted to open savannah in north-western Tanzania and south-western Kenya , where it is a vector of both human and animal trypanosomiasis in wildlife reserves and in neighbouring conservation areas used by pastoralists . Despite the challenge to minimize disease transmission through effective management of the vector in the presence of abundant wildlife reservoirs , little has been done to test the efficacy of modern tsetse traps or targets for controlling G . swynnertoni . We made field tests in the Serengeti and Maasai Mara to determine the most visually-attractive , long-lasting and practical object that induces the strongest landing response in G . swynnertoni . Fly landings were twice as high on 1 m2 blue-black targets as on pyramidal traps when equivalent areas of these devices were covered with adhesive film . Furthermore , blue leg panels in either cloth or plastic and blue or blue-black-blue cloth targets under half the size of traditional targets captured tsetse at equivalent numbers per unit as the latter . These smaller targets and leg panels show promise as cost-effective devices for management of G . swynnertoni populations as they can be used for both control ( insecticide-impregnated cloth ) and monitoring of this species ( rigid plastic with insect glue or adhesive film ) .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"infectious",
"diseases",
"veterinary",
"diseases",
"pest",
"control",
"biology",
"zoology",
"veterinary",
"science",
"agriculture"
] |
2013
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Standardizing Visual Control Devices for Tsetse Flies: East African Species Glossina swynnertoni
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Actins are highly conserved proteins and key players in central processes in all eukaryotic cells . The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species . Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic . We show that actin I cannot complement actin II in male gametogenesis , suggesting critical structural differences . Cryo-EM reveals that Plasmodium actin I has a unique filament structure , whereas actin II filaments resemble canonical F-actin . Both Plasmodium actins hydrolyze ATP more efficiently than α-actin , and unlike any other actin , both parasite actins rapidly form short oligomers induced by ADP . Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties . Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro . In vivo , this chimera restores gametogenesis in parasites lacking actin II , suggesting that stable filaments are required for exflagellation . Together , these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties , implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties .
Actins are the most abundant and among the most conserved proteins in eukaryotic cells and play indispensable roles in a plethora of key cellular events , including muscle contraction , cell division , shape determination , transport , and cell motility [1] , [2] . Actins are highly conserved in opisthokonts with <10% divergence between yeast and man . The six mammalian actin isoforms differ from each other by a maximum of 6% of the sequence , and are virtually identical across species . Nevertheless , these subtle differences are enough to determine isoform-specific functions [3] . Common to most actins is their capacity to form long filaments . However , in a number of phylogenetically distinct organisms , such as Trypanosoma and Plasmodium spp . , actin filaments have not been observed [4] , [5] . Unlike other members of the phylum Apicomplexa , which comprises single-celled eukaryotic intracellular parasites , the malaria parasites have two actin isoforms , which at the sequence level are <80% identical with canonical ( opisthokont ) actins and each other . This is a remarkable difference , considering the near identity among canonical actins ( Fig . S1 ) . An important question is how this divergence at the amino-acid level translates into different structures – and how this , in turn , influences polymerization . Most studies on apicomplexan actins have concentrated on their role in gliding motility , a unique mode of migration , essential for the parasite to infect new cells . However , like in other eukaryotes , parasite actins must have several cellular functions . Actin polymerization is indispensable for gliding and likely involved in host cell invasion and egress [6]–[8] . Despite evidence for this crucial role of filamentous actin , long filaments have only been visualized in Theileria [9] , which appears not to use actin filaments for host cell invasion [10] . The presence of regular actin filaments in Plasmodium is uncertain [11]–[13] . In vitro , apicomplexan actins form short , ∼100-nm long filaments , which undergo rapid treadmilling [14]–[17] . Recently , specific antibodies revealed filament-like structures in motile forms of Plasmodium [12] , [13] . Toxoplasma gondii actin , which is 93% identical to Plasmodium actin I , has been reported to polymerize at concentrations 10-fold lower than canonical actins [15] , and most recently , it has been proposed to polymerize in an isodesmic manner without a lag phase or a critical concentration , which is unique among all actins or actin homologs studied to date [18] . Yet , most of the cellular actin is present as monomers [19] , implying that filaments occur only transiently , and polymerization is under tight control of regulatory proteins or governed by distinct properties of the monomer . On the other hand , it has been estimated that 2/3 of Plasmodium actin in merozoites – the infective blood stage form , which does not exhibit gliding motility – could be present as short filaments [20] . Plasmodium actin I is abundant and expressed throughout the life cycle of the parasite , whereas actin II is present only in the gametocytes and mosquito stages [21]–[24] , including sporozoites [25] , the highly motile form of the parasite , transmitted to the vertebrate by the mosquito . Actin I is an indispensable part of the parasite motor machinery responsible for the unique gliding motility of the parasite . Actin II has at least two functions in the mosquito stages , as revealed by reverse genetics analyses . It is required both during male gametogenesis and in the zygote stage [23] , [24] . However , no clear molecular function has been assigned for actin II . To understand the properties of the divergent Plasmodium actins , we have determined their monomer crystal structures and analyzed their filament assembly using electron microscopy ( EM ) . We show that , unlike in any other cell reported so far , the two isoforms differ substantially from each other in their ability to form filaments and that both oligomerize in the presence of ADP . Their functional uniqueness is further highlighted by the finding that Plasmodium actin II has a distinct role in male gametogenesis that cannot be complemented by actin I . Finally , we show that a chimera of Plasmodium actin I and canonical actin can form long filaments and , importantly , restores the function of actin II in gametogenesis .
The most peculiar property of apicomplexan actins is their apparent inability to form long , stable filaments . This is a fundamental difference to all actins studied so far , and in the lack of structural information , the reasons for the poor polymerizability are not understood . It has been shown by atomic force microscopy that the dimensions of jasplakinolide ( JAS ) -stabilized P . falciparum actin I filaments , purified from merozoites , are different with respect to their helical symmetry from canonical actins [26] . However , the structure of actin II filaments has not been studied before . We visualized the structures formed by both Plasmodium actin isoforms using EM ( Fig . 1 ) . When polymerized in the presence of ATP in high-salt conditions at room temperature overnight , actin I forms only short , irregular structures of approximately 100 nm in length ( Fig . 1 A and F ) , while actin II forms significantly longer filaments ( average length 650 nm ) ( Fig . 1 B , C , F ) . In the presence of JAS , which in vitro stabilizes actin filaments , both actin I and II form long , rather straight filaments ( Fig . 1 D–F ) . To evaluate whether the helical assemblies of the two Plasmodium actins are different , we used cryo-EM . Filaments of both parasite actins were embedded in vitreous ice ( Fig . 2 ) . First , we inspected the averaged power spectra derived from segments of 330 and 56 filaments for actin I and II , respectively . These look virtually identical because they can only be compared at 1/60 Å−1 resolution ( Fig . 2 A ) . We then characterized the structure of the filaments in real space and performed k-means classification of helical segments [27] . Inspection of the classes allows a direct measurement of the cross overs or half-pitch of the two-start helix , which represent the distance the filament requires to undergo a 180° rotation . For actin I and II , cross-over distances cluster around 406±16 Å and 364±10 Å , respectively ( Fig . 2 B ) , which is confirmed from Eigen images that represent the half-pitch of the two-start helix ( Fig . 2 C ) . Hence , actin II has symmetry parameters identical to α-actin , for which a cross-over distance of 371 Å has been reported [28] , while actin I possesses a significantly longer cross-over distance , which is in agreement with the earlier work performed on actin I using atomic force microscopy [26] . To understand whether the longer cross-over distance is a result of a change in the helical rise and/or the helical rotation , we determined the low-resolution 3D structure of actin I ( Fig . 3 A ) using single-particle based helical reconstruction [29] , [30] . We refined the helical symmetry ( Fig . 3 B ) and determined the low-resolution filament structure at 25-Å resolution ( at FSC 0 . 5 cutoff , Fig . 3 C ) . This showed that the cross-over distance change is mainly due to a change of helical rotation from −166 . 6° [28] to −167 . 5° , which corresponds to the predicted rotation change if the helical rise remains constant at 27 . 7 Å . Despite the obvious difference in helical symmetry , at the current resolution , the cryo-EM structure of the actin I subunit looks very similar to the canonical actin filament ( Fig . 3 A ) . This is the first time that such large differences in the properties of filaments have been observed for actin isoforms of any species . Thus , higher resolution is needed to further characterize the molecular interactions giving rise to the different polymerization propensities and the observed helical rotation changes . The divergent polymerization properties of Plasmodium actins in vivo may be partly accounted for by differences in the activities of the actin-binding proteins , which in these parasites are also poorly conserved and have partially divergent functions compared to canonical counterparts [31]–[38] . However , differences in the actin monomer structure must be responsible for the observed differences in filament structure in vitro and also for interactions with regulatory proteins and , thus , functional differences in vivo . Therefore , high-resolution structures are required for understanding the biological and molecular differences of the parasite actins compared to their canonical homologs . Such information will also aid us in evaluating the suitability of Plasmodium actins as drug targets . We set out to determine the crystal structures of P . falciparum actin I and P . berghei actin II . The sequence identities between the counterparts from P . falciparum and P . berghei are 99% for actin I and 92% for actin II . The gelsolin G1 domain was used to stabilize the monomers and facilitate crystallization of both actins [39] , [40] . The actin I structure was refined to a resolution of 1 . 3 Å and actin II to 2 . 2 Å . These high-resolution structures allow for a very detailed comparison of the Plasmodium actins with each other and with other actins ( Figs . 4 , S2 , and S3 ) . Although Plasmodium lacks a gelsolin homolog , the mammalian G1 is bound between subdomains 1 and 3 in both Plasmodium actins , similarly to other actin–G1 complexes [39] , [41] ( Fig . S2 D and E ) . For all comparisons , we have used canonical actin structures that have also been determined in complex with G1 , in order to rule out structural rearrangements caused by gelsolin binding . In most canonical actin structures , the C terminus is folded as an α-helix , which interacts with the bottom part of subdomain 1 . This is true also for Plasmodium actin II ( Fig . 4 A ) . In actin I , however , the C terminus turns towards solvent and is disordered ( Fig . 4 B ) . The large hydrophobic cleft between subdomains 1 and 3 is defined as a ‘hotspot’ for regulatory protein binding [42] ( Fig . 4 A and B ) . A smaller hydrophobic patch in the direct vicinity of the C terminus is , in addition , involved in binding at least profilin [43] . In actin I , the large hydrophobic residues in this smaller cleft , including Trp357 , have adopted different conformations compared to canonical actins ( Fig . 4 B ) , possibly influencing the binding of profilin , which we have previously proposed to bind actin in a different manner in Plasmodium compared to opisthokonts [31] . In actin II , these hydrophobic residues , like the C terminus , are in the canonical conformations . All in all , despite the obvious similarity to other actins , especially actin I shows appreciable structural deviations , in particular in regions involved in binding of regulatory proteins [42] . This may provide possibilities for structure-based drug design targeted at the Plasmodium actin–regulatory protein interfaces . A key question concerning the properties of the major Plasmodium actin isoform , as well as other apicomplexan actins , is why they , unlike all the extensively studied actins , form only short , unstable filaments . Comparing the crystal structures of the monomers to the recently determined high-resolution cryo-EM structures of canonical actin filaments [28] , [44]–[46] can provide clues to answer this question . In canonical F-actin , the axial interactions , meaning the longitudinal contacts between the actin monomers in each of the two protofilaments , are tight and mainly electrostatic , as revealed by cryo-EM studies [28] , [45] . The most important axial interactions are discussed below . The DNase I binding ( D- ) loop in subdomain 2 ( residues 39–61 in actin I ) is one of the most important regions for polymerization and , in the filament , inserts into the hydrophobic cleft between subdomains 1 and 3 of the neighboring monomer [28] , [45] , [47] , [48] . In both Plasmodium actin crystal structures , the D-loop is disordered , and the tip of it is not visible in the electron density maps ( Figs . 4 and S3 ) . Interestingly , the most notable changes in the D-loop sequence concern the first and the last residues of a segment ( residues 42–49 ) that forms a short α-helix in some G-actin structures with ADP bound [49] and has been modeled as a helix also in one of the recent F-actin structures [45] . This part of the D-loop inserts deep into the neighboring monomer in the filament , contacting the so-called proline-rich loop ( residues 109–115 ) . Residue 42 , which is a glutamine or threonine in most other actins , is a proline in both Plasmodium actins . A proline restricts the conformation of the chain and , although also known as a ‘helix breaker’ , is often also seen as the first residue in α-helices . At the C terminus of this segment , residue 49 , which is a glycine in canonical actins , is a glutamate in both Plasmodium actins . These replacements at both ends of this segment would be expected to increase the helical propensity of the tip of the D-loop . In canonical F-actin , Thr325 in the loop between α-helix 11 and β-strand 18 ( residues 323–327; Fig . 4 A and B ) in subdomain 3 of one monomer interacts with Glu242 in the loop connecting β-strands 15 and 16 in subdomain 4 ( residues 242–247; Fig . 4 A and B ) of the neighboring monomer [28] . In actin I , this loop and the threonine side chain have turned away from the optimal position for this interaction ( Figs . 4 B and S3 ) . In actin II , Ser325 is positioned such that hydrogen bonding to the apposing glutamate can be easily achieved . The difference in the conformation can be explained by the substitutions Y280F and M284K in actin I compared to canonical actins and actin II . The third main site contributing to axial interactions is formed by the loop connecting helices 9 and 10 ( residues 284–290 in subdomain 3; Fig . 4 A ) inserting between subdomains 2 and 4 of the neighboring monomer . This area is almost fully conserved in both parasite actins , the only substitution being that of Met283 by a lysine ( 284 ) in actin I . All in all , the largest differences in the axial contacts concentrate to the D-loop and concern equally both Plasmodium actins . Smaller differences in subdomain 3 may , however , partly explain the different polymerization propensities of the two parasite actin isoforms . The lateral contacts between the two protofilaments concern mainly interactions between subdomains 1 and 4 and the beginning of the D-loop in subdomain 2 interacting with the so-called hydrophobic loop ( residues 263–275 ) ( Figs . 4 A–B , S1 , and S2 D–E ) between subdomains 3 and 4 of the apposing monomer [28] , [45] , [48] . In a recent high-resolution cryo-EM filament structure [45] , Arg206 in subdomain 4 interacts with Ser271 of a neighboring monomer . In actin I , Ser271 is replaced by Ala272 , and Lys207 and Glu188 are involved in a short hydrogen bond with salt-bridge character ( Fig . 4 C ) . A similar interaction occurs between Arg206 and Asp187 in Latrunculin A-bound α-actin [50] . In the absence of latrunculin , the distance between these residues in α-actin is longer and the geometry suboptimal for a salt bridge ( Fig . 4 C ) . Latrunculins prevent actin polymerization , presumably by limiting the flexibility of subdomains 2 and 4 [50] , [51] , and the salt bridge seen in the latrunculin-actin structure , similar to Plasmodium actin I , may be one reason for this . The longer side chain of Glu188 in actin I compared to Asp187 in canonical actins may facilitate the interaction with Lys207 . Actin II has Tyr187 in place of Asp187/Glu188 , which affects the orientation of Arg206 . Actin II , furthermore , has Cys272 in the place of Ser271 of canonical actins , allowing for hydrogen bonding upon polymerization . At the N terminus of the D-loop , Arg40 and His41 of canonical actins are replaced by lysine and asparagine in actin I and lysine and methionine in actin II . In the filament , Arg40 forms a salt bridge with Glu271 [28] , and the replacement to lysine can subtly weaken this interaction . His41 , in turn , interacts with Ser266 [28] . Although the asparagine in actin I can also contribute to a hydrogen bond , the interaction may be weaker due to the shorter side chain . In F-actin , Lys114 in the proline-rich loop ( residues 109–115 in subdomain 1 , following actin I numbering ) forms a salt bridge with Glu196 of a neighboring monomer [28] . Uniquely , the neighboring residue is Gly115 in actin I ( Fig . 4 D ) , which can have a large effect on the mobility of the proline-rich loop . Actin II has a non-glycine residue ( threonine ) at this position , similarly to canonical actins ( alanine or serine ) . Taken together , especially residues involved in lateral contacts , crucial for the stability of the filaments , are altered in Plasmodium actin I but more conserved in actin II . The most important differences concern subdomain 4 , the D-loop , and the proline-rich loop . The conformations of all these affect each other allosterically and can also modulate the ATPase activity [52]–[54] . Because of the observed differences in sequence , conformation , and helical symmetry between Plasmodium and canonical actins and the importance of the D-loop-mediated contacts for polymerization , we constructed a chimera , in which the entire D-loop of actin I was replaced with that of α-actin . Strikingly , this chimera in the absence of any stabilizing agents forms filaments with an average length of 1 . 6 µm , which is longer than either actin I or II filaments ( Figs . 5 A–B and 1 F ) . However , the appearance of the chimera filaments is not as regular as the filaments formed by canonical actins or actin II . To further characterize the filaments formed by the actin I–α-actin chimera , we analyzed electron cryo-micrographs of JAS-stabilized chimera filaments and subjected them to the same classification and symmetry analysis as outlined above ( Fig . S4 ) . Based on the determined half-pitch distance and the symmetry analysis , we conclude that the symmetry parameters of the chimera filaments are in close agreement with the wild-type actin I filaments . Therefore , the symmetry parameters appear to be retained from the wild-type actin I , and thus , the D-loop of canonical actin merely confers increased filament stability . The crystal structure reveals that the 3D structure of the chimera in monomeric form is very similar to that of wild-type actin I ( Figs . 5 C and S3 ) . However , there are some notable differences . The D-loop in the chimera is slightly more ordered . In addition , the C-terminal helix is visible and can be superimposed with the C terminus of actin II and most canonical actins . The Trp357 side chain in the hydrophobic patch is not flipped as in wild-type actin I ( Figs . 4 B and 5 C ) . This observation is in line with earlier reports on allostery between the conformations of the D-loop and the C terminus [55] , [56] and demonstrates the ability of even large residues in the hydrophobic core of actin to move , which is a requirement for the rearrangements taking place in the monomer upon polymerization . In addition to residues already discussed , a notable difference between the D-loop of Plasmodium actin I and canonical actins is at position 54 , which has a tyrosine in opisthokont actins and Plasmodium actin II but phenylalanine in actin I . In the chimera , the OH group of Tyr54 is hydrogen bonded to the main chain of Lys51 and could also interact with the tip of Lys62 ( Fig . 5 C ) . In wild-type actin I , Phe54 has moved away from Lys62 and seems to push Lys51 to a slightly different conformation . In actin II , Tyr53 ( corresponding to Phe54 in actin I ) is able to interact with Lys61 ( Lys62 in actin I ) , but otherwise , this region is rather different in conformation compared to both actin I and canonical actins . This is due to conformational changes in subdomain 4 , involving Thr203 , Arg206 , and seemingly originating from the bulky side chain of Tyr187 , which is replaced by aspartate in canonical actins and glutamate in actin I , as discussed above . According to a recent filament structure [45] , Tyr54 becomes stacked between the side chains of Asp52 and Lys62 of the same monomer and its only possible rotamers would allow an OH–π interaction with the phenyl ring of Tyr170 of the neighboring monomer . The side chain of Phe54 in actin I is not able to participate in this kind of an interaction due to the missing hydroxyl group . Thus , the replacement of tyrosine by phenylalanine at this position may affect both the rigidity of the D-loop in the monomeric state as well as the stability of the filament . The state of the nucleotide-binding pocket can be described using two parameters: ( i ) the “phosphate clamp” , which is the distance between the Cα atoms of Gly15 and Asp157 , and ( ii ) the “mouth” , which is the distance between the Cα atoms of Gln59 and Glu207 [46] . The phosphate clamp distance in Plasmodium actins does not differ significantly from other actins . However , the mouth of the binding pocket is significantly more closed in both parasite actins: 9 . 85 Å in actin I and 9 . 04 Å in actin II , compared to an average of 10 . 87±0 . 4 Å in 9 other actin–G1 structures used for comparison . In three recent high-resolution F-actin structures [28] , [45] , [48] , the mouth distance varies between 7 . 88 and 9 . 66 Å . Both parasite actins reach the more closed conformation by slightly different means . In actin I , the largest differences in conformation to canonical actins are in subdomain 4 and in actin II , subdomain 2 ( Figs . 4 and S3 ) . Both structures reported here have calcium and ATP bound to the nucleotide-binding cleft between subdomains 2 and 4 ( Figs . 4 and 6 ) . In the nucleotide-binding residues , there is one interesting difference in actin I compared to canonical actins and actin II; residue 17 , which is hydrophobic ( methionine/leucine ) in all canonical actins and actin II , is an asparagine in actin I ( Fig . 6 A and B ) and also T . gondii actin . This side chain is close to the α- and β-phosphates of ATP . In actin I , the distance of the Asn17 Nδ2 atom to the β-phosphate O1 atom ( ∼3 . 75 Å ) is too long for hydrogen bonding in this conformation . However , together with its own main chain N , that of Gly16 , and Nζ of Lys19 , the Asn17 side chain could form an oxyanion hole to stabilize a negative charge on the β-phosphate O1 atom ( Fig . 6 A ) . In addition , it could interact with the α-phosphate . The Asn17 side chain is flexible in the crystal structure , as evident from the electron density maps and B factors , and based on the shape of the electron density as well as anisotropic ellipsoids , seems to move in concert with active-site water molecules as well as the nearby Tyr338 , which is in a double conformation . A catalytic mechanism based on a nucleophilic attack of a water molecule activated by His162 and Gln138 has been proposed for ATP hydrolysis in actin [57] . The reaction itself is rather simple , containing only proton transfer steps . The complication arises from the fact that G-actin – currently the only form of which atomic-resolution information can be achieved – is a poor catalyst , and conformational changes upon polymerization are needed for achieving the catalytically competent conformation of the active site . Water 39 ( actin I numbering ) has been proposed to be the nucleophile initiating the reaction , and depending on the bound metal , the position of this water changes [57] . In actin I , this water is 4 . 75 Å away from the γ-phosphate , and the angle between the β-γ bridging O , Pγ , and water 39 is 152 . 4° , which is amenable for a nucleophilic attack ( Fig . 6 B ) . The catalytic site water structure in actin II is different compared to actin I ( Fig . 6 C ) , as the presumed catalytic water ( 147 in the actin II structure ) has moved towards His161 and Pro109 and has weak electron density and a high B factor . The distance of this water to Pγ is 5 . 43 Å , and it is in an almost in-line position ( 162 . 4° ) . In fact , according to the electron density and the distances to neighboring atoms , this water may be a second conformation of water 357 , which is directly hydrogen bonded to His161 . Together , these differences in the active site architectures between the two parasite actins and compared to other actins indicate that the catalytic activity and the exact mechanism of ATP hydrolysis may differ in them , likely resulting also in differences in polymerization . Given the structural differences in the catalytic sites , we set out to test if the Plasmodium actins differ from each other and canonical actins in their ability to hydrolyze ATP and/or release phosphate . We first measured phosphate release in the presence of Mg2+ ( Fig . 6 D ) . It should be noted that , as phosphate release at least in canonical actins is much slower than hydrolysis , this method gives only indirect information about the hydrolysis rate . In these conditions , both parasite actins release phosphate faster than α-actin . Of the two parasite actins , actin I has a slightly higher rate ( ∼2 times higher than actin II ) . Curiously , the chimera with the α-actin D-loop is approximately 3-fold more active than wild-type actin I and 23-fold more active than α-actin . We also made two point mutations to actin I; G115A , which we predicted to affect the flexibility of the proline-rich loop and , thus , the rate of hydrolysis , and F54Y , which we hypothesized might affect the rigidity of the D-loop in monomeric state . Neither of these mutants formed long filaments without JAS ( Fig . 1 F ) . In the presence of Mg2+ , the F54Y mutant shows identical behavior to wild-type actin I . G115A , however , has a reduced rate , similar to actin II . Also the kinetics differ from each other in the different actins . Whereas actin I and the point mutants release phosphate in a linear way , α-actin , actin II , and the chimera display more complex kinetics , having an initial very short , faster , non-linear phase , followed by a linear phase . We next measured phosphate release in the Ca2+-bound , presumably mainly monomeric , forms ( Fig . 6 D ) . As expected , α-actin showed an even lower release of phosphate in the Ca2+-bound compared to the Mg2+-bound form . Both actin I and actin II have activities equal compared to each other and approximately 5-fold higher than α-actin . The chimera releases phosphate 3-fold less in the presence of Ca2+ than with Mg2+ , but the rate is still significantly higher than that of muscle actin or both wild-type parasite actins with Ca2+ . Of the point mutants , F54Y has practically no activity with Ca2+ ( identical to α-actin ) , whereas G115A is slightly more efficient in the presence of Ca2+ than Mg2+ . Altogether , these data show that the Plasmodium actins have a different mechanism of ATP hydrolysis and/or subsequent phosphate release compared to canonical actins , which are poor catalysts in the monomeric form and adopt the catalytic conformation only upon polymerization , which is a prerequisite for non-equilibrium polymerization kinetics enabling directional growth [58] . We were also able to pinpoint amino acid residues responsible for these differences . In order to evaluate the oligomeric state of the parasite actins in the presence of ATP/ADP and different ions , we used native PAGE . With ATP bound , actin I spontaneously forms short polymers ( from tetramers up to 11–12-mers ) in ∼48 h when stored on ice ( Figs . 7 and S5 ) . Actin II stays mainly monomeric over the same period of time , although minute amounts of oligomers ( dimers–octamers ) appear . Interestingly , in the presence of ADP , oligomerization starts instantly , and the majority of both actins is oligomeric ( dimers–10-mers ) immediately after a 1-h hexokinase treatment at 298 K to remove ATP . After incubation of the actins at 298 K for 1 h without hexokinase , only minute amounts of oligomers can be visualized for actin I , and no visible oligomerization of actin II takes place ( data not shown ) . After 48 h , the proportion of larger oligomers of the ADP forms is much higher , and monomers as well as lower oligomers are practically non-existent . The formation of oligomers is not caused by oxidation , as using even a large excess of the reducing agent TCEP in the sample does not reduce the amount of oligomerization ( Fig . 7 A ) . Both ATP and ADP forms of α-actin remain monomeric in the same conditions . However , short oligomers of ATP-α-actin have been reported below the critical concentration for polymerization [59] . Because the distribution , when separated on a gel , does not necessarily reflect the equilibrium between different species in solution , we also used dynamic light scattering ( DLS ) to visualize the size distribution and polydispersity of the actin mono- and oligomers in solution over time ( Fig . S6 ) . The resolution of DLS is far from that of the native gel assay , and it is only possible to detect size differences of approximately 5–6 fold . Therefore , e . g . monomers , dimers , and trimers will appear as a single , polydisperse peak . 6 h after purification , actin I is seen in mainly two separate peaks of average hydrodynamic radii of approximately 2 and 6 nm ( Fig . S6 A ) . 2 nm would be very close to the expected hydrodynamic radius of the monomer . After 11 h , nearly all of actin I is in particles with a radius of ∼5 nm ( Fig . S6 B ) . As time goes by , the distribution becomes divided between particles of below 3 nm ( close to a monomer ) and larger oligomers with an average radius of 11–12 nm ( Fig . S6 C and D ) . The polydispersity of the sample after 11 h is very high , indicating that the sample contains a mixture of monomers and small oligomers , and the polydispersity diminishes again , as the sample gains a multimodal distribution , indicating that the smallest oligomers disappear over time , leaving behind a pool of monomers in addition to the higher oligomers , consistent with our native PAGE data . As seen also in the native gels , actin II retains a higher fraction of monomers over 48 h , but also gains a fraction of significantly higher oligomers , which are , however , infrequent and very heterogeneous in size ( Fig . S6 E–H ) . In order to probe the effects of Mg2+ and K+ ions on the oligomerization behavior , we also performed native PAGE in the presence of two concentrations ( 1 and 5 mM ) of MgCl2 as well as 5 mM MgCl2 and 50 mM KCl ( Fig . S7 ) . In the presence of ATP , Mg2+ slightly reduces the amount of the short oligomers for both actin I and II compared to the Ca2+ forms ( Fig . S7 A and B ) . However , some actin I is visible at the bottom of the well at the top of the gel , which would imply filaments too long to enter the gel . This could not be seen in the Ca2+ gels for either the Plasmodium proteins or α-actin , but was much more pronounced for α-actin with Mg2+ . In the presence of ADP , there is a clear shift towards longer oligomers in actin I , and after 48 h , part of actin I stays in the well , not entering the gel in the presence of 5 mM MgCl2 both with and without KCl ( Fig . S7 C and D ) . Thus , Mg2+ alone seems to be sufficient for polymerization . We used genetically modified parasites to address the question whether the observed structural differences translate into different properties of the proteins in vivo . While it is not possible to delete actin I due to its essential functions , a knock-out of the actin2 gene has been done , resulting in a block of male gametogenesis [23] , [60] . We reasoned that a replacement of actin2 with actin1 would display the mutant phenotype if the two actin isoforms have different biological functions , while restoration of gametocyte development would indicate a similar function . Male gametogenesis in the malaria parasite is a unique event , involving the formation of flagellar gametes . This event , called exflagellation ( Fig . 8 A and Video S1 ) , is easily scored under the microscope , allowing us to use it as a quantitative method . In our approach , actin1 was expressed under the control of the actin2 flanking regions . We used a recipient line , in which the complete open reading frame ( ORF ) of actin2 had been deleted . Therefore , these parasites do not exflagellate [60] . This line was separately transfected with two constructs , both aiming at integration into the actin2 locus . The complementation construct ( act2com ) restored the actin2 ORF , which allowed expression of the cognate gene comparable to wild type . In the replacement construct ( act2rep ) , a fragment corresponding to the actin1 ORF was used instead of the actin2 ORF . The constructs were otherwise identical and were integrated in the locus via a single crossover homologous recombination event in the 5′ flanking region of actin2 ( Fig . 8 B ) . In both cases , clonal lines were obtained . We compared the act2com and act2rep parasite lines with wild-type parasites in the exflagellation assay ( Fig . 8 C ) . In the wild-type and act2com parasites , the number of exflagellation events was similar , indicating that complementation with actin2 restored the function of the gene . However , while some normal exflagellation events were detected also in the act2rep parasites , the numbers were significantly reduced compared to the act2com parasites ( Fig . 8 C ) , strongly suggesting that actin II has unique functions , which actin I cannot fulfill during male gametogenesis . As it became apparent that actin I polymerization properties in vitro could be altered by exchanging its D-loop to that of α-actin , we decided to investigate if this modification would also have an impact on the in vivo function of actin I . We produced transgenic parasites using the same strategy as described above; inserting the actin I–α-actin D-loop chimera into the actin2 locus , producing the act1chi parasites ( Fig . 8 B ) . Surprisingly , this revealed that the exchange of the D-loop had a remarkable impact on exflagellation ( Fig . 8 C ) . In the act1chi parasites , exflagellation was significantly increased compared to the act2rep strain and restored to values close to the act2com strain . These data show that the D-loop has a critical role in the function of these actins , but actin II , as shown by the structural data , has acquired other properties that contribute to its higher filament stability . Furthermore , and to our surprise , it seems that the molecular function of actin II may be dependent on the ability of the protein to form filaments .
The two Plasmodium actins differ in their polymerization propensities , filament stability , and filament helical symmetry – the hallmark of canonical F-actin . The second , stage-specific actin isoform of Plasmodium that forms long filaments with canonical F-actin symmetry is unique among Apicomplexa . Curiously , at the sequence level , actin II is as divergent from Plasmodium actin I as it is from all other actins . It has been suggested [23] , and our structural data support the view , that the actin2 gene has arisen only after the diversion of Plasmodium from other Apicomplexa , and the protein seems to have gained a higher filament stability independent of the evolution of higher eukaryotic actins . In canonical actins , polymerization is tightly coupled to ATP hydrolysis , such that structural rearrangements upon polymerization enable the active site to adopt a conformation optimal for catalysis [67] . Two key factors have been described necessary for achieving the catalytically competent conformation upon the transition from monomeric to filamentous state . These are: ( i ) a rotation of the outer domain ( subdomains 1 and 2 ) , resulting in flattening of the monomer and ( ii ) bending down of the proline-rich loop in subdomain 1 [45] , [48] . The Plasmodium actins hydrolyze ATP also in the monomeric form , releasing phosphate more efficiently than canonical actins , and oligomerize readily in the presence of ADP , which is a fundamental difference to all other actins characterized , and must be a result of different atomic structures . Interestingly , Plasmodium actin I has a unique glycine at the end of the proline-rich loop . This allows more flexibility for this loop , which apparently increases the catalytic rate in the presence of magnesium but , surprisingly , has an opposite effect in the calcium-bound form ( Fig . 6 D ) . Also the more closed conformations of subdomains 2 and 4 in the parasite actins may facilitate ATP hydrolysis but also reduce the conformational change – or flattening – required upon insertion of the monomer into the filament . An interesting difference that may also contribute to catalysis is Asn17 close to the α- and β-phosphates of ATP in the active site . Intriguingly , the bacterial actin homolog MreB [68] shares this residue with Plasmodium actin I , whereas canonical actins and also Plasmodium actin II have a hydrophobic residue at this position . Thus , this asparagine may be a relict from an early , polymerization incompetent ancestor . The structural features described above may explain the parasite actins' unconventional response to ADP . Surprisingly , the state of the nucleotide seems to determine polymerization propensity , but not in the same way as in canonical actins . The tight link between ATP hydrolysis and polymerization in higher eukaryotes has probably been refined during the hundreds of millions of years after the diversion of Apicomplexa . Our data and a recent report proposing an isodesmic polymerization mode for apicomplexan actins [18] suggest that the same has also happened for allosteric regulation of conformational changes taking place upon polymerization . However , it is clear that higher resolution data on the Plasmodium actin filaments are needed in order to find out what kind of conformational changes the parasite actins undergo upon polymerization and what is the arrangement of the protomers in the filament , leading to the altered symmetry compared to canonical F-actin . On the other hand , the distribution of oligomers , as seen on the native gels and DLS ( Figs . 7 , S5 , and S6 ) , suggests that polymerization may involve a nucleation step , the nucleus being either a dimer or trimer , which are the species that disappear early in the process . Thus , we hypothesize that the ADP state may favor nucleation , making ATP hydrolysis a rate-limiting step for polymerization . The D-loop plays a key role in the conformational changes upon polymerization as well as the conformation and stability of F-actin [28] , [45] , [47] , [48] . Both previous work [26] and our EM analyses reveal differences in the helical architecture of actin I compared to α-actin . In the crystal structures , several regions important for intra-filament contacts in canonical actin filaments show substantial differences between the parasite and opisthokont actins , and the polymerization propensity and filament stability are overall likely a sum of numerous atomic details in the monomers . Yet , the sequence of the α-actin D-loop alone is sufficient to restore the ability of actin I to form long filaments , without altering the symmetry compared to the JAS-stabilized wild-type actin I filaments . Thus , whereas the longitudinal contacts by the D-loop are important for stability , the shape and symmetry of the filaments are determined by other factors . Actin II shows us that stability can be obtained by other means than the D-loop , probably involving lateral interactions . In addition to the differences we have described in the residues involved in lateral contacts , a candidate responsible for increased stability is residue 200 , which is a glycine in Plasmodium actin I and T . gondii actin [69] but serine or threonine in canonical actins as well as Plasmodium actin II and Theileria actin [9] , all of which form long filaments . It has been reported that the double mutant G200S/K270M in T . gondii actin leads to an increased filament length when using phalloidin-labeled filaments [69] . However , we were not able to visualize long filaments of this mutant of Plasmodium actin I in polymerizing conditions without JAS ( data not shown ) , indicating that several small changes are cumulatively responsible for the increased stability of actin II filaments . The tip of the D-loop can adopt a helical conformation , albeit it is disordered in the vast majority of all G-actin structures , and appears mainly intrinsically disordered in solution in all nucleotide states of G-actin [70] . The likely higher helical propensity of the D-loop in Plasmodium actins may affect polymerization and filament stability in at least two different ways . If the helical conformation is more likely to occur in the filamentous form , this might actually facilitate polymerization , which would be in line with the proposed low critical concentration [15] or isodesmic model for polymerization of parasite actins [18] . However , it has also been proposed that the helical form occurs only transiently in the filament or that it is favored in the ADP form and leads to filament destabilization [54] , [70] . In this way , a higher helical propensity would contribute to the lower stability of the parasite actin filaments . Tyrosine hydrogen bonds can contribute substantially to protein stability [71] . Tyr54 is a phosphorylation target and plays a regulatory role in many actins [72]–[74] . For Dictyostelium actin , phosphorylation of this tyrosine increases the critical concentration and controls cell shape changes and spore formation [72]–[74] . In Mimosa pudica L . , a contact sensitive plant , where actin is heavily phosphorylated , tyrosine phosphatase inhibitors inhibit the fragmentation of actin filaments during leaf bending [75] . In addition to affecting binding to other proteins , phospho-Tyr54 stabilizes the D-loop conformation [74] . Upon polymerization , this region undergoes a large conformational change , and it seems that the OH group of Tyr54 may be involved in stabilizing interactions [45] that the Phe54 side chain could not fully compensate for . Only 11 of over 300 known actin sequences contain a phenylalanine at this position , and no other substitutions are known . Most of these 11 sequences are actins from Plasmodium or Trypanosoma , both species where actin filaments have not been observed in vivo . Despite the apparent importance of tyrosine at this position for normal actins , a single mutation to phenylalanine in Dictyostelium actin does not affect its polymerization properties [74] . In line with this , we also could not observe long filaments of the actin I F54Y mutant ( Fig . 1 F ) . However , the large effect of the F54Y mutation on the phosphate release rate of actin I ( Fig . 6 D ) suggests that this residue , indeed , may significantly affect the conformation and flexibility of the D-loop . Together , the above described structural properties may lead to a higher polymerization propensity but also lower filament stability in the parasite actins by lowering the energy barrier of the transition between monomeric and filamentous forms . Yet , the fact that the replacement of the D-loop alone is sufficient for stabilizing the filaments formed by actin I , while retaining their unique symmetry , is surprising , taking into account how similar the D-loops of the two Plasmodium actins with different stabilities are . This implies that , starting from an unstable filament forming ancestor , actin II has reached its present form mainly using other means than the D-loop for gaining additional filament stability . Male gametogenesis is a complex , rapid series of cellular events including escape from the host cell , three mitotic divisions , and axoneme assembly , leading to the formation of eight flagellar and highly motile gametes from each gametocyte within 10–20 min from activation . Both actin isoforms are present in male gametocytes of P . berghei [23] , but their function in these events is not understood . Actin II is not expressed in the asexual blood stages [23] . Its deletion blocks male gametogenesis , and therefore , these mutant parasites cannot be transmitted through the mosquito [23] . Still , it has not been possible to pinpoint the exact role of actin II . We show that the function of actin II cannot be complemented by actin I , proving distinct molecular functions for the two actins and suggesting that their unique structures and the differences in their ability to form filaments directly translate into different functional characteristics in vivo . By generating transgenic parasites expressing the actin I–α-actin chimera , we found that this mutant protein was able to function almost as well as actin II in vivo . This strongly confirms the in vitro experiments and supports the notion that the D-loop has a significant role in determining the polymerization properties of the parasite actins . Furthermore , we can hypothesize that the reason two actins evolved in Plasmodium , was the need to have actins with different propensities to polymerize in cells lacking a large repertoire of actin-binding proteins . Another example of distinct general and reproductive actin isoforms can be found in plants , where it was recently shown that animal cytoplasmic but not muscle actins can take over the functions of the plant vegetative actins [76] . Remarkably , also three actins from single-celled protists could carry out the same tasks , suggesting that the properties required for fulfilling the cytoplasmic actin functions during spatial development in multicellular organisms were present already early on in the evolutionary history . However , it seems that the polymerization properties of both Plasmodium and all other actins have evolved separately , starting from a poorly polymerizing ancestor . It would be interesting to see if either of the Plasmodium actins can support spatial development in either plants or animals . The current hypothesis is that actin I in Plasmodium is required for gliding motility , and the filaments involved need to be short and short-lived . Our data support this , as actin I forms only very short polymers . For the suggested role of actin I in gliding , the formation of long , stable filament seems undesirable [69] . Actin II clearly is able to form long filaments , which may be needed for functions specific to actin II within the mosquito stages , although such functions have not yet been specified . Intriguingly , Plasmodium appears to be the only apicomplexan parasite that has faced the evolutionary pressure for acquiring a second actin isoform that forms stable , long filaments . Our data provide a structural basis for understanding the different functional properties of the two actin isoforms of Plasmodium spp . These structures represent the , so far , most divergent and primitive actins characterized , and we show that the two isoforms have the most unique biochemical properties , structures , and biological functions of all known actin isoforms . High-resolution structural information will serve as a starting point for understanding these functions in detail and for evaluating the suitability of parasite actins and actin-binding proteins as drug targets .
Purification of G1 was performed as described [40] . Endogenous pig skeletal muscle α-actin was purified as described [36] , [77] . P . falciparum actin I ( PlasmoDB PF3D7_1246200 ) and P . berghei actin II ( PlasmoDB PBANKA_103010 ) were expressed in Sf21 cells at 300 K , as described before [36] . A chimera , where residues 40–61 of the P . berghei actin I were replaced by the corresponding residues from α-actin , was cloned into pFastBac HT A ( Invitrogen ) and expressed in the same way as the wild-type actins . Two point mutations ( G115A and F54Y ) were introduced to actin I by incorporating the corresponding mutation to the 5′ end of the primers . The parental plasmid was cleaved with DpnI and recirculated with the T4 DNA ligase . The protein coding sequences were confirmed by DNA sequencing . The purification of the wild-type actin–G1 complexes was performed as described [40] . The chimera–G1 was also purified as described before for the two wild-type actins [40] , except that HEPES ( pH 7 . 5 ) was used in the lysis buffer , and size exclusion chromatography was performed in 10 mM HEPES ( pH 7 . 5 ) , 50 mM NaCl , 5 mM dithiothreitol ( DTT ) , 0 . 2 mM CaCl2 and 0 . 5 mM ATP . Peak fractions containing the chimera–G1 were pooled and concentrated to 5 . 6 mg ml−1 for crystallization . The purification of all the actin variants without G1 was performed essentially as described [40] except for a few modifications , as listed . For actin I , the lysis was carried out in 10 mM HEPES ( pH 7 . 5 ) , 5 mM CaCl2 , 250 mM NaCl , 1 mM ATP , 5 mM β-mercaptoethanol , 15 mM imidazole , and size exclusion chromatography was performed in 15 mM HEPES ( pH 7 . 0 ) , 0 . 5 mM ATP , 5 mM DTT , and 0 . 2 mM CaCl2 . The pH of the lysis buffer for actin II was 8 . 7 , and size exclusion chromatography was performed in 25 mM Tris-HCl ( pH 7 . 5 ) , 0 . 5 mM ATP , 5 mM DTT , and 0 . 2 mM CaCl2 . For the chimera , lysis was carried out in 20 mM HEPES ( pH 7 . 5 ) , 5 mM CaCl2 , 250 mM NaCl , 1 mM ATP , 5 mM β-mercaptoethanol , 15 mM imidazole , and size exclusion chromatography was performed in 15 mM HEPES ( pH 7 . 0 ) , 0 . 5 mM ATP , 5 mM DTT , and 0 . 2 mM CaCl2 . For DLS and filament length measurements , size exclusion chromatography was performed in 5 mM HEPES ( pH 7 . 5 ) , 0 . 5 mM ATP , 2 mM DTT , and 0 . 2 mM CaCl2 . DLS was measured using a Wyatt DynaPro platereader-II and 15 or 30 µl of actin I and II at concentrations between 8 . 5–24 µM at 298 K . The measurements were performed in triplicate and the samples stored at room temperature between the measurements . ADP-actin was prepared by incubating 50 µl of 10 µM actin with 1–2 mg of hexokinase-agarose beads ( Sigma-Aldrich , #H-2653 ) in 15 mM HEPES pH 7 . 5 , 1 mM ATP , 1 mM tris ( 2-carboxyethyl ) phosphine ( TCEP ) , 0 . 2 mM CaCl2 , 2 mM D-glucose for 1 h at 298 K . As a control reaction , Plasmodium actins I and II were incubated in identical conditions without D-glucose and hexokinase and subsequently run on native PAGE . The residual ATP contamination in ADP stocks was removed by treating them in a similar fashion . Native PAGE was performed using a running buffer of 25 mM Tris-HCl ( pH 8 . 5 ) , 195 mM glycine , 0 . 5 mM ATP or ADP , and 0 . 1 mM CaCl2 or MgCl2 . The sample buffer consisted of 25 mM Tris-HCl ( pH 8 . 5 ) , 195 mM glycine , 10% ( v/v ) glycerol ( final concentrations ) . Actin samples were loaded at a concentration of 6 . 7 µM in a volume of 10 µl . Commercial TGX 4–20% gradient gels ( Biorad ) were pre-run for 30 min at 277 K , 100 V before applying the samples . Samples were run for 7 h using the same voltage settings and temperature , with corresponding nucleotides and divalent cations in the running buffer . The gels were stained the next day with Coomassie Brilliant Blue R250 . Relative mobilities were determined by measuring the distance of the bands from the top of the image and dividing this value by that of the monomeric band . In the absence of a reference monomeric band in ADP-ActI ( 48 h ) , the absolute value from ATP-ActI ( 0 h ) was used as a reference . The absolute mobilities of the other visible bands in these images had a difference of <2 . 5% . Gel images were processed and band intensities extracted using ImageJ [78] . A rolling ball background subtraction was applied before manually extracting the intensities . Actin samples were prepared for the phosphate release assay by treating 10–15 µM purified actin with DOWEX 1X8 to remove nucleotides and free phosphate . After the removal of the nucleotide and phosphate , ATP was replenished by adding a small volume of a concentrated stock solution . Buffer controls were treated in a similar fashion , in order to reset the level of free phosphate and nucleotide compared to the samples . The concentration to be used for determining the release rate was measured from the nucleotide-free solutions in order to reduce the effect of pipetting errors . After the DOWEX treatment , samples were divided in triplicate wells of a UV-transparent 96-well plate ( Corning ) containing reagents from the EnzChek Phosphate Release Assay ( Molecular Probes ) without using the reaction buffer , which contains MgCl2 at a final concentration of 1 mM . For calcium measurements , the final reaction contained 1 mM CaCl2 and 0 . 1 mM MgCl2 . The total omission of MgCl2 was not possible , since the coupled enzyme requires magnesium . For magnesium measurements , the respective concentrations were 0 . 13 mM CaCl2 and 1 mM MgCl2 . Formation of the 2-amino-6-mercapto-7-methylpurine from the coupled reaction was measured as absorbance at 360 nm with a kinetic interval of 60 s over a period of 5 h at 298 K . The total measurement volume was 200 µl . Phosphate release rates were calculated from linear parts of the plot ( 100 to 200 min ) using GraphPad PRISM 5 . 03 . Crystallization and diffraction data collection of both wild-type actin–G1 complexes has been described [40] . The chimera–G1 complex was crystallized similarly , and the final crystallization condition contained 100 mM Tris-HCl ( pH 8 . 0 ) , 8% ( w/v ) polyethylene glycol ( PEG ) 20 000 , and 2% ( v/v ) dioxane . Before flash-cooling in liquid nitrogen , the crystal was shortly soaked in 100 mM Tris-HCl ( pH 8 . 5 ) , 14% ( w/v ) PEG 20 000 , 2% ( v/v ) dioxane , 0 . 5 mM ATP , 50 mM NaCl , 0 . 2 mM CaCl2 , and 10% ( w/v ) PEG 400 . A diffraction data set to 2 . 5-Å resolution was collected on a Pilatus 6M detector at the beamline P11 , PETRA III ( DESY ) , Hamburg , using a wavelength of 0 . 92 Å at 100 K . The data ( Table 1 ) were integrated with XDS [79] and scaled with XSCALE [79] using XDSi [80] . The actin II–G1 structure was solved by molecular replacement with Phaser [81] using the α-actin–G1 complex as a search model ( PDB code 1P8Z [82] ) . For actin I–G1 and chimera–G1 , the actin II and actin I in complex with gelsolin , respectively , were used as molecular replacement models . The refinement was carried out with PHENIX . refine [83] and manual model building in Coot [84] , and structure validation using the MOLPROBITY server [85] . For actin I , actin II , and chimera–G1 complexes , 99 . 8% , 99 . 8% , and 99 . 4% of the amino acids , respectively , were in the allowed regions of the Ramachandran plot . The final electron density maps as well as data and refinement statistics are presented in Fig , S2 A–C and Table 1 . The structure figures were prepared using PyMOL and Chimera [86] . Actin ( 7–13 µM ) was polymerized overnight at room temperature . Polymerization was induced by adding 1/10 volume of 10× polymerization buffer [50 mM Tris-HCl ( pH 8 . 0 ) or HEPES ( pH 7 . 5 ) , 500 mM KCl , 20 mM MgCl2 ( in cryo-EM 40 mM MgCl2 ) , 50 mM DTT , and 10 mM ATP] with or without 5–7 µM JAS . In order to concentrate the filaments , actin II and the chimera in F-buffer were spun for 45 min at 435 , 000 g , and remaining pellet was resuspended into polymerization buffer . 2–3-µl aliquots of the samples were diluted in the polymerization buffer before applying them on glow-discharged grids ( CF-300CU , Electron Microscopy Sciences ) and stained with 1% ( w/v ) uranyl acetate or potassium phospho-tungstate ( pH 7 . 0 ) . The grids were examined with Tecnai G2 Spirit ( 100 kV ) or FEI Tecnai F20 microscopes ( 200 kV ) . Filament lengths were measured using ImageJ [78] . Many of the longest ( >1 µm ) measured filaments are fragments , as both ends were not always visible in the images . Polymerized samples were applied in 3-µl aliquots onto freshly glow-discharged holey carbon grids ( Quantifoil R 2/2 ) at 295 K and 70% humidity and vitrified in liquid ethane using a Leica EM GP vitrification robot . Specimens were held in a Gatan 626 cryoholder maintained at 93 K for imaging in a FEI Tecnai F20 microscope operated at 200 kV . Micrographs were recorded under low dose conditions on a Gatan Ultrascan 4000 CCD camera at a magnification of 69 , 000 to give a final pixel size of 2 . 21 Å . The contrast transfer function ( CTF ) of the micrographs was determined using CTFFIND [87] . A total of 330 ( actin I ) , 56 ( actin II ) and 457 ( chimera ) filaments were selected using e2helixboxer . py from the EMAN2 suite [88] . For classification , segments were excised using a mean step size of 30 Å and an additional random shift along the helix between -15 and 15 Å to avoid high-resolution artifacts in the class average power spectra introduced by regularly shifted images . The segments were further corrected for their CTF by phase flipping , and aligned to the vertical axis . This resulted in 4 , 581 segments for actin I , 968 for actin II , and 8 , 052 for the chimera actin . Two-dimensional ( 2D ) classification of helical segments was performed using the SPARX k-means algorithm [27] . The segments were iteratively classified and aligned against a subset of class-averages chosen based on their quality with a total of four iterations . At each cycle , multiple copies of the chosen references were created by applying integer y-shifts ranging from −15 Å to +15 Å in order to be able to reduce the Y-shift search range during alignment to less than half of the step size in order to avoid summation of successive images on a filament shifted at the same axial position . The total number of class averages used to measure the cross-over distance was 40 for actin I and the chimera actin , and 20 for actin II . In addition , Eigen images were calculated and the corresponding pitch distances were measured . For 3D structure determination of actin I filaments , 2 , 182 segments were excised using a regular step size of 70 Å , convolved by their respective CTF and further reconstructed as described [30] using the software SPRING [89] . In addition , symmetry refinement was performed using the IHRSR method [90] by systematically varying the initial helical rises and azimuthal rotations from 26 to 30 Å ( step 1 Å ) and from 164 to 170° ( step 1° ) , respectively . More specifically , 25 iterations of refinement were computed with SPIDER , using a solid cylinder of 100 Å in diameter as a starting model . The symmetry parameters were refined with the hsearch program after the second refinement iteration , using a step size of 0 . 03 Å for helical rise and of 0 . 05° for azimuthal rotation . The actin2 complementation and replacement constructs were made in a derivative of the pL0006 vector , which encodes human DHFR conferring resistance to the drug WR99210 [91] , [92] . The design of the constructs is described in detail elsewhere [24] , and the three different constructs were produced following the same strategy . Briefly , 2 . 7 kilobase pairs of the promoter and 728 base pairs of the 3′-flanking region of the P . berghei actin2 gene were amplified from gDNA and cloned into the vector . For the act2rep construct , P . bergei actin I complete ORF including start and stop codon was amplified from gDNA and cloned between the actin2 promoter and the 3′ flanking region of actin2 . The same strategy was followed for the act2com construct using the P . berghei actin2 ORF and the act1chi construct . The plasmids were linearized before transfection of the recipient act2−::mCherry parasite line [60] . Parasites were cloned as described [93] . Correct integration was verified by PCR genotyping and Southern blotting . Exflagellation was scored after diluting blood from an infected mouse in exflagellation medium [23] and incubating the samples for 10–20 min at 292 K . The exflagellation events were counted under a light microscope . The structure factors and coordinates for all three crystal structures have been submitted to the PDB under the codes 4cbu , 4cbw , and 4cbx . The actin I EM map has been deposited to the EMDB under the accession code EMD-2572 . Figure S1 shows an alignment of apicomplexan and canonical actin sequences . Figure S2 shows the electron density maps around the ATP-binding site of the Plasmodium actins and the chimera and the gelsolin complexes for actin I and II in two orientations . Figure S3 depicts root mean square deviations between Plasmodium and canonical actin structures . Figure S4 shows the cryo-EM analysis of the actin I–α-actin chimera filaments . Figure S5 shows native PAGE analysis of the Plasmodium actins in the calcium-bound form . Figure S6 shows the DLS analysis of the oligomerization of the parasite actins over time . Figure S7 shows native gels of the Plasmodium actins in the magnesium-bound form . Video S1 shows an exflagellation event of a male P . berghei gametocyte .
|
Malaria parasites have two actin isoforms , which are among the most divergent within the actin family that comprises highly conserved proteins , essential in all eukaryotic cells . In Plasmodium , actin is indispensable for motility and , thus , the infectivity of the deadly parasite . Yet , actin filaments have not been observed in vivo in these pathogens . Here , we show that the two Plasmodium actins differ from each other in both monomeric and filamentous form and that actin I cannot replace actin II during male gametogenesis . Whereas the major isoform actin I cannot form stable filaments alone , the mosquito-stage-specific actin II readily forms long filaments that have dimensions similar to canonical actins . A chimeric actin I mutant that forms long filaments in vitro also rescues gametogenesis in parasites lacking actin II . Both Plasmodium actins rapidly hydrolyze ATP and form short oligomers in the presence of ADP , which is a fundamental difference to all other actins characterized to date . Structural and functional differences in the two Plasmodium actin isoforms compared both to each other and to canonical actins reveal how the polymerization properties of eukaryotic actins have evolved along different avenues .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biochemistry",
"cytoskeletal",
"proteins",
"protein",
"chemistry",
"cell",
"motility",
"actin",
"filaments",
"protein",
"interactions",
"cell",
"biology",
"molecular",
"complexes",
"proteins",
"protein",
"structure",
"biology",
"and",
"life",
"sciences",
"molecular",
"cell",
"biology",
"biophysics",
"molecular",
"biology"
] |
2014
|
Structural Differences Explain Diverse Functions of Plasmodium Actins
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The emergence of novel respiratory pathogens can challenge the capacity of key health care resources , such as intensive care units , that are constrained to serve only specific geographical populations . An ability to predict the magnitude and timing of peak incidence at the scale of a single large population would help to accurately assess the value of interventions designed to reduce that peak . However , current disease-dynamic theory does not provide a clear understanding of the relationship between: epidemic trajectories at the scale of interest ( e . g . city ) ; population mobility; and higher resolution spatial effects ( e . g . transmission within small neighbourhoods ) . Here , we used a spatially-explicit stochastic meta-population model of arbitrary spatial resolution to determine the effect of resolution on model-derived epidemic trajectories . We simulated an influenza-like pathogen spreading across theoretical and actual population densities and varied our assumptions about mobility using Latin-Hypercube sampling . Even though , by design , cumulative attack rates were the same for all resolutions and mobilities , peak incidences were different . Clear thresholds existed for all tested populations , such that models with resolutions lower than the threshold substantially overestimated population-wide peak incidence . The effect of resolution was most important in populations which were of lower density and lower mobility . With the expectation of accurate spatial incidence datasets in the near future , our objective was to provide a framework for how to use these data correctly in a spatial meta-population model . Our results suggest that there is a fundamental spatial resolution for any pathogen-population pair . If underlying interactions between pathogens and spatially heterogeneous populations are represented at this resolution or higher , accurate predictions of peak incidence for city-scale epidemics are feasible .
Novel respiratory pathogens continue to pose substantial public health challenges , not least because of the risk that large epidemics may overwhelm key health care resources such as vaccination stockpiles and intensive care facilities . Recent epidemics of concern include: SARS [1] , influenza [2]–[4] , H7N9 [5] , [6] and MERS [7] , [8] . During an epidemic it is important to accurately predict the impact of the epidemic over different spatial scales , where scale refers to the size of the region being monitored; such as a hospital , city , country or globally . Intervention policies should be defined relative to this spatial scale , for example taking account of how long it will take to vaccinate a whole city or to distribute a treatment country-wide . Those making decisions about intervention strategies need a clear understanding of the underlying epidemic process , so as to anticipate the magnitude and timing of peak incidence at their scale of interest and to effectively control the epidemic . Spatially explicit transmission models are used frequently to increase understanding of the spread of epidemics caused by pathogens which transmit between individuals close in space . For example: influenza [9]–[11] , measles [12]–[14] , and smallpox [15] , [16] have all been represented by spatially explicit epidemic models . All of these examples can be thought of as metapopulation models in which the population of interest is represented as a collection of sub-populations located in space , for example households [17]–[19] , airports ( GLEaM [20] ) or districts/states [21] . The advantages of these models are that they can capture complicated mobility and mixing patterns and heterogeneous population density , without the complexity of an individual-based model . Also , model output can be easily reported for specific populations , such as counties or cities . It is known that heterogeneity both in population density and typical mixing behaviour heavily influence disease spread . Both of these are defined according to the resolution of the population representation , where resolution defines the number and size of the pixels making up the “image” of the population within the model . A pixel is the smallest single component of an image . A high resolution representation will divide the region into many small pixels; a lower resolution uses fewer , larger pixels ( Fig . S1 ) . The resolution chosen is usually decided by the data available: for example , population and travel data may be defined at the ward or county level only . The level of mixing between individuals in distinct pixels is defined by mobility models , these are often fitted to travel data from censuses . Also , sometimes , resolution is limited by computational capacity . The concepts in our paper require precise definitions of the terms: scale , resolution and pixel . The literature using these three words is somewhat ambiguous with the terms resolution and scale sometimes used interchangeably . Therefore , for clarity , we have included explicit definitions at first use of the words ( above ) and in Table 1 .
We implemented a generic metapopulation model with arbitrary spatial resolution ( see Methods ) varying from approximately ( 30″ by 30″ , the smallest unit representation ) upwards ( Fig . S1 ) . We generated a theoretical population density in a region with total population just over 4 million and of size approximately ( 49×49 pixels ) . The region had three ‘urban’ areas where population density was generated using a 2-dimensional bivariate Gaussian and a ‘rural’ area , generated from a uniform distribution , Fig . 1G . We used this formulation to simulate the spread of a pathogen representative of influenza , with an SIR-like natural history , assuming that the generation time was 2 . 6 days and the basic reproductive number was 1 . 8 . The epidemic was seeded with 10 individuals in a central region ( Fig . 1G ) , simulations were repeated 25 times at each resolution . The within-pixel contact rate was fixed for all pixels . Mobility between pixels was represented by a kernel with an offset power function . A kernel defines the relative probability of travelling between two pixels . The offset power function is an adaptation of the gravity model . The gravity model states that an individual's probability of mixing in a pixel different to their home pixel is inversely proportional to the distance apart of the pixels , to some power . The offset power function adds in an offset distance parameter , which means that pixels closer together than this distance mix fully . See Methods for a full definition of the kernel and the resulting mobility model . Initially we considered three different kernels: we used an offset of 2 km and three different powers giving low , medium and high contact between pixels ( the power , , was −6 , −4 and −2 respectively ) , Fig . S2 . The highest mobility kernel is in line with kernels fitted to commuter data in the UK and US [9] . However , our review of data on travel patterns found that only 15% of an average individual's journeys are commuting , making up just 19% of the total distance an average individual travels each year [22] ( Table S1 ) . Commuting data also excludes key at-risk groups – the under 17 s and over 70 s – who have lower mobility travel patterns compared to the 18–69 population [22] . Therefore , we explored more restrictive kernels than those estimated using commuting data to reflect shorter distances travelled and lower frequency travel in the most at-risk populations and the regular non-commuting travel of the wider population . We confirmed that the overall cumulative attack rate ( CAR ) for our model was independent of the mobility kernel and the model resolution ( Fig . 1A–F ) . This was by design: the model was constructed such that with the assumption of mass action mobility ( the rate of contact between two groups is proportional to the size of each of the groups ) the epidemic was identical at every resolution . This means that the next generation matrix at any resolution and for any mobility has the same spectral radius: was the same at all resolutions and contact levels and the local and global s were the same . A full proof that was constant with respect to resolution is given in the Text S1 in Supporting Information S1 , and is similar to that in Ref [23] . Because was constant , if the mobility was such that there was contact between every pair of pixels , the final epidemic size was the same across all resolutions and in every pixel . If mobility was restrictive enough that some pixels were never infected the final CAR reflected this restriction . The full proof that attack rates were constant with respect to resolution is in the Text S2 in Supporting Information S1 , and is similar to those in Refs [24] , [25] . For the theoretical population density , the existence of a fundamental spatial resolution was apparent: at resolutions lower than this threshold , system-wide peak incidence was substantially over-estimated , obtaining a high peak incidence and fast spread similar to that obtained in a fully mixed model ( the lowest resolution ) . However , at the fundamental resolution and above , consistent estimates of the peak attack rate were obtained ( Fig . 1H ) . This was increasingly evident as mobility became more and more restricted: for the most localised mobility assumptions ( low power ) , peak incidence in the fully mixed case was nearly double that at the highest resolution . At high resolutions , multiple small pixels containing low numbers of individuals and with a high heterogeneity in population size slowed the epidemic spread; resulting in a long epidemic duration and a low peak incidence compared to low resolution model scenarios . Increased mobility reduced the effect of resolution on the epidemic trajectory . At medium mobility , peak incidence increased with decreasing resolution but there was no distinct threshold . At the highest mobility , peak incidence was unaffected by resolution: the high level of contact between pixels facilitated the quick spread of the epidemic , indicated by a short epidemic duration and a high peak incidence at every resolution ( Fig . 1A–C ) . Resolution and mobility remained important when the model was constructed with real population densities . We repeated the analysis ( using the same three kernels ) for four regions selected from LandScan data [26]: Guangzhou , Rio de Janeiro , Delhi and New York ( Fig . 2A ) . The smallest LandScan unit is approximately 1 km2 ( 30″ by 30″ ) in size . The effect of resolution was most evident when mobility was more restricted , as with the theoretical population . In Guangzhou , Rio and New York , changing the spatial resolution had a significant effect on the peak incidence when mobility was at a low to medium level , though the effect was less clear in Delhi ( Fig . 2B–E ) . The Delhi region had the largest total population size and the highest mean population density of all regions we considered ( Table S2 and Fig . S3 ) . Therefore , even at low mobility the numbers mixing will be relatively high , meaning that the disease spread will not be as restricted as it would be in a less densely populated region . We used Latin Hypercube Sampling ( LHS ) [27] to determine whether the patterns we saw with the illustrative mobility kernels could be generalised within a wider parameter space of mobility functions . We varied the kernel parameters: the power , , between −6 and −2 ( as discussed earlier , this selection gave a wide range of mobility levels ) and the saturation distance between 1 and 10 km , choosing from a log scale ( so smaller distances are more likely ) . We tested 50 parameter sets chosen using the LHS technique [27] , with 10 separate realisations of each set for each region ( variation in results from the stochastic model was low - see confidence intervals for 25 repeats in Fig . 2 for example ) . Kernels for the 50 sets are plotted in Fig . S4 . The LHS results confirmed that the effect of resolution is most important in populations which are less mobile , Fig . 3 . As mobility decreased ( a combination of the offset and the power in the kernel ) the difference in peak incidence between the lowest and the highest resolution increased . This was particularly true in Guangzhou , Rio and New York , but in Delhi the effect was reduced ( due to Delhi having a very large population in comparison to the other regions ) . Recently it has been suggested that the movement of individuals depends not only on the source and destination cities , but also on the population density of the surrounding area [28] . This model is called the radiation model and has been proposed as a distinct alternative to the gravity model . However , we calculated the actual number of individuals moving between pixels ( the flux ) and found the radiation model flux to be very close to the offset gravity model of medium mobility , particularly at the highest resolution , Fig . 4 . Indeed the radiation model is always bounded by the three gravity models we use and our LHS models explore a large space around these . More generally , gravity-like models have been implemented with a number of different normalisation assumptions , some of which produce population flux patterns very similar to the radiation model [15] , [17] .
When managing epidemics it is desirable to know the size and duration of the epidemic and the magnitude and timing of the peak incidence over the spatial scale of interest [4] , [29] , [30] . This scale of interest may be a city , a region or a whole country . Resources such as treatment , vaccinations and diagnostic tests will take time to be deployed over this scale and it can take time to develop and generate enough of these resources for the whole affected population [31] , [32] . Accurate predictions about the magnitude and timing of peak incidence would greatly enhance the ability of public health officials to effectively limit the impact of epidemics . We have shown how the representation of population interactions can impact model estimates of key epidemic outcomes . We examined the effect of the resolution of the population density on the model predictions of epidemic spread over the scale of interest . We refer to resolution as defining the number and size of the individual pixels dividing the region; higher resolution representations use a higher number of smaller pixels . Our results imply that for plausible population densities and mobility patterns , fundamental resolutions exist for specific pathogens such that the detail of the population and their interactions must be represented faithfully if accurate epidemic trajectories are to be estimated . The impact of model resolution was clear in models of less mobile populations: our results indicate that at lower mobility , low resolution representations overestimated the peak incidence , obtaining a high peak incidence and fast spread similar to that obtained in a fully mixed model . However , sufficiently high resolution representations gave lower and later peak incidences because of the delaying effect of multiple small pixels . Indeed at low mobility , clear thresholds existed for the resolution of the theoretical population density , such that models with resolutions below the threshold over-estimated the system-wide peak incidence . Similar thresholds existed for real population densities: Guangzhou , Rio , New York and Delhi . Increased mobility reduced the effect of resolution on the epidemic . The kernels which were most affected by resolution were those which gave a lower mobility than that identified by commuting data ( Table S1 ) . Generally children are considered to cause the majority of transmission of pathogens like flu and measles , because their level of age group assortative mixing is very high [33] , [34] . Children also travel less far than working adults [22] . Together , these imply that a kernel for children is likely to be more restrictive than those defined by commuting data alone . Therefore , our results indicate that the correct specification of population interactions and sufficient spatial resolution is particularly relevant for epidemics such as measles and flu - those in which children play a large role . Although we have considered age effects implicitly by including lower mobility levels than are reported for commuting data ( Table S1 ) , the explicit representation of age within a similar modelling framework may lead to additional insight . For example , transmission dynamics at different scales may be driven by different age groups: the behaviour of more mobile adults may be disproportionately important in the seeding of nearby pixels . However , the slower than expected within-country spatial spread during 2009 [35] suggests that for pandemic influenza , population sub-groups with reduced mobility likely do define the fundamental resolution . We have chosen to represent the real biological process by a high resolution metapopulation model . Although we have not been able to push the model to resolutions higher than 1 km by 1 km , we suggest it is reasonable to assume , for the mobility kernels considered here , that the thresholds observed for peak incidence would not change substantially were we to approach the resolution of an individual-based model . The model used here was intended specifically to test only the changing resolution of the disease transmission process . By design we did not want to assume that transmissibility was intrinsically higher or lower in different parts of the population . In future work , we hope to calibrate this model structure using actual disease incidence data and ( after a minor modification to the definition of the force of infection ) test for the possibility that population density affects transmissibility . Although it is somewhat reassuring that estimates of peak incidence are biased upwards if resolution is too low , the epidemic duration is underestimated . In order to avoid the effects of incorrect model specification , where possible , spatial resolution should be treated in a similar manner to temporal resolution in fixed-time-step models: neither the doubling nor halving of spatial resolution should have a substantive effect on key model outputs .
We defined a spatially explicit meta-population model as follows ( similar to Ref . [21] ) . A given region of known population density was represented as pixels , such that each pixel ( index ) is the same spatial size but the number of individuals in the pixel ( ) varied according to location . Mixing between and within each pixel was determined by a mobility model , represented by a matrix such that an entry was equal to the probability that for an individual from pixel , given that the individual made a contact , this contact was with an individual from pixel ( mobility was defined using a kernel , discussed later ) . The rate at which susceptible individuals in pixel became infected depended on ( 1 ) their risk of infection from those in pixel , ( 2 ) the risk of infection from infected individuals in pixel who travelled to , ( 3 ) the risk of infection that susceptible individuals from encountered when they travelled to . Therefore , the force of infection or the average rate that susceptible individuals in pixel became infected per time-step was: ( 1 ) where was the total number of pixels and for any pixel , was the number of individuals , was the number of infected individuals and infectious contacts were made with other individuals present in the pixel with rate . Note that is the same across all pixels; in future it may be of interest to vary the transmissibility across pixels ( so moves into the sum in Eqn . ( 1 ) as ) . The system of difference equations for a pixel in the stochastic SIR model was ( with the condition that all classes hold a whole number of individuals ) : ( 2 ) where was the number of individuals in pixel , state ( S , I or R ) that experienced the event – infection or recovery – in time-step . We ignored death in this model as we considered fairly short timescales and a non-fatal strain of influenza . Each time-step , the number of individuals experiencing each event ( ) that occurred in pixel and state , with a population was determined in the following way: We used a mobility model to determine the relative frequency of potentially infectious contact . This was represented as a matrix with entries , defined as the probability that for an individual from pixel , given that the individual made a contact , this contact was with an individual from pixel , so: ( 3 ) where was the total population in pixel , was the interaction kernel defining the effect of the distance between pixels and on the contact between them . The kernel defines the relative probability of travelling between two pixels and not the absolute flux , similar to [15] , [17] . The factor normalised and ensured that the rows sum to 1 . The matrix was used in the calculation of force of infection , Eqn . ( 1 ) . We used a variation of the offset power function for the kernel ( similar to [9] , [15] ) : ( 4 ) where was the distance below which the kernel function saturated , we used . The power determined the mixing between pixels , this was varied to give a range of mobilities but was always less than 0 . The next generation matrix , , for the model with pixels and force of infection ( Eqn . ( 1 ) ) , can be defined ( similar to [21] ) : ( 5 ) where was the time spent infected ( which depended on recovery rate such that , same for all pixels ) , was the number of individuals in pixel , infectious contacts were made with other individuals present in the pixel with rate and was the mobility matrix defined earlier . Then was equal to the spectral radius of this matrix [36] , [37] . For this model , , i . e . was independent of resolution and mobility; see Text S1 in Supporting Information S1 for full derivation .
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Fundamental spatial processes such as individuals' interactions and movement are not sufficiently well understood and yet they define the transmission of infectious diseases through populations . Spatial models of epidemics represent the region of interest ( such as a city or country ) as a collection of spatial units . To anticipate the magnitude and timing of peak incidence and to predict demand on health care resources in the region a clear understanding is needed of the relationship between the resolution of the representation ( number and size of the pixels ) , the population interactions and the epidemic trajectories . We used a spatially explicit meta-population model of disease transmission to demonstrate that thresholds existed such that models with too low a resolution overestimated peak incidence , implying that ill-defined models may result in incorrect predictions . However , the results suggest that if population interactions are represented in sufficient detail , accurate estimates of peak demands on key health care resources are feasible .
|
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"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
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"infectious",
"diseases",
"medicine",
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"health",
"sciences",
"population",
"modeling",
"epidemiology",
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] |
2014
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The Spatial Resolution of Epidemic Peaks
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Vector control is the only widely utilised method for primary prevention and control of dengue . The use of pyriproxyfen may be promising , and autodissemination approach may reach hard to reach breeding places . It offers a unique mode of action ( juvenile hormone mimic ) and as an additional tool for the management of insecticide resistance among Aedes vectors . However , evidence of efficacy and community effectiveness ( CE ) remains limited . The aim of this systematic review is to compile and analyse the existing literature for evidence on the CE of pyriproxyfen as a vector control method for reducing Ae . aegypti and Ae . albopictus populations and thereby human dengue transmission . Systematic search of PubMed , Embase , Lilacs , Cochrane library , WHOLIS , Web of Science , Google Scholar as well as reference lists of all identified studies . Removal of duplicates , screening of abstracts and assessment for eligibility of the remaining studies followed . Relevant data were extracted , and a quality assessment conducted . Results were classified into four main categories of how pyriproxyfen was applied: - 1 ) container treatment , 2 ) fumigation , 3 ) auto-dissemination or 4 ) combination treatments , –and analysed with a view to their public health implication . Out of 745 studies 17 studies were identified that fulfilled all eligibility criteria . The results show that pyriproxyfen can be effective in reducing the numbers of Aedes spp . immatures with different methods of application when targeting their main breeding sites . However , the combination of pyriproxyfen with a second product increases efficacy and/or persistence of the intervention and may also slow down the development of insecticide resistance . Open questions concern concentration and frequency of application in the various treatments . Area-wide ultra-low volume treatment with pyriproxyfen currently lacks evidence and cannot be recommended . Community participation and acceptance has not consistently been successful and needs to be further assessed . While all studies measured entomological endpoints , only two studies measured the reduction in human dengue cases , with inconclusive results . Although pyriproxyfen is highly effective in controlling the immature stages of dengue transmitting mosquitoes , and–to a smaller degree–adult mosquitoes , there is weak evidence for a reduction of human dengue cases . More well designed larger studies with appropriate standardised outcome measures are needed before pyriproxyfen is incorporated in routine vector control programmes . Additionally , resistance to pyriproxyfen has been reported and needs investigation .
Over the past five decades , the global burden of dengue is estimated to have increased massively: Bhatt et al . [1] postulated that in 2010 there were 96 million apparent , and 294 million unapparent infections worldwide , with 22 , 000 registered dengue-related deaths reported in 2014 [2] . Transmission of dengue is through infective bites of female Aedes aegypti ( L . ) ( Diptera: Culicidae ) mosquitoes and , to a lesser extent , of Ae . albopictus ( Skuse ) . The immature stages of the Aedes mosquitoes are found in water filled containers [3] . In the absence of anti-viral medication and with the first commercially available vaccine not yet widely available for public health use [4] , vector control remains the cornerstone for dengue prevention [5 , 6] . Due to their behaviour , adult mosquitoes transmitting dengue are difficult to attack and larviciding as well as larval source reduction are often the first choice of intervention . Larvicides have most often been implemented against Ae . aegypti as it breeds almost exclusively in domestic water containers . Ae . albopictus uses both , artificial and natural breeding sites [7] and is therefore more difficult to tackle . Pyriproxyfen is an insect growth regulator ( IGR ) with a slow-acting larvicidal activity against a broad spectrum of public health insect pests [8] and it is being used extensively worldwide both in public and private settings . Acting on the endocrine system of insects by mimicking the juvenile hormone , pyriproxyfen hinders molting and subsequently inhibits reproduction . In addition , it causes morphological and functional aberrations in emerging adults , such as decreased fecundity and fertility . Due to its very low mammalian toxicity [9] , pyriproxyfen is approved by the World Health Organization ( WHO ) for the treatment of potable water against mosquitoes [10] . Pyriproxyfen has been studied extensively in experimental , i . e . controlled laboratory or semi-field settings , with evidence of efficacy against immature Aedes spp . [11 , 12] . Yet , in field application studies , pyriproxyfen demonstrated mixed outcomes regarding its effectiveness as well as persistence . To date , no systematic review of the scientific literature has been undertaken to examine the evidence for the effectiveness of pyriproxyfen against dengue vectors . Therefore , the objective of this study is to review systematically the available literature for evidence on the community effectiveness ( CE ) of pyriproxyfen as a vector control method reducing Ae . aegypti and Ae . albopictus populations and dengue transmission .
The following inclusion criteria were used: 1 ) Studies providing original research dealing with the CE of pyriproxyfen—alone or in combination with other chemical vector control products . 2 ) As for study types , included were any cRCTs or randomised controlled trials ( RCT ) ; non-RCTs ( nRCT ) only if they were relevant to the research question and using a control , e . g . quasi-randomised controlled trials ( quasi-RCTs ) , intervention control trials , controlled before and after studies . Unlike in RCTs , allocation in quasi-RCTs is performed in a way that is open to systematic bias , i . e . chances of being in one group or another are not equal . In cRCTs , pre-existing groups of participants are allocated to ( or against ) an intervention . Intervention control studies use methods designed to examine efficacy or effectiveness of an intervention in a group but do not use randomisation . 3 ) Any study that applied pyriproxyfen in the field—defined as any community or environment where dengue vectors naturally occur—was considered CE and included in the analyses . Efficacy studies , defined as trials performed under laboratory conditions were excluded . Of the studies that undertook both methods , only the CE component was considered . Inclusion criteria included the above-mentioned study types , to give a broader picture of existing studies , since vector-control studies have varying designs and information may be useful . Two researchers independently carried out the literature search until 01 August 2016 with no starting time limit . The search was conducted in English , but articles were not excluded if the full text was not available in English . The search strategy was applied to the following seven databases to locate peer-reviewed studies: PubMed , Web of Science , EMBASE , LILACS , WHOLIS and Cochrane . In addition , grey literature using Google Scholar has been searched . Search terms were divided into three broad categories , including 1 ) disease relevant terms , 2 ) vector relevant terms and 3 ) intervention relevant terms . For the disease category , the terms were: Dengue , Dengue haemorrhagic fever , Dengue shock syndrome , along with the abbreviations DF ( dengue fever ) , DHF ( dengue haemorrhagic fever ) , DS ( dengue syndrome ) , and DSS ( dengue shock syndrome ) ; for the vector category: Aedes , Aedes aegypti , Aedes albopictus; and for the intervention category: pyriproxifen , and insect growth regulator . Once screened for duplicates by author , title , journal and publication date , eligible studies were screened against the inclusion criteria . At first , titles and abstracts were screened by two independent reviewers , in a second step articles were reviewed in full and relevant information was extracted into the evidence table ( Table 1 ) . A third reviewer was available for the potential case of disagreement between the two independent reviewers . Studies were assessed for quality using the assessment of multiple system reviews ( AMSTAR ) [14] . For the purpose of this review , the overall quality of studies was not used to exclude studies , but as a tool for evaluating the impact of the reported outcomes . A comparative analysis of the main study outcomes was conducted , using the quality of each individual study as a weighing tool . The use of randomisation , the calculation of sample sizes and the size of the unit of allocation all impacted the weight individual studies were assigned . Finally , analytical categories were developed based on the method of pyriproxyfen application . These categories were: 1 ) container treatment , 2 ) fumigation , 3 ) auto-dissemination , and 4 ) combination of pyriproxyfen with adulticides . As for 1 ) , ‘container treatment’ is for the purpose of this review defined as any intervention performed by using any kind of Aedes spp . infested containers ( Table 2 ) .
The included studies were published between 2005 and 2014 ( Table 1 ) . One was in Thai the others in English . Seven of the studies were conducted in Central or South America ( four in Argentina [21 , 22 , 23 , 24] , one in Colombia [25] , two in Peru [26 , 27]; four in Southeast Asia ( one in Cambodia [28] , two in Thailand [29 , 30] , one in Vietnam [31] ) ; two in the USA [32 , 33] , two in Martinique [34 , 35] , and two in Europe ( Italy [36] , Netherlands [38] ) . Information on potential confounding factors such as the socio-economic status of residents [23 , 25 , 28 , 31 , 33] or housing construction [22 , 28] was not systematically reported . Weather conditions , either historical or during the intervention period , were reported in six of the studies [26 , 28 , 31 , 32 , 33 , 35] . No study incorporated a specific economic analysis or provided cost estimates for consideration . The most common study design was nRCT , used in 12 studies . Three were RCTs , one quasi-RCT and one cRCT . Regarding the vectors , Ae . albopictus alone was studied in two of the included studies [33 , 36] , one study [32] tackled both , Ae . aegypti and Ae . albopictus , both laboratory-reared . The remaining 14 studies looked at Ae . aegypti only . Ten studies [21 , 23 , 24 , 25 , 27 , 28 , 32 , 33 , 34 , 35] assessed the feasibility and efficacy of container treatment . Two studies were RCTs [25 , 35] , the others intervention control studies . Four studies were performed in an urban environment [21 , 23 , 25 , 27] , with the largest study covering an entire town [25]; the other six [24 , 28 , 32 , 33 , 34 , 35] were conducted in villages and rural areas . In six of the studies [21 , 23 , 24 , 28 , 32 , 35] , pyriproxyfen had a significant ( 82–100% EI ) and long lasting ( up to 8 months ) effect . However , the two RCTs reported less positive results with significant effects for 4 weeks only . However , it must be considered that one RCT [25] measured catch basin positivity , making comparison with the other studies difficult . It also had several additional constraints such as moderate Aedes indices , low pupae/person indices from the start , inability to reach the intended sample size , and the emergence of a dengue epidemic during the intervention . The other RCT [35] and one nRCT [34] reported treatment persistence of only 4 weeks , though initial larval densities were significantly reduced . One study [21] found a variable larvicidal effect in 20 containers and attributed this to larvicide dilution , though none of the other container studies reported efficacy limitations with increasing container sizes . Notably , Sihuincha [27] treated different sizes of water tanks successfully for five months despite a high turnover of the treated water . Regarding area-wide ULV application , two studies [23 , 32] recommend this method of application over a wide range of container sizes , while one [33] found it not suitable for either larval habitat treatment or auto-dissemination . The two studies that did report good results with ULV application [23 , 32] used pyriproxyfen combined with permethrin . As for persistence in container treatment , slow-release formulations had the best results . Sihuincha [27] reported a mortality rate >80% over five months with application by gauze bags , Seng [28] reported similar effects over eight months with resin strands and Seccacini [24] reported 100% EI at six months using pyriproxifen-impregnated O-rings . A shorter persistence of four weeks was reported from a large field trial with granules in water in Martinique [35] , while the longest duration of effective EI through ULV treatment was 35 days [23] , and the shortest only two weeks [32] . Two studies [21 , 22] examined the use of fumigant canisters . Both used a combination of pyriproxyfen and permethrin and reported a significant inhibitory effect on adult emergence of Ae . aegypti as well as on BI . However , the size of the effect and persistence ( less than 9 weeks ) was limited . Adding outdoor ULV application of permethrin increased the effectiveness of the intervention . Six studies evaluated auto-dissemination . The only RCT examining auto-dissemination [30] found significant results only in their BGS trap counts . However , these are known to be the most sensitive when counts are low from the start [39] . The remaining studies [26 , 33 , 36 , 37] , using intervention control designs , primarily demonstrated that this approach is efficacious and that it can be applied easily and at low costs . Caputo [36] performed field experiments with wild Ae . albopictus and reported an inhomogeneous product transfer to different sentinel sites . However , it was rightly pointed out that if the approach is applied to reduce Ae . albopictus adult densities , the mosquitoes themselves will disseminate the larvicide to the most attractive ( i . e . most productive ) natural breeding sites . Devine [26] used 0 . 5% pyriproxyfen concentration in 1 l plastic containers and demonstrated overall reductions of Ae . aegypti adult emergence of 49–84% , as opposed to 7–8% in controls . In another experiment [36] with the same pyriproxyfen concentration , the overall reduction of Ae . albopictus adult emergence was 20 . 8% , as opposed to 2 . 4% mortality in controls . Given that the LC50 reported for Ae . albopictus ( 0 , 11 ppb; [39] ) is about 10 times higher than that reported for Ae . aegypti , this is a promising result . Seven of the reviewed studies combined pyriproxyfen with interventions targeting adults as well . They did not stratify the effects by product but rather as combined results . Five studies evaluated pyriproxyfen in combination with permethrin [21 , 22 , 23 , 29 , 31] , one in combination with the fungus B . bassiana [35] , and one with Spinosad [34] . Two of these studies were RCTs [22 , 31] . Both found significant changes through their respective interventions . While both used a combination of pyriproxyfen with permethrin , their application differed ( fumigation versus EcoBio-block S ) and so did their measured outcome values ( BI versus CI and seroprevalence studies ) . Direct comparison is difficult but it can be summarised that they had good results that lasted for 9 weeks ( fumigation plus ULV ) and 5 months ( EcoBio-block S ) , respectively . Similar results were reported in the non-RCTs . In summary , the combination of adulticidal and larvicidal products can increase effectiveness by simultaneously controlling adults and larvae , and by expanding persistence [22] . Only one study found and reported on resistance of pyriproxyfen . Marcombe [35] demonstrated Ae . aegypti being tolerant against pyriproxyfen presumably due to cross-resistance with temephos , as pyriproxyfen had never been used on the island of Martinique before . Three of the included studies [22 , 25 , 28] described community perceptions and uptake of the interventions . Seng [26; Cambodia] reported overwhelmingly positive perceptions after an initial period of concern . Harburguer [22; Argentina] found the community capable and ready to participate in a mosquito control programme by using non-professional control tools ( fumigant tablet ) . However , the authors also highlight the community’s reluctance to take part in training workshops , even though most applied the tablet while only 16% attended the workshop . The third study ( [25]; Colombia ) describes the importance of engaging and empowering local field staff regarding design and operation of entomological surveillance activities .
This systematic review of the CE of the juvenile hormone mimic pyriproxyfen against Aedes spp . presents evidence suggesting that pyriproxyfen can effectively control the adult emergence of immature stages of dengue vector mosquitoes in a variety of real world habitats . If the most productive breeding sites are identifiable and accessible–e . g . catch basins , water storage containers—direct treatment by monthly application appears to be the most effective and feasible with controlled-release formulations having strong and long-lasting effects . With regards to efficacy , inconsistent results were presented [11 , 27] . These are most probably the result of differences between strains , formulations , and experimental conditions . There is a clear evidence that pyriproxyfen effectively inhibits Aedes adult emergence at concentrations of <1 ppb . As to the methods of application , the evidence is highly variable . For container treatment , the effectiveness of pyriproxyfen seems to depend on factors such as the material of which the individual containers are made and the local environmental conditions . For example , Vythilingam [40] reported much higher levels of sustained residual activity in plastic tubs than in earthen jars , while Schaefer [41] and Glare [42] demonstrated a lower stability of pyriproxyfen at higher temperatures . In addition , there seems to be a natural inter-individual variation between containers [35] . There are contradictory reports on whether different container sizes play a relevant role , probably to be addressed by the amount of pyriproxyfen used . Overall , the available evidence shows that a targeted treatment with a slow-release ( e . g . granular ) or a long-lasting ( e . g . resin strands ) formulation can yield an adequate EI for up to 34 weeks . However , more research is needed to define the lowest effective concentrations for each formulation and the frequency by which the treated containers should be replenished . Studies should be performed with standard containers in field trials to avoid accurate estimates of mosquito density being obscured by random variation among individual containers . Also , further work is needed to determine the impact of environmental conditions ( UV light , dilution , temperature , etc . ) on effectiveness and persistence . Regarding ULV application , only interventions with a product combination ( e . g . permethrin ) showed a significant effect , suggesting that ULV treatment with pyriproxyfen alone cannot be recommended . Some authors discussed the potential reasons for the failure of ULV and hypothesised that a ) container openings were too small for pyriproxyfen to enter; b ) treatment surfaces were too small to cover a significant number of breeding sites; c ) extreme weather conditions ( first year very dry , second year higher than usual precipitation ) adversely impacted the larvicidal effect; d ) cryptic habitats of Ae . albopictus not accessed or e ) the slow mode of action of pyriproxifen was not considered in the follow up evaluation [32 , 33] . Space spraying–here as fumigation—has shown its effect only indoors , and it should be considered as part of a multi-intervention approach . According to Devine [26] , auto-dissemination could be an interesting approach to reach elusive breeding sites . Sihunicha [27] showed that an exposure of 30 min to water containing 0 . 003 g a . i . /m2 pyriproxyfen allowed for horizontal transfer of effective larvicidal pyriproxyfen doses to untreated environments . The same study reports that subsequent eclosion of eggs was decreased by 70–90% . Different ovitraps have been designed and their effectiveness tested . Extraordinarily low doses of pyriproxifen ( Ae . aegypti: LC50 = 0 . 011 ppb ( 11 ) , 0 . 012 [27] , 0 . 0039 ppb [43]; Ae . albopictus: LC50 = 0 . 11 ppb [45] ) are needed for this approach [27 , 44] . The herein reviewed studies reported maximum distances of 150 m ( 28 2013 ) and 200 m [45] travelled by the mosquito from the treatment sites . These are in line with Marini et al . [46] who demonstrated that gravid female Aedes spp . travel 50–200 m from the release sites . Kaufmann et al . [47] reported longer distances of up to 3 km . Further studies are needed to prove and improve the auto-dissemination strategy under field conditions . Specifically , methods of application , concentrations and frequencies of treatment need to be clarified . Controlled-release/long-lasting formulations had a good effect with much longer persistence than other treatments . In order to improve adherence in community based approaches , such interventions should be preferred . They may even be more efficient as re-treatment would have to be less often and , therefore , the overall operation is more cost-effective as less professional personnel is involved . Those studies that combined the application of pyriproxyfen with different adulticidal products clearly showed that effectiveness can be increased by simultaneously controlling adults and larvae , and by expanding persistence [22] . From the presented studies , there is insufficient evidence to determine what impact the level of motivation in a community could have on vector control . This question warrants further investigation in larger prospective studies . Such trials are also needed to assess whether and how communities can be motivated and control efforts sustained . The majority of the included studies were from South and Central America , yet , Bhatt [1] postulates that Africa’s dengue burden is nearly equivalent to that of the Americas ( i . e . 16 ( 11–22 ) million infections annually , representing , 16% of the global total ) . Also , India is estimated to contribute 34% ( i . e . 33 ( 24–44 ) million infections per year ) of the global total dengue infections . Further evidence is required on the effectiveness of pyriproxyfen in Africa and India to understand the influence of local environmental and societal factors . Concluding , although pyriproxyfen is highly effective in killing the larvae of dengue transmitting vectors , and–to a smaller degree–also adult mosquito stages , evidence for the reduction of human disease transmission is weak . Lack of evidence is primarily due to small sample sizes , inappropriate study designs and lack of relevant , standardised outcome measures . Before issuing specific recommendations for the routine use of pyriproxyfen as a larvicide in dengue control programmes these research gaps must be addressed . Additionally , cross-resistance to pyriproxyfen has previously been reported by Macoris-Andrighetti [48] and , should it be verified , would have operational consequences for future dengue vector control . It therefore requires further attention in future studies as well as public health programmes .
|
There is evidence that pyriproxyfen may effectively reduce the density of immature mosquito stages when applied to identified breeding sites . Various formulations are commercially available , and easy to use without a health threat to the user . However , questions remain regarding its use as a single agent in a community setting . Considering its mode of action , it would not be the product of choice for use in an acute outbreak setting . However , for a sustainable community approach , especially slow-release pyriproxyfen formulations seem promising , because they are the longest lasting choice . The analysis suggests , that combination with a second vector control chemical , preferably an adulticide tackling different stages of mosquito development , increases the efficacy of pyriproxyfen and prolongs the duration of a single application . This systematic literature review clearly shows that there is a need for further studies , preferably utilising cluster-randomised controlled ( cRCT ) designs , to investigate the community effectiveness of pyriproxyfen and to link entomological outcomes to human dengue transmission .
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2017
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Community effectiveness of pyriproxyfen as a dengue vector control method: A systematic review
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Enzootic strains of Venezuelan equine encephalitis virus ( VEEV ) have been isolated from febrile patients in the Peruvian Amazon Basin at low but consistent levels since the early 1990s . Through a clinic-based febrile surveillance program , we detected an outbreak of VEEV infections in Iquitos , Peru , in the first half of 2006 . The majority of these patients resided within urban areas of Iquitos , with no report of recent travel outside the city . To characterize the risk factors for VEEV infection within the city , an antibody prevalence study was carried out in a geographically stratified sample of urban areas of Iquitos . Additionally , entomological surveys were conducted to determine if previously incriminated vectors of enzootic VEEV were present within the city . We found that greater than 23% of Iquitos residents carried neutralizing antibodies against VEEV , with significant associations between increased antibody prevalence and age , occupation , mosquito net use , and overnight travel . Furthermore , potential vector mosquitoes were widely distributed across the city . Our results suggest that while VEEV infection is more common in rural areas , transmission also occurs within urban areas of Iquitos , and that further studies are warranted to identify the precise vectors and reservoirs involved in urban VEEV transmission .
Members of the Venezuelan equine encephalitis virus ( VEEV ) complex are arboviruses belonging to the Alphavirus genus of the Togaviridae family . First identified among equines in the 1930s [1] , VEEV-associated human disease was not recognized until 1943 [2] , [3] , although epidemiological data suggest that outbreaks may date back to the 1920s [4] . VEEV subtypes cause a wide clinical spectrum of disease ranging from undifferentiated fever to severe neurological symptoms , with a case fatality rate of 1–4% [5] . Two transmission cycles have been identified: an enzootic cycle , maintained among rodent reservoirs in forest habitats , and an epizootic cycle that causes high rates of mortality in horses as well as epidemics among human populations [4] . These cycles are typically associated with distinct subtypes of the VEE virus complex: subtypes IAB and IC with equine epizootics , subtypes ID , IF , and II–VI with the equine avirulent enzootic cycle [4] , [6] , and subtype IE with both enzootic and equine-virulent transmission cycles [7] , [8] , [9] . Despite disparate serological and clinical phenotypes some enzootic and epizootic subtypes are highly genetically conserved . In particular , strains of the enzootic subtype ID ( Columbia/Venezuela genotype ) show less than 0 . 5% divergence from epizootic IAB and IC subtypes at the amino acid level [10] , [11] , [12] . Based on this genetic conservation , epizootic strains have been proposed to emerge periodically from progenitor strains continuously maintained in an enzootic forest cycle . Accordingly , a single amino acid change within the E2 envelope gene has been shown to confer an epizootic phenotype on an enzootic VEEV strain [10] , [11] , [12] , [13] . Geographically , members of the VEEV antigenic complex have been restricted to tropical and sub-tropical regions of the Western Hemisphere , with VEEV complex isolates reported from Argentina through the southern United States . The majority of human VEEV infections have occurred during large outbreaks in Central America and northern South America , most notably in Colombia and Venezuela [14] , [15] . In Peru , multiple human epidemics and equine epizootics have occurred on the Pacific coastal plain , possibly due to introduction of epizootic virus from Ecuador . At present , all evidence suggests that the epidemiology of VEEV on the west coast of Peru has been associated exclusively with the epizootic subtype IAB [3] , [16] , [17] , [18] . In contrast , in the Amazon Basin to the east of the Andean mountains VEEV isolates have been restricted to enzootic ID , IIIC , and IID subtypes . The first cases of human VEEV infection in this region were reported in 1994 when Peruvian Army personnel were deployed to an area near Iquitos [18] , [19] . Human VEEV cases have been documented near Iquitos continuously since then [6] , [19] ( TJK , unpublished data ) . Based on entomological studies carried out in the nearby village of Puerto Almendras and the Otorongo Military Base from 1996–2001 [20] , mosquitoes from the Culex ( Melanoconion ) group have been incriminated as the local sylvatic VEEV vector in rural areas , consistent with results from Panama , Colombia , and Venezuela [4] , [5] , [21] , [22] , [23] . While potential vectors of enzootic VEEV have also been periodically detected within urban neighborhoods [24] , [25] , the possibility for urban transmission of enzootic VEEV has not been systematically addressed . The city of Iquitos represents the interface between the Amazon forest and a densely populated urban environment , and therefore a potential bridge between enzootic transmission cycles and potential peridomestic urban transmission . In 1990 , the Naval Medical Research Center Detachment ( NMRCD ) initiated a clinic-based surveillance program to determine the etiologies of febrile illness within Iquitos as well as nearby villages . Herein we report evidence of a 2006 outbreak of febrile illness associated with enzootic VEEV infection detected by the NMRCD surveillance program . Following the outbreak , a seroprevalence survey was carried out in three Iquitos neighborhoods where acute human cases were identified as well as in a control neighborhood where acute cases were not reported during the 2006 outbreak . Additionally , a series of mosquito collections were conducted both during and after the outbreak to characterize potential urban VEEV vectors . The primary objective of this article is to evaluate the evidence for peri-domestic VEEV transmission within the city of Iquitos , Peru .
The study was conducted in the Loreto Department in Peru in the city of Iquitos located 120 meters above sea level in the Amazon forest ( 73 . 2°W , 3 . 7S° ) . This site has been described in detail previously [24] , [25] , [26] , [27] , [28] . Briefly , Iquitos is a geographically isolated population within the Amazon forest , accessible only by river or air travel . The major industries of Iquitos are small business , fishing , oil , lumber , tourism and some agriculture [27] . The climate is tropical , with an average daily temperature of 25°C and year-round precipitation totaling 2 . 7 meters . Daily temperature and precipitation data for 2000–2006 from a weather station located at the Iquitos airport were retrieved from http://lwf . ncdc . noaa . gov/cgi-bin/res40 . pl ? page=climvisgsod . html ( Figure S1 ) . River levels surrounding the city change dramatically due to runoff from the eastern side of the Andes mountains , increasing by up to 10 m ( 108–118 m ) between the “vaciente” ( May–November ) and “creciente” ( December–April ) [29] . Information for daily river levels was obtained from the local water plant ( Figure S1 ) . Serological and entomological surveys described in the study were initially targeted to three areas of Iquitos based on the residences of VEEV-infected patients detected through a clinic-based surveillance system during 2006 ( Figure 1 ) . The neighborhoods included in this study were Bella Vista Nanay ( San Pedro , Nuevo Bellavista , Acción Católica , Nuevo Amanecer , and San Valentín ) located in the northern-most section of the city; Belén , located in the eastern part of the city along the Itaya River; and three sites along Avenida Participación in the San Juan District ( Las Mercedes , San Pablo de la Luz , and 26 de Febrero ) . Common attributes of the Bella Vista , Belén , and San Juan sites include seasonal flooding , and proximity to rivers , lowland humid tropical forest , and open farmland . The area surrounding Iquitos has experienced varying degrees of deforestation , but patches of both primary and secondary growth trees are found on opposite banks of the three surrounding rivers . The habitat observed in all three neighborhoods is rather homogeneous . Species diversity , including a wide variety of aquatic plants ( Onagraceae , Pontederiaceae , Araceae families ) and abundance was highest in the Avenida Participación neighborhoods , followed by Bella Vista Nanay , and finally Belen which had the highest density of housing and port activity ( Table S1 for species list ) . The Bella Vista site is seasonally flooded by the blackwater Nanay River , whereas the Belen and San Juan sites are located on the silty and sediment-rich whitewater Itaya River . The Allpahuayo National Reserve and “Ell Huayo” Botanic Garden are located ∼25 km to the south of the city where both feral mammalian and forest mosquito species have been well characterized [30] , [31] . In addition to the three neighborhoods with active VEEV cases during 2006 , we obtained blood samples from residents in designated a control area where no increase in VEEV activity was detected . This area included 22 blocks in north central parts of Iquitos where previous dengue studies had been conducted ( ACM , unpublished data ) . This geographically diverse group of blocks was easily accessible to our research team because of our previous studies there , and represented a contrast to the 3 study neighborhoods . Overall , these control neighborhoods were of higher socio-economic condition [29] ( Morrison unpublished data ) and located several blocks from the river whereas the other study areas were adjacent to the river . Municipal records obtained from “La asociación de viviendas inundables y desarrollo humano de Punchana” and “El Programa de Emergencia Social Productivo Urbano” indicate that the neighborhoods in BellaVista Nanay and Avenida Participación ( “New” ) were all established since 1998–2003 whereas the Belen and control blocks ( “Old” ) have been registered since 1943 and have existed prior to that date . We will present data from 3 separate studies . First we will describe a bimodal outbreak of febrile illness attributed to VEEV infection that began in 2005 and culminated with a significant increase in cases in the first half of 2006 . This notable increase in human cases stimulated NMRCD to carry out a cross-sectional seroprevalence study in three neighborhoods where VEEV cases had been detected within the city limits of Iquitos , as well as a series of entomological studies to document the abundance of and VEEV infection rates in potential mosquito vectors . Below we describe in detail the methods associated with each sub study . Since 2000 , NMRCD has been conducting syndromic surveillance in 11 Government Health Centers and Hospitals ( 9 urban and 2 rural ) . This study , entitled “Surveillance and Etiology of Acute Febrile Illness , ” was approved by the NMRCD Institutional Review Board ( NMRCD . 2000 . 0006 ) . Trained health workers are stationed in each location 0700-1300 . All acute , undifferentiated , febrile illness cases ( i . e . temperature greater than or equal to 38°C for 7 days duration or less ) seen by a health center physician were referred first to the national Malaria program where they are tested for Malaria by a thick smear and then to our surveillance program . For inclusion into the study , in addition to fever , patients needed to report one or more of the following symptoms: headache; muscle , ocular and/or joint pain; generalized fatigue; cough; nausea; vomiting; sore throat; rhinorrhea; difficulty breathing; diarrhea; bloody stools; jaundice; dizziness; disorientation; stiff neck; petecchiae; ecchymoses; bleeding gums or nose . Children younger than five years of age were included if they presented with hemorrhagic manifestations indicative of dengue hemorrhagic fever ( DHF ) , including bleeding gums or nose , petecchiae , bloody stool or hematemesis . Written informed consent was obtained from adults greater than 18 years of age . For minors , written consent was obtained from parents , and assent was obtained from participants ages 8–17 . For participants unable to read and sign the consent form , a witness was present to testify to oral consent . Demographic data , residential address , medical history , and clinical features for each patient were obtained using a standard questionnaire . During the acute phase of illness blood samples were obtained from each patient , and when possible , convalescent samples were obtained 10 days to 4 weeks later for serological studies . In addition , axial temperature , blood pressure , and respiratory rate were recorded , and in most facilities a tourniquet test was performed . Exclusion criteria included a clear focus of infection ( i . e . respiratory , gastrointestinal , urinary tract ) . Acute and convalescent samples were tested for a range of arboviruses including VEEV . Diagnoses were considered confirmed if they met the following criteria: clinical diagnosis along with laboratory confirmation ( isolation of virus from the sample , identification by RT-PCR , or 4-fold increase in IgM antibody titers ) . Patients' residences were located using existing GIS data for Iquitos [26] and confirmed by study team members . In response to the notable increase in VEE cases observed in early 2006 , serological surveys were initiated in three neighborhoods with high VEEV activity between January–June 2006 and on blocks that had participated in a previous dengue cohort study ( 22 city blocks located in 7 geographic zones ) located in the districts of Maynas , San Juan and Punchana . Surveys were conducted between early November and mid-December of 2006 . The post-outbreak study protocol was reviewed and approved by the NMRCD Institutional Review Board ( PJT . NMRCD . 001 ) . Trained phlebotomists ( 10–22 in total ) working in two person teams were assigned individual maps and proceeded door to door to explain the study and recruit participants . Participation was offered to all individuals ≥5 years of age . If the residents agreed to participate , the consent and assent forms were signed before samples were obtained . Written informed consent was obtained from participants older than 18 years , and from parents of participants younger than 18 . In addition , assent was obtained from participants 8–17 years of age . If participants were unable to read and sign the consent form , oral consent was obtained and documented in the presence of a witness . Each participant was asked a series of questions about their homes , as well as travel histories and illnesses during the previous year . Younger children ( <14 years ) were interviewed with their parents . Blood samples were obtained using standard aseptic techniques using a vacutainer tube and 21–23 gauge needles . All blood samples were tested for anti-VEEV antibodies using IgG and IgM ELISAs . All samples that tested positive by IgG ELISA were further evaluated for anti-VEEV antibodies by the plaque reduction-neutralizing test ( PRNT ) . Two adult mosquito collection methods were used for this study . First , standard household surveys were carried out as previously described [24] , [25] . In these surveys adult mosquitoes were collected using a backpack aspirator both inside and outside the house . Mosquito collections were concentrated in areas with high numbers of human VEEV cases during the 2006 outbreak . Second , CDC light traps baited with CO2 were placed outdoors between 1800-0600 h on four continuous nights in Bella Vista Nanay , Belen and in San Juan neighborhoods of Las Mercedes , San Pablo de la Luz and 26 de Febrero . Adult mosquitoes were identified to species [32] on dry ice and sorted into plastic vials by species for storage at −70°C for later testing for VEEV RNA by RT-PCR . Proportions were compared using a chi-square test using the FREQ procedure in SAS ( SAS Version 8 , 1999 , SAS Institute Inc . , Cary , NC . ) . Risk factors for infection with VEEV were evaluated by logistic regression using LOGISTIC in SAS . Models were constructed with the dichotomous dependent variable: PRNT positive for VEEV antibody at a titer of ≥1∶60 and the following independent variables: age ( adult , child ) , occupation , travel history ( report of multiple day trips outside Iquitos ) , and animals ( on property ) .
From 2000–2004 the NMRCD febrile surveillance program detected up to four VEEV cases per month with annual totals ranging from 10–14 cases ( Table 1 ) . In 2005 , however , 15 cases were identified in June and July , with an annual total of 27 . Fifteen of these cases came from rural clinics Zungaracocha ( 10 cases ) and Quistococha ( 5 cases ) . An additional five cases came from Hospital Apoyo , which serves patients from the entire Department of Loreto; three of these five maintained residence outside of Iquitos . In 2006 , there were 63 confirmed cases of VEEV infection captured in the febrile surveillance study ( Table 1 ) , representing a 5-fold increase in the number of cases from the 2000–2004 average . Of these 63 cases , 29 were identified by IgM seroconversion , and 34 were identified by IFA and RT-PCR . The partial PE2 nucleotide sequence was determined for a subset of the viral isolates and compared to previously characterized VEEV strains; all sequenced isolates were found to belong to the enzootic Panama/Peru ID subtype ( Figure S2 ) , closely related to previous isolates from the region [6] . Of the 63 cases detected in 2006 , 60 were detected from February to July , with the peak occurring in April and May . In both 2005 and 2006 , VEEV activity was concentrated during the first half of the year , as river levels were increasing to a peak in April and May . Precipitation levels were higher during January–March in both 2005 and 2006 when compared to 2000–2004 and in 2006 river levels were higher than previous years . Sixty of the VEEV-infected individuals reported to public health centers , and three were Peruvian Navy personnel reporting to a military health center . Health facilities in Belen , Bella Vista Nanay and San Juan were the urban centers with the highest 6-year and 2006 VEEV case totals ( Table 1 ) . The demographic information and travel history of the civilian cases observed in 2006 are summarized in Table 2 , with comparison to other febrile patients reporting to public health centers during the same year . No statistically significant differences were found between VEEV patients and other patients in gender , travel history , or occupation ( data not shown ) . Compared with other febrile patients , a higher percent of VEEV patients reported residences outside of urban Iquitos ( Table 2; χ2 = 10 . 2 , df = 1 , P< . 005 ) . Despite this bias , the majority of VEEV patients resided within the city ( 44 , 73 . 3% ) and did not report history of travel within the 30 days preceding their illness ( 53 , 88 . 3% ) .
Based on data from a clinic-based febrile illness surveillance program , transmission of enzootic VEEV subtypes has been well documented in the Iquitos area of northeastern Peru at consistent but low levels since the early 1990s [6] , [19] , [42] . In this study we report an outbreak of human VEEV infections during the first half 2006 detected through the NMRCD surveillance program , with the majority of patients residing within city limits . In response to this outbreak , we conducted an antibody prevalence study and mosquito collections within urban areas of Iquitos , targeting neighborhoods with large numbers of cases during the 2006 outbreak . The prevalence of VEEV antibody exceeded 18% in all areas , and known vectors of the disease were identified across the city . To our knowledge , this is the first antibody survey for enzootic VEEV in an urban population . Enzootic subtypes of VEEV have been thought to be maintained primarily in sylvatic cycles of tropical and sub-tropical forests . Consistent with this idea , we found acute VEEV infection to be more common among febrile patients residing outside of Iquitos , adjacent to the rain forest . Furthermore , we found multi-day travel and forest-related occupations to be statistically significant risk factors for VEEV antibody positivity . However , while transmission may be higher in rural areas , several lines of evidence suggest that transmission occurs within the urban areas of Iquitos as well . First , while forest-related occupation was a significant risk factor , these occupations comprised only a very small percentage of the total . In previous studies , enzootic VEEV clinical disease has been most frequently reported in adult males due to this association with high-risk forest occupations [43] . In contrast , we found no correlation in this study between gender and VEEV antibody status or acute VEEV infection . Second , consistent with prior reports [6] , the majority of the acute human cases detected in the passive surveillance program maintained residence within the city proper and did not report recent travel . Third , nearly 70% of VEEV-antibody positive survey respondents did not report recent travel , and day trips were not strongly associated with antibody positivity . It should be noted that our study might underestimate respondents' exposure to the forested zones surrounding Iquitos , as recall bias is likely a limitation in obtaining accurate travel history information . Furthermore , our antibody prevalence study did not adequately control for migration into the city . Specifically , we did not determine the length of residence at the current address for study participants or the location of previous residence . Especially in older age groups , infection before establishing residence in Iquitos must be considered . Despite these limitations , the existing information suggests that some level of VEEV transmission occurs within urban Iquitos . While our data suggest that enzootic VEEV strains are transmitted in urban areas , the exact mechanism is unclear . One possibility is the existence of a self-perpetuating endemic cycle established within urban Iquitos . Alternatively , urban cases of enzootic VEEV may be caused by repeated introductions of the virus from local forests , either by infected vectors or by infected hosts . Enzootic VEEV has been isolated from spiny rats ( Proechimys spp . ) in the region [6]; however , within the city limits of Iquitos rodent fauna appears to be mostly limited to Rattus ratus , Rattus norvegicus and mus musculus [44] . Iquitos is geographically isolated within the Amazon forest , and the distances between natural forest cycles and the city may well be within the natural home range of vectors and reservoirs . Within 20 km of the city there is a diversity of mammalian fauna , including rodents ( Proechimys spp . , Oryzomys spp . , Neacomys spp . , and Dasyprocia spp . ) , marsupials ( Phliander spp . , Marmosops spp . , Micoureus spp . , Caluromys spp . , Metachirus spp . , and Monodelphis spp . ) , bats ( Platyrihinus spp . , Artibeius spp . , Sturnira spp . , and Carollia spp . ) and sloths ( Choloepus spp . and Bradypus spp . ) In addition to sylvatic rodents , various species of waterfowl are readily infected by enzootic VEEV [41] , [45] . Such birds , if found to be amplifying reservoirs , could quickly expand the geographic and ecological distribution of the virus . Delineating the precise mechanism of urban transmission will require identification of relevant vectors and reservoir hosts currently infected and circulating within the city . Mosquitoes from the Culex ( Mel . ) species have been previously implicated as the primary vectors of enzootic subtypes of VEEV [40] , [46] , [47] . In this study , we found that Culex ( Mel . ) species are present throughout the city and abundant at certain times of the year . Most notably , Cx . ( Mel . ) ocossa , the vector of ID VEEV in Panama [41] , was observed in significant numbers . We have also identified the presence of other genera within urban Iquitos that have been previously incriminated as potential vectors for enzootic VEEV . For example , in our study blocks we detected Psorophora ( especially cingulata ) , Mansonia and Coquillettidia species , which have been shown to be competent vectors in the laboratory [48] , [49] . Additionally , Aedes aegypti has been shown to be a competent vector of both enzootic [50] and epizootic VEEV in the laboratory [51] , [52] , [53] , [54] and is present throughout the city of Iquitos . While feeding preference , as well as temporal and spatial distribution , argues against a role for Aedes aegypti in VEEV transmission cycles in Iquitos , the possibility warrants further examination . In the current study , we tested a wide range of mosquito species collected from CDC traps for the presence of VEEV RNA . We were unable to detect VEEV in any of the mosquito species tested; however , these studies were conducted at least four months after the last 2006 case was detected in the passive surveillance study . Furthermore , in previous studies VEEV infection rates in mosquitoes have been found to be very low [16] , [20] . Turell et al . , for example , recovered 25 VEEV isolates from 245 , 053 Culex ( Mel . ) spp . specimens [20] . In that study , 14 of 25 VEEV isolates were from Culex ( Mel . ) gnomatos; in our study , only one Culex ( Mel . ) gnomatos specimen was collected . To clearly define urban transmission patterns within Iquitos , prospective studies of potential vectors , including VEEV isolation and abundance pattern characterization , are needed during seasons of high incidence . Irrespective of species , there is compelling indirect evidence linking mosquito exposure within the city to prior VEEV infection . First , Bella Vista Nanay and San Jaun had higher mosquito abundance in both household surveys and CDC light-trap collections than the two neighborhoods with lower seroprevalence rates . Furthermore , mosquito net use was significantly more common in neighborhoods with higher seroprevalence than in those with lower seroprevalence rates . The fact that mosquito-net use was a risk factor for previous infection VEEV may seem counterintuitive , but it is also a proxy for intensity of exposure to mosquito bites . In areas where biting intensity is higher more individuals use nets out of necessity . This association needs to be interpreted in the context of the specifics of mosquito net use ( eg . the precise time of day that are people protected ) and condition . Many respondents mentioned bathing in the river at sunset; Culex mosquitoes are crepuscular and thus would have access to people during the dusk hours regardless of nighttime mosquito net use . Overall , it is clear that exposure to potential mosquito vectors occurs in all areas of the city , but this evidence indicates that exposure is highest in the areas with the highest seropositivity . The cause for the 2006 outbreak of VEEV infections is unclear . It is interesting to note that the 2006 outbreak inside the city was preceded by a spike in cases just outside the city in 2005 . There are several possibilities that might explain both increases . Vector abundance may have increased due to cyclical weather patterns , increasing rates of transmission in the forest , with a subsequent urban spillover . Furthermore unusually high annual river levels occurring in early 2006 may have increased competent vectors within the city ( i . e . , Cx . ( Mel . ) ocossa ) leading to the observed urban VEEV cases . Alternatively , human encroachment on forest areas , due to activities such as agriculture and logging , may have increased human contact with established enzootic transmission cycles and altered vector and reservoir distribution . Mutations in circulating sylvatic viruses might have led to greater potential for human infection and disease , and thus an increase in clinical cases . In light of the genetic similarity between enzootic and epizootic strains of VEEV , the possibility of humans as productive hosts , as well as the potential for emergence of an epidemic strain in urban areas , needs to be considered .
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Venezuelan equine encephalitis ( VEE ) is a mosquito-borne viral disease often causing grave illness and large outbreaks of disease in South America . In Iquitos , Peru , a city of 350 , 000 situated in the Amazon forest , we normally observe 10–14 VEE cases per year associated with people traveling to rural areas where strains VEE virus circulate among forest mosquitoes and rodents . In 2006 we detected a 5-fold increase in human VEE cases , and many of these patients had no travel history outside the city where they lived . In response to this outbreak , we decided to determine if potential carrier mosquitoes were present within the city and if city residents had been previously exposed to the virus . We found that mosquitoes previously shown to transmit the virus in other locations were present—in varying amounts based on location and time of year—throughout Iquitos . A large percentage of the human population ( >23% ) had antibodies indicating past exposure to the virus . Previous VEE infection was associated with age , occupation , mosquito exposure , and overnight travel . Our data represent evidence of transmission of a forest strain of VEE within a large urban area . Continued monitoring of this situation will shed light on mechanisms of virus emergence .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"public",
"health",
"and",
"epidemiology",
"virology",
"public",
"health",
"and",
"epidemiology/epidemiology",
"virology/emerging",
"viral",
"diseases",
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"epidemiology/global",
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"diseases"
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2008
|
Venezuelan Equine Encephalitis Virus in Iquitos, Peru: Urban Transmission of a Sylvatic Strain
|
A complex program of translational repression , mRNA localization , and translational activation ensures that Oskar ( Osk ) protein accumulates only at the posterior pole of the Drosophila oocyte . Inappropriate expression of Osk disrupts embryonic axial patterning , and is lethal . A key factor in translational repression is Bruno ( Bru ) , which binds to regulatory elements in the osk mRNA 3′ UTR . After posterior localization of osk mRNA , repression by Bru must be alleviated . Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression , separate from the full complexity of osk regulation . This assay reveals a form of translational activation—region-specific activation—which acts regionally in the oocyte , is not mechanistically coupled to mRNA localization , and functions by inhibiting repression by Bru . We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo . Loss of dimerization does not disrupt repression , as might have been expected from an existing model for the mechanism of repression . However , loss of dimerization does impair regional activation of translation , suggesting that dimerization may constrain , not promote , repression . Our work provides new insight into the question of how localized mRNAs become translationally active , showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization .
Localized mRNAs function in many biological settings to facilitate region-specific protein synthesis [1–3] . Translational repression of these mRNAs helps restrict distribution of the encoded proteins , an essential property if the protein has adverse effects at inappropriate locations . In addition , translational repression could be a prerequisite for mRNA localization , if the act of translation interferes with that process . With repression comes the need for translational activation , either by disrupting repression or by a separate mechanism . The Drosophila oskar ( osk ) mRNA is subject to an extensive program of regulation and provides a model for elucidation of the mechanisms of repression , localization and activation , and how these events are coordinated [4] . The Osk protein , whose distribution during oogenesis is restricted to the extreme posterior region of later stage oocytes , acts as a posterior determinant responsible for posterior patterning of the embryos and formation of the embryonic germline ( reviewed in [5] ) . In the absence of Osk , the abdominal segments are missing and no germ cells form [6] . Conversely , mis- or overexpression of Osk , such that the protein is not tightly restricted to the posterior of the oocyte , leads to a reorganization of the embryonic body plan and ectopic formation of germ cells [7 , 8] . Thus , proper deployment of Osk is critical . The osk mRNA is present from the earliest stages of oogenesis , transcribed in nurse cells and rapidly transported into the oocyte . As oogenesis proceeds , osk mRNA persists in the oocyte , and is transiently enriched near the anterior at stage 8 before assuming its final position at the extreme posterior of the oocyte [9 , 10] . It is only at this point that substantial levels of Osk protein accumulate [11–13] . The absence of Osk protein at earlier stages , and from unlocalized osk mRNA , is due to translational repression . A key player in repression is Bruno ( Bru; encoded by the aret gene ) , which binds to multiple sites in the osk 3′ UTR . Mutation of these sites leads to excess Osk activity and precocious Osk protein [11] . Translational repression by Bru has been recapitulated using in vitro assay systems from Drosophila tissues [14 , 15] . Two models have been proposed for the mechanism of repression by Bru . In one model , the events that occur at the mRNA 5′ cap , a structure bound by eIF4E , are targeted . During cap-dependent initiation , eIF4E binds to eIF4G , resulting eventually in assembly of a functioning ribosome . The Cup protein also binds to eIF4E , using the same site bound by eIF4G . Another interaction of Cup is with Bru , leading to the model: Cup is recruited to an osk mRNA by Bru , binds to the eIF4E bound to the 5′ cap of that mRNA , and thus blocks the required eIF4E/eIF4G interaction [16] . In the other model for Bru-dependent repression , Bru promotes osk mRNA oligomerization and formation of large silencing particles , which are proposed to be inaccessible to ribosomes [17] . In this model Cup is also involved , but not the ability of Cup to bind eIF4E . Oligomerization of osk mRNA is also promoted by direct RNA dimerization and formation of large RNP complexes by Polypyrimidine Tract Binding protein ( PTB ) [18 , 19] . After the osk mRNA has been localized to the posterior pole of the oocyte at stage 9 , translational repression must be overcome [11–13] . This could be achieved in different ways , with or without the need for specific regulatory elements . At one extreme , and requiring no activation elements , is inactivation or degradation of the repressors . At the other extreme , the repressors would remain in place and functional , but independent forms of activation ( mediated by further regulatory elements ) would overcome repression by exerting more powerful positive influences on translation . Just as for repression , there appear to be multiple contributions to activation: a variety of proteins and regulatory sequences have been implicated , with no unifying model for how their actions collectively lead to activation [11–13 , 20–26] . Here we characterize the interactions of Bru , using in vitro assays to map protein-binding domains and sites of phosphorylation , and designing mutations that affect the interactions . A simplified in vivo system , focusing on Bru-mediated regulation in the absence of much of the complex regulation of osk mRNA , reveals one form of translational activation and provides concrete insights into its mechanism .
A GST pull-down assay was used to test for the ability of Bru to dimerize . Full-length Bru was expressed as a fusion to GST , and incubated with Bru bearing a His6 tag . Following affinity purification of GST::Bru with glutathione sepharose beads , copurification of His6::Bru was tested by Western blot analysis using the anti-His6 antibody . By this assay , Bru did dimerize while His6::Bru did not bind to GST alone ( Fig . 1A ) . To map the domain of Bru responsible for dimerization , deletion derivatives of Bru ( Fig . 1B ) were tested in the GST::Bru pull-down assay . The three RRMs all function in RNA binding [27] , so we focused on the other domains . Deletion of the Bru amino-terminal domain ( aa1–146 , Δ1–146 ) eliminated binding to GST::Bru , while deletion of most of the linker domain between RRMs 2 and 3 ( aa334–416 , Δ334–416 ) had no effect . The amino-terminal domain was not only required for dimerization with Bru , but was also sufficient: the isolated domain bound GST::Bru ( Fig . 1A ) . The ability of Bru to dimerize provides an explanation for how Bru oligomerizes osk mRNA: a molecule of Bru bound to one osk mRNA could dimerize with a second molecule of Bru bound to a different osk mRNA . With the many Bru binding sites in the osk mRNA 3′ UTR [11 , 26] , formation of large , highly interconnected protein-RNA assemblies is possible . This suggests that the proposed use of osk mRNA oligomerization as a mechanism of translational repression [17] would rely on Bru dimerization . A second Bru interaction , with Cup , provides the basis for the other proposed mechanism of translational repression , in which Bru recruits Cup to the osk mRNA [16] . A GST::Cup pull-down assay was used to monitor interaction with Bru . As expected , full-length Bru ( Bru+ ) bound GST::Cup . Deletion of either aa1–146 or aa334–416 of Bru had no dramatic effect on binding , but deletion of both domains eliminated binding . Just as for Bru dimerization , the isolated amino-terminal domain was sufficient for binding to GST::Cup ( Fig . 1A , B ) . Our evidence that the amino-terminal domain of Bru is essential for dimerization and contributes to Cup binding suggested that this domain is likely to play an important role in repression . To test this prediction we established an in vivo tethering assay , in which translation of a GFP-MS2 reporter mRNA was monitored . The 3′ UTR of the GFP-MS2 mRNA includes multiple copies of the bacteriophage MS2 stem loop , a binding site for the MS2 coat protein ( MCP [28] ) . Forms of Bru were expressed as fusions to MCP to direct binding to the reporter mRNA . Both the reporter mRNA and tethered Bru proteins were expressed in Drosophila ovaries using the UAS/GAL4 system . The GFP-MS2 reporter by itself was expressed throughout the germline cells of the egg chamber ( Fig . 2A ) . Coexpression of tethered Bru dramatically reduced the GFP level ( 10 fold; Fig . 2B , F ) . The strong reduction in GFP expression from tethered Bru was accompanied by a reduction in GFP-MS2 mRNA level ( 1 . 7 fold; Fig . 2G ) . Therefore , in this assay Bru both repressed translation and reduced mRNA stability , although repression was the stronger effect ( compare Fig . 2F and 2G ) . Testing Bru mutants in the tethering assay revealed that deletion of the amino-terminal domain led to a substantial increase in GFP , although not to the level in the absence of repression ( Fig . 2C , F ) . Deletion of the linker domain had no strong effect ( Fig . 2D , F ) . The combination of deleting both the amino-terminal domain and the linker domain was no stronger than deleting the amino-terminal domain alone ( Fig . 2F , H ) , but the range of fluorescence intensities was greater ( error bars in Fig . 2F ) and some egg chambers had weaker repression ( Fig . 2E ) . Both mutants with enhanced GFP also had slightly elevated GFP-MS2 mRNA ( Fig . 2G ) , although the changes were strongest at the protein level ( Fig . 2F , H ) . The differing activities of the mutant proteins were not due to differences in their expression ( S1A Fig . ) . Thus , the mutants impaired both activities of Bru monitored in this assay: translational repression and destabilization of mRNA . To allow translation of the osk mRNA once it has been localized to the posterior pole of the oocyte , there must be a release from repression . How this is accomplished is not known , but one possibility is that Bru is post-translationally modified to change its activity . To ask if Bru is phosphorylated , the conventional approach of testing for phosphatase-dependent changes in electrophoretic mobility of the protein was used . In untreated ovary extract , Bru appeared by Western blot analysis as a major band , with a faint lower-mobility band . Treatment with phosphatase eliminated the weak band . By contrast , addition of phosphatase inhibitors enhanced the minor band , consistent with the interpretation that this small fraction of Bru is phosphorylated ( Fig . 3A left ) . To make a more compelling case for phosphorylation , we also tested the MCP::HA3::Bru 1–146 protein from above , which at 32 kDa is substantially smaller than Bru ( 64 kDa ) and thus might display a larger change in mobility from phosphorylation . This was indeed the case , and the difference between the major Bru band and the slower migrating fraction was more dramatic ( Fig . 3A right ) . Bru phosphorylation was also analyzed by phosphate-affinity SDS-PAGE with the acrylamide-pendant Phos-tag , which separates different phosphoprotein isoforms [29] . Using this approach , multiple , different phosphorylated species could be detected ( S2A Fig . ) . The NetPhosK 1 . 0 and KinasePhos prediction programs were used to identify candidate phosphorylation sites in Bru . Both report multiple sites for many different kinases , although none of the candidate sites had scores suggesting a high probability of phosphorylation ( S1 Table ) . Nevertheless Bru is phosphorylated , and so even the sites with modest scores remain as candidates . Several amino acids are predicted to be targets for Protein Kinase A ( PKA ) , an interesting option since alteration of PKA activity affects osk expression pattern and embryonic body patterning [30] . To evaluate PKA , in vitro phosphorylation assays were performed using the PKA catalytic subunit and full-length Bru . Bru was strongly phosphorylated , while BSA ( a negative control ) was not ( S3A Fig . ) . By contrast , neither Casein Kinase I ( CK1 ) nor Calmodulin-dependent Protein Kinase II ( CaMKII ) , which share a part of the recognition motif of PKA [31–33] , supported detectable phosphorylation of full-length Bru ( S3B Fig . ) . To map the sites of phosphorylation , Bru deletion proteins from above ( Fig . 1B ) were used as substrates for PKA . Deletion of the amino-terminal domain greatly reduced phosphorylation , and the isolated domain was itself phosphorylated . Deletion of the linker region did not reduce phosphorylation , nor did it enhance the effect of deleting the amino-terminal domain ( Fig . 3B ) . Thus , PKA phosphorylates the amino-terminal domain of Bru . To identify sites of phosphorylation , we performed a tandem mass spectrometry ( MS/MS ) analysis of Bru phosphorylated in vitro . Although four candidate sites are predicted in the amino-terminal domain , only phosphoserine at position 7 ( S7 ) was identified with high confidence and no ambiguity ( Fig . 3D ) . S88 could not be tested since aa36–119 was undetectable due to a low coverage of MS/MS ( see Materials and Methods ) , and a majority of peptides containing either S4 or T135 was detected as unphosphorylated . Nevertheless , there is still a possibility of weak phosphorylation below the limit of detection at either S4 or T135 . Mutation of S7 to alanine ( S7A ) substantially reduced phosphorylation by PKA . Because the S7A mutant retained a low level of phosphorylation , we also tested mutations in the other predicted sites , either alone or in combinations . Of the mutants tested , S4A/S7A/T135A was most resistant to phosphorylation ( Fig . 3C ) . The mutants , except for S4A , were also tested in the context of full-length Bru , and similar results were obtained ( S2B Fig . ) . Since the amino-terminal domain of Bru is essential for repression , the potential phosphorylation of one or more residues within this region might inhibit or enhance repression . We therefore asked if phosphomimetic mutations would interfere with Bru protein interactions mediated by the amino-terminal region and implicated in repression . Pull-down assays were performed with GST::Bru and GST::Cup , using Bru mutants with phosphosilent alanine ( A ) or phosphomimetic glutamate ( E ) substitutions at one or more of the three residues that affect phosphorylation by PKA: S4 , S7 , and T135 . None of the phosphosilent mutants showed reduced binding to GST::Bru ( Fig . 4A ) or GST::Cup ( Fig . 4B ) , demonstrating that mutation of the affected residues did not inherently disrupt the protein interactions . By contrast , the phosphomimetic mutants altered interactions . S7E significantly reduced dimerization , and the S4E/S7E double mutant retained only a very low level of dimerization . Including the T135E mutation did not obviously further reduce dimerization by S7E ( in S7E/T135E ) , but did reduce dimerization in the triple mutant ( S4E/S7E/T135E ) to below the level of detection ( Fig . 4A and 4C ) . Bru binding to Cup was less sensitive to the phosphomimetic mutations . S7E did not reduce binding , and the double mutation combinations caused only modest defects . Even the S4E/S7E/T135E triple mutant retained detectable binding ( Fig . 4B and 4C ) . Because the S4E/S7E/T135E triple mutant eliminated detectable Bru dimerization , repression dependent on this interaction is expected to be disrupted . The prediction is less clear for Cup-dependent repression , given the residual Bru/Cup interaction . Our analysis of PKA phosphorylation of Bru in vitro suggests that PKA may also modify the protein in vivo . Testing this prediction has proven to be challenging , with no conclusive answer . This is due in part to failure in making an antibody against the phospho-S7 peptide . Nevertheless , the Bru mutants defective in dimerization provide useful tools to test the importance of this interaction for Bru function . As one such test , we made use of the tethering assay to monitor translational repression . Notably , none of the mutants tested , including the S4E/S7E/T135E triple mutant that prevented Bru dimerization and impaired the Bru/Cup interaction , showed any substantial decrease in repression ( Fig . 5 ) . Likewise , there were no substantial changes in reporter mRNA levels ( Fig . 5H ) . MCP is known to dimerize [34] . This property could substitute for Bru dimerization , and thus neutralize the effect of the dimerization-defective mutations . To address this possibility , we tested another tethering system which relies on binding of a bacteriophage lambda N peptide ( which does not dimerize ) to the boxB stem-loop RNA [35] . In this case , the reporter mRNA was GFP with 6 copies of the boxB sequence in the 3′ UTR ( GFP-boxB ) , and the Bru proteins were expressed as fusions to the λN peptide . Just as with the other system , tethered Bru repressed translation of the reporter mRNA ( compare S4A and S4B Fig . ) . Notably , the S4E/S7E/T135E triple mutant did not affect repression ( S4C , D Fig . ) , confirming that dimerization is not required for translational repression . Cooperative binding is a common strategy to enhance affinity for a substrate . Dimerization of Bru might facilitate cooperative binding to RNA , and if so , the mutations inhibiting dimerization are expected to impair RNA-binding activity of Bru . A UV-crosslinking assay was used to test Bru proteins for their ability to bind the osk 3′ UTR AB region RNA , which has multiple Bru binding sites [11] . The two mutants most strongly defective in dimerization , S4E/S7E and S4E/S7E/T135E , showed compromised RNA binding ( S5A Fig . ) . After quantitation , normalization for protein levels and statistical analysis of three independent experiments , both mutants were considered to have a significant change in their RNA-binding ability when compared to their ala-mutant ( dimerization-competent ) counterparts ( S5B Fig . ) . Although the reduction in RNA binding is modest , it is possible that this change could contribute to a reduction in Bru activity in vivo by weakening the interaction with target mRNAs , such as osk . The tethering assays were useful for monitoring , in isolation from much of the complex program of osk regulation , translational repression by Bru . However , these assays have limitations . First , any region-specific change in repressive activity might be missed , as the reporter protein is diffusible . Second , since the RNA-binding activity of Bru was not required in the assay , a possible disruption of repression by reduced RNA-binding affinity would not be detected . As an alternate approach to address these limitations we wanted to maintain the use of a simplified system which focuses on repression by Bru , but allows detection of regional differences in translation under conditions where Bru binds directly to the mRNA . Our approach has two components . The first , described in the following paragraphs , is the development of an appropriate reporter mRNA . The second , described in the next section , is manipulation of the aret gene , which encodes Bru , to introduce the mutations that disrupt Bru dimerization . A requirement for the reporter mRNA is that the encoded protein be anchored , such that its site of synthesis is revealed . The Osk protein is itself anchored to the oocyte cortex , with the amino-terminal domain of the Long Osk isoform required for this function [36] . To determine if this domain would anchor GFP , the UAS-osk1–534::GFP transgene ( which includes nucleotides 1–534 of the osk mRNA , and thus the first 173 amino acids of Long Osk ) was tested . While GFP alone appears diffuse throughout the germline cells ( Fig . 6A ) , Osk::GFP is highly enriched at cortical regions of the oocyte and at cell boundaries in the nurse cells ( Fig . 6B ) . Addition of the osk 3′ UTR to this transgene ( in UAS-osk1–534::GFP-osk3′UTR ) restricts expression to the posterior pole of the oocyte , where the mRNA is localized . Notably , the protein from osk1–534::GFP-osk3′UTR mRNA remains concentrated in a narrow crescent at the posterior pole , and does not diffuse extensively along the oocyte cortex ( Fig . 6C ) . Therefore , the amino-terminal domain of Osk provides a useful anchoring domain to reveal the site of translation . The reporter mRNA also needs to be subject to Bru-dependent repression , and with Bru binding directly to the mRNA . Just as in the tethering assay , we wanted to avoid the complexity of the full program of osk regulation . This can be achieved through the use of the osk 3′ UTR AB region , which contains multiple Bru binding sites [11 , 26] . Addition of this region to a GFP reporter confers efficient repression in the ovary [26] , and as expected repression was dependent on the Bru binding sites ( below ) . Combining these two required features for the reporter mRNA , the UAS-osk1–534::GFP-AB transgene encodes an anchored GFP protein whose translation will be repressed by Bru . Characterization of the osk1–534::GFP-AB reporter mRNA confirmed the expected translational repression , but also revealed a novel pattern of region-specific translational activation . During previtellogenic stages of oogenesis the osk1–534::GFP-AB mRNA was strongly repressed throughout the egg chamber , with no detectable GFP signal ( Fig . 6D ) . Translation of osk1–534::GFP-AB mRNA continued to be strongly repressed in nurse cells at later stages of oogenesis . However , starting at stage 7/8 a faint Osk::GFP signal was detected in the oocyte ( Fig . 6E ) . The intensity of the signal increased at later stages and , strikingly , Osk::GFP accumulated in a weak gradient extending from the posterior pole ( Fig . 6F , G ) . This mRNA lacks localization signals required for posterior localization of osk mRNA [37 , 38] , and , not surprisingly , we found no detectable posterior localization of the reporter mRNA ( Fig . 6H , I ) . Therefore , translation of the reporter was activated independent of association with the mRNA localization machinery . There are two options to explain the posterior gradient of the anchored Osk::GFP fusion protein . One is that activation of translation occurred in a posterior gradient essentially the same as that displayed by Osk::GFP . Alternatively , activation of translation could have occurred just where pole plasm assembles at the posterior pole of the oocyte ( from the fraction of the mRNA located in that region by chance ) , followed by diffusion of Osk:GFP from that site to create the extended gradient we observe . However , as shown above , when we selectively expressed the same Osk::GFP fusion protein just at the posterior pole from a localized mRNA , it remained there and did not form a gradient ( Fig . 6C ) . Thus , the gradient of Osk::GFP produced by the unlocalized transgene mRNA must have formed by translational activation in a broad and graded domain: highest at the posterior pole , extending with diminishing strength along almost the entire length of the oocyte , and virtually undetectable at the anterior margin of the oocyte . These results reveal a spatially-restricted form of translational activation , which we call region-specific activation . This form of activation could inhibit Bru-mediated repression . Alternatively , activation could be independent , and simply superimposed on repression . To distinguish between these options we tested a version of the transgene , UAS-osk1–534::GFP-AB all- , with the Bru binding sites mutated . The mutations disrupted repression , allowing translation of the Osk::GFP protein , which appeared in nurse cells and oocytes ( Fig . 6J ) . If activation inhibits repression by Bru , in the absence of repression there would be no detectable activation and the Osk::GFP protein should be present at similar levels along the anteroposterior axis of the oocyte . By contrast , if activation is independent of Bru , it should still occur when the Bru binding sites are mutated , leading to a higher level of Osk::GFP in the more posterior portion of the oocyte . We detected no posterior enhancement of Osk::GFP when repression was disrupted ( Fig . 6J ) . Thus , region-specific activation of translation must act by inhibiting the repressive function of Bru . For the assay system showing region-specific activation of translation , Bru is provided by the endogenous gene . To test dimerization-defective Bru in this assay , we used homologous recombination ( HR ) [39] to exchange exons that encode the amino-terminal region of Bru ( Fig . 7A ) . The replacements were wild type ( aret+ ) , S4A/S7A/T135A ( aret3ala ) , or S4E/S7E/T135E ( aret3glu ) . Loss of aret function leads to an early arrest of oogenesis , with no oocyte specified [40] . Females in which the HR replacement alleles provided the only copy of aret all displayed normal progression through oogenesis , indicating that the mutations did not substantially alter aret function . Protein levels for the different alleles were similar ( S6 Fig . ) . Each of the proteins showed the normal distribution of Bru ( S6 Fig . ) , including the granular cytoplasmic appearance due to association with nuage and sponge bodies [41] . Embryos obtained from these females were tested for patterning defects . Although misregulation of osk expression—either too little or too much Osk activity—causes striking patterning defects [6–8] , no such defects were found for any of the aret HR alleles . The only phenotype detected was for the aret3glu mutant , and was unrelated to any known osk defect: an increase in the proportion of embryos that fail to develop ( Fig . 8J ) . To ask if preventing dimerization of Bru influenced the regional activation of translation , expression of the osk1–534::GFP-AB mRNA was monitored in the engineered aret-mutant ovaries ( Fig . 7B-G ) . Repression in nurse cells was similar for all genotypes , consistent with the absence of any repression defect in the tethering assays ( Fig . 7B-D ) . Both aret3ala and aret3glu mutants reduced the degree of posterior activation , with the strongest effect from the Bru dimerization- defective aret3glu mutant: the fraction of oocytes with a strong posterior gradient was about half that of aret+ , and over a third of the oocytes had no detectable posterior gradient ( Fig . 7H ) . Thus , the aret3glu mutant substantially disrupted regional activation . Because this mutant , in effect , enhanced repression , these results are consistent with the failure to detect any loss of repression in the tethering assays . The aret3ala and aret3glu mutants were also tested for an effect on Osk expression . Because defects in activation of osk mRNA translation can be most pronounced late in oogenesis [26] , this analysis included the use of a genomic osk::GFP transgene for detection of Osk::GFP after deposition of the vitelline membrane ( which is impermeable to antibodies ) , as well an epitope tagged osk transgene ( oskHA ) for detection at earlier stages . There was no defect in repression in stage 8 egg chambers ( Fig . 8A-C ) , as expected from the tethering assay results . Likewise , we did not detect any difference in Osk expression pattern among the three replacement lines in stage 9 ( Fig . 8D-F ) or later ( Fig . 8G-I ) egg chambers , consistent with the absence of patterning defects ( Fig . 8J ) and suggesting redundancy in mechanisms of translational activation .
To better understand translational regulation of osk mRNA by Bru , we characterized Bru protein interactions and developed in vivo assays to monitor Bru activity . Bru binds Cup , an interaction that is essential for at least one model of repression [16] . Here we have shown that Bru dimerizes , an interaction very likely to contribute to a second model of repression . In that model , the oligomerization of osk mRNA by Bru creates silencing particles , in which osk transcripts are made inaccessible to the translation machinery . The evidence for this model comes from an in vitro assay using purified Bru , which oligomerizes RNAs containing many Bru binding sites [17] . Because no other macromolecules are present in the assay , dimerization of Bru appears to be the only plausible option for oligomerization of the RNAs . The alternative is for the different Bru RNA-binding domains ( there are three RRMs [27 , 42 , 43] ) to bridge different RNAs , a kinetically unfavorable option: after the initial binding of one Bru domain to an RNA ( a second order reaction ) , the subsequent binding of another Bru domain would strongly favor association with the same RNA ( first order ) as opposed to another RNA ( second order ) . Furthermore , only one of the RRMs provides a high degree of binding specificity [43] , and so specificity of oligomerization would be low ( and inconsistent with the observed binding specificity of the intact protein [11 , 43] ) if different domains bound to different RNAs . We found that the amino-terminal domain of Bru is essential for dimerization . The same domain was sufficient for Cup binding , and deletion of both the amino-terminal and linker domains was required to eliminate Cup binding . These Bru/Cup interaction data using a GST pull-down assay contrast with those from the yeast two-hybrid assay , where the linker domain was shown to be both necessary and sufficient for Cup interaction , while the amino-terminal domain was neither necessary nor sufficient [16] . We do not know why there are differences . In both assays Bru is present as a protein fusion , and it may be that the different additional protein domains—GST in the one assay , and a transcriptional activation domain in the other assay—differentially constrain Bru binding . To explore the role of dimerization on Bru activity in vivo , two types of simplified assays were used , both of which monitor the activity of Bru independent of much of the rest of the complex regulation of osk mRNA . In one type of assay , Bru was tethered to a reporter mRNA whose repression was monitored . For this assay , a variety of Bru mutants were used , both deletions and point mutations . In this assay the amino terminus of Bru was shown to be essential for repression , while the point mutants that prevent Bru dimerization and reduce Bru binding to Cup had no effect on repression . Therefore , Bru dimerization was not essential for repression . Notably , the tethering assay also revealed that Bru binding reduced stability of the reporter mRNA , in addition to translational repression . Regulation by Cup is known to alter mRNA stability in cultured cells [44] , and our results are consistent with that property . The second type of simplified in vivo assay made use of a reporter mRNA designed to display the pattern of translation within cells , not simply the total level of the encoded protein . Strikingly , Bru-dependent repression of the reporter mRNA was alleviated within the oocyte in a graded fashion , with peak translation at the posterior pole of the oocyte and extending with decreasing efficiency most of the distance to the anterior of the oocyte . Thus , this reporter revealed one form of translational activation—region-specific activation—for which concrete conclusions about mechanism can be drawn: activation is regional , but not narrowly restricted to the site at which osk mRNA is localized; activation is not mechanistically coupled to mRNA localization; and activation involves the inhibition of Bru repression . To test the requirement for Bru dimerization in region-specific activation , homologous recombination was used to introduce point mutations into the aret locus . The new alleles can be used with no concerns about appropriateness of expression or artifacts from use of fusion proteins . Notably , the mutations preventing dimerization interfered with region-specific activation , suggesting that dimerization acts to disrupt repression , opposite of what might be expected for the silencing particles model of repression . Region-specific activation disrupts or alters Bru activity , which could involve a direct effect on Bru , or be mediated by a regulatory element which could indirectly affect Bru . A direct effect on Bru could entail , for example , phosphorylation . We have shown that PKA phosphorylated Bru in vitro , and PKA plays a positive role in Osk expression [30] . PKA is involved in a posterior signal transduction event in the oocyte [45] , and could thus act in the region where activation occurs ( although there has been no demonstration of the site of PKA activity ) . However , phosphomimetic mutations of the sites at which PKA phosphorylates Bru in vitro did not promote activation , and instead diminished activation . Nevertheless , phosphorylation or another post-translational modification remains as a likely option for region-specific activation , as observed for other repressors of localized mRNAs in which this modification reduces RNA binding affinity [46–48] . The mutations mimicking phosphorylation of Bru also reduced RNA binding affinity , but by a modest degree and the significance of this effect is uncertain . It is noteworthy that region-specific activation was not tightly confined to the extreme posterior of the oocyte . This very strongly indicates that the factors involved are not restricted to the germ plasm that assembles in a crescent at the posterior pole . If region-specific activation relies on a regulatory element , it must lie within the osk sequences of the osk1–534::GFP-AB mRNA , either the 5′ osk sequences or the 3′ UTR AB region . There has been no indication of a role for the AB region in translational activation . The 5′ osk sequences have been reported to include a translation activation element which mediates posterior-specific inhibition of Bru-mediated repression [21] . However , recent work shows that the 5′ element is not essential , and that the evidence for it functioning only at the posterior of the oocyte is based on an incorrect assumption ( M Kanke and PMM , submitted ) . Multiple factors and cis-acting elements have been implicated in translational activation of osk mRNA [11–13 , 20–26] , and it is clear that the region-specific mechanism identified here is not the only form of activation . Indeed , the mutant aret alleles which disrupt regional activation of the osk1–534::GFP reporter mRNA have no detectable effect on regulation of endogenous osk mRNA . This is most likely due to redundancy , with the loss of one form of activation being obscured by persistence of other types of activation . Redundancy in activation mechanisms is not unexpected: there are multiple contributions to repression of osk mRNA translation [11 , 16 , 19 , 49–51] , and the same might be expected for activation . Furthermore , different mechanisms could be required to overcome different forms of repression . Redundancy presents a substantial challenge , in part because it makes identification of factors and regulatory elements difficult . Each different type of activation may need to be characterized in isolation , before an understanding of the overall process can be obtained . Another major challenge in understanding translational activation concerns the question of whether an activation mechanism serves as a prerequisite for translation , or is used in a spatially restricted manner . Addressing that question with in vitro systems would be extremely difficult , as spatial differences within the oocyte are lost in the process of preparing an extract . Our strategy of monitoring the pattern of translation in vivo provides a solution , and may be useful for analysis of other forms of activation .
w1118 flies were used as the wild type . Transgenic fly stocks were established by standard methods . Expression of the UAS transgenes was driven by the nosGAL4VP16 [52] or matα4-GAL-VP16 driver [53] , as indicated . The P[UAS-GFP-MS218] and P[UAS-GFP-boxB6] transgenes are similar to UASp-GFP-312 [54] , but with 18 copies of the MS2 binding sites or 6 copies of the boxB binding site instead of mi-312 targets . P[UAS-MCP::HA3::bru2–3-] shares the same synthetic 5′ UTR as UASp-GFP-312 , fused to MCP , 3 copies of the HA epitope , and bru cDNA bearing point mutations in RRM2 ( K239A , F241A ) and RRM3 ( N521A , F523A ) [27] . Mutations in the bru sequences were introduced using restriction sites or PCR . P[oskT140::HA] and P[oskT140::GFP] are based on a genomic DNA fragment which provides complete osk function and regulation [10] , and have either 3 copies of the HA epitope tag or mGFP6 [55] inserted after T140 . To make P[UAS-osk1–534:GFP] ( which encodes a fusion protein with the first 173 amino acids of Long Osk ) , P[UAS-GFP] [26] was modified by addition of osk sequences that include 20 nt of the 5′ flanking region and the first 534 nt of the mRNA ( the 5′ UTR and the coding region for amino acids 1–173 ) . Derivatives with the osk 3′ UTR AB region [11] , a version of the AB region with Bru binding sites mutated ( all- ) [26] , and with the entire osk 3′ UTR were made as described [26] . For homologous recombination of aret , a 2 . 1kb region ( 12 , 270 , 445–12 , 272 , 531; R5 . 48 ) including the first two protein-coding exons that encode the amino terminus of female Bru ( up to aa193 ) was targeted . Details are provided in the Supplemental Materials . GST::Bru was constructed by subcloning full-length bru cDNA into pGEX-2TK ( GE Healthcare ) . GST::Cup577–947 was a gift from Robin Wharton [56] . Bru proteins for binding assays were tagged at the amino terminus with six histidine residues provided by the pET-15b ( Novagen ) vector and used for purification , and made use of the same mutations as in the UAS-MCP::HA::bru transgenes . GST fusion proteins were expressed in CodonPlus ( Stratagene ) E . coli , after induction with IPTG . Pelleted cells were resuspended in ice-cold GST lysis buffer ( 50mM Tris-Cl pH8 . 0 , 150mM NaCl , 1mM EDTA , 1mM DTT , 2mg/ml lysozyme , and 0 . 1% IGEPAL-CA-630 ) supplemented with protease inhibitors ( Complete , Mini , EDTA-free protease inhibitor cocktail tablet ( Roche ) ) , and extracts were prepared as previously described [27] . For His6 tagged proteins , extracts were prepared with His lysis buffer ( 50mM NaH2PO4H2O pH8 . 0 , 300mM NaCl , 20mM imidazole , 0 . 01% β-Mercaptoethanol , and 2mg/ml lysozyme ) supplemented with protease inhibitors . 250μl Ni-NTA Agarose ( Quiagen ) in 50% slurry was added per 1ml lysate , and the reaction was incubated for 1–2 hr at 4°C on a rotator . The lysate-Ni-NTA mixture was then loaded into a disposable column equilibrated with the His lysis buffer to remove flow-through and washed with increasing concentrations of imidazole in His lysis buffer ( up to 40mM ) . The proteins were eluted with 250mM imidazole in His lysis buffer . Glycerol was added to the supernatant to 20% final volume and extracts were stored at -70°C . Equivalent amounts of GST::Bru , GST::Cup or GST was first immobilized on Glutathione Sepharose 4B ( GE Healthcare ) prepared in 50% slurry in binding buffer ( 50mM Tris-Cl pH7 . 5 , 150mM NaCl , 10% glycerol , 1mM DTT , and 0 . 1% IGEPAL-CA-630 ) supplemented with protease inhibitors , by incubating with extract overnight at 4°C on a rotator . The beads were spun down , washed and resuspended in binding buffer to make 50% slurry . Then 20μl of this slurry was incubated with ~100ng of each of the N-terminally His6-tagged Bru proteins in 80μl reaction containing binding buffer for 2–3 hr at room temperature with rotation . The beads were spun down , washed with binding buffer , and boiled in 5μl 2X SDS loading buffer to elute the bound proteins . Eluates were separated by SDS-PAGE and analyzed by Western blotting . The mouse anti-His antibody ( ABGENT ) diluted at 1:2000 and alkaline phosphatase-conjugated goat anti-mouse antibody ( Applied Biosystems ) diluted at 1:5000 were used to detect Bru proteins . Phosphorylation reactions ( 20μl ) contained ~250ng of purified substrate , 1–2 unit of recombinant PKA catalytic subunit , CK1 or CaMKII ( all from NEB ) , and 0 . 2mM [γ-32P]ATP ( adjusted to 250μCi/μmol , Perkin Elmer ) in kinase buffer ( buffer for PKA: 50mM Tris-Cl pH7 . 5 , 100mM KCl , 5mM MgCl2 , and 2 . 4mM DTT; buffers for CK1 or CaMKII provided by NEB ) supplemented with protease inhibitors . Reactions were incubated at 30°C for 30 min and terminated by addition of 3X SDS loading buffer . Proteins were separated by SDS-PAGE , and gels were dried ( Bio-Rad ) and exposed to a Phosphor Screen ( Molecular Dynamics ) for 12 hr . The screen was then analyzed with a Typhoon laser scanner ( GE Healthcare ) . RNase protection assay was performed as previously described [26] , except that quantitation was done using ImageJ . Ovaries from young females , fed on yeast for 3–4 days , were dissected and extract was prepared as previously described [11] in ice-cold lysis buffer ( 50mM Hepes pH7 . 9 , 150mM KCl , and 1% IGEPAL-CA-630 ) supplemented with protease inhibitors . Reactions ( 20μl ) contained 10–15μg of ovary extract in phosphatase buffer ( 50mM Hepes pH7 . 9 , 100mM NaCl , 10mM MgCl2 , and 1mM DTT ) , and where indicated , one or more of the following components were added: 1–2 units of alkaline phosphatase ( calf intestinal , NEB ) , 1 . 6M beta-glycero phosphate , and 40mM sodium vanadate . Reactions were incubated at 30°C for 1 hr and terminated by addition of 3X SDS loading buffer . Proteins were separated by SDS-PAGE and analyzed by Western blotting . For phosphate-affinity SDS-PAGE using acrylamide-pendant Phos-tag ( WAKO ) , 50μM Phos-tag and 200μM MnCl2 were added to both stacking and separating gels in solution . Ovaries were processed for imaging as described [26] , with an alternate protocol for detection of Bru [57] . Primary antibodies were used at the following dilutions: mouse anti-HA ( Covance ) , 1:1000; rat anti-Bru , 1:500 . Secondary antibodies , used at 1:800 , were Alexa Fluor 488 goat anti-mouse ( Invitrogen ) and Cy5 goat anti-rat . DNA was stained with TO-PRO-3 Iodide ( 642/661 , Invitrogen ) diluted 1:1000 . Samples were mounted in Vectashield ( Vector Labs ) and analyzed with a Leica TCS-SP laser scanning confocal microscope . Quantitation of GFP levels was done using the Macnification software from images obtained using a single plane of focus . The average green fluorescence was sampled from four different regions in the nurse cell cytoplasm of each of 10 to 11 , stage 5 or 6 egg chambers . Antibodies for western blotting were mouse anti-Bru ( AN , unpublished ) ( 1:8000 ) , mouse anti-HA antibody ( Covance ) ( 1:1000 ) , mouse anti-αTubulin ( Sigma ) ( 1:1000 ) and rat anti-Cup219 [58] ( 1:2000 ) . Alkaline phosphatase-conjugated secondary antibodies were used at 1:5000 ( anti-mouse; Applied Biosystems ) or 1:10000 ( anti-rat; Sigma ) . The osk 3’ UTR AB probe was transcribed using MAXIscript Kit ( Ambion ) and uniformly labeled with [α-32P]UTP ( 800Ci/mmol , Perkin Elmer ) . UV cross-linking assay was performed as described [11] , except that 10X binding buffer consisted of 60mM Hepes pH7 . 9 , 300mM KCl and 20mM MgCl2 , and was supplemented with protease inhibitors ( Roche ) . ~500ng of purified recombinant Bru proteins was used . After electrophoresis of cross-linked adducts , gels were dried ( Bio-Rad ) and exposed to a Phosphor Screen ( Molecular Dynamics ) for 12 hr . The screen was then analyzed with a Typhoon laser scanner ( GE Healthcare ) .
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Proteins are often enriched to specific regions within cells via localization of mRNAs . This phenomenon serves a variety of roles , both bringing together factors involved in particular cellular processes to enhance their efficiency , and in restricting proteins that could do harm if deployed at inappropriate positions . In the latter situation , translational repression prevents expression before mRNA localization , and there must be activation mechanisms to inhibit or override repression . How the processes of mRNA localization and translation are coordinated is not well understood , in part because cellular extracts prepared to study mechanisms in vitro do not retain the spatial information present in the intact cell . We developed an in vivo assay to monitor the pattern of translation in the Drosophila oocyte , where several patterning determinants must be localized to specific regions . Using this assay , we showed that repression of translation by the Bruno protein is inhibited , and we could visualize when and where this occurs during oogenesis . Regional activation occurs not only at the site of mRNA localization , but more broadly in a graded fashion , and it does not require an activation element in the mRNA . We also show that Bruno dimerizes , and that dimerization is important for translational activation .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
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Region-Specific Activation of oskar mRNA Translation by Inhibition of Bruno-Mediated Repression
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We use >250 , 000 cross-over events identified in >10 , 000 bovine sperm cells to perform an extensive characterization of meiotic recombination in male cattle . We map Quantitative Trait Loci ( QTL ) influencing genome-wide recombination rate , genome-wide hotspot usage , and locus-specific recombination rate . We fine-map three QTL and present strong evidence that genetic variants in REC8 and RNF212 influence genome-wide recombination rate , while genetic variants in PRDM9 influence genome-wide hotspot usage .
Reciprocal recombination between homologues fulfills an essential mechanistic role during meiosis in most organisms [1] , [2] . It is required for proper bivalent alignment on the metaphase I plate preceding disjunction and segregation at anaphase I . Correct segregation of the full chromosome complement demands tight , sex-specific control of the number of cross-overs ( CO ) per arm , as well as of their position relative to chromosomal landmarks ( centromeres and telomeres ) and other CO ( in the case of multichiasmatic meioses ) [3] , [4] . Failures in this process underlie aneuploidies affecting as many as 5% of human oocytes [5] . At the population level , recombination affects the rate of creation and loss of haplotypes with cis-configured favorable alleles , placing second order selection pressure on modifiers of global and/or local recombination including inversions [3] . Components of the recombination apparatus are well described in yeast and C . elegans , but remain largely undefined in most other organisms including mammals [4] , [6] . One strategy to identify such components is to positionally clone the genes and variants that underlie inherited variation in recombination phenotypes . Genome-wide recombination rate ( GRR ) is characterized by considerable inter-individual variation , which is in part inherited [7]–[10] . Genome-wide association studies ( GWAS ) have identified several loci influencing GRR in human [11]–[13] . These include the 17q21 . 31 inversion [11] , as well as the RNF212 gene harboring common variants with antagonistic effects on GRR in males and females [12] . Of note , women's recombination rate correlates positively with reproductive success9 . In human , ∼80% of CO events map to ∼10–20% of the genome , encompassing >25 , 000 recombination hotspots [3] , [14]–[16] . Hotspot usage differs considerably between individuals [17] and this was shown to involve variation in cis-acting hotspot-triggering sequences [18] , as well as in the trans-acting PRDM9 H3K4 trimethyltransferase and hotspot regulator [19]–[22] . Recombination hotspots and their PRDM9 regulator undergo accelerated evolution ( explained in part by the self-destructive drive of hotspot motifs due to biased gene conversion ) [18] , [21] , [23] , [24] , and PRDM9 has been identified as a hybrid sterility gene in the mouse [25] . Genome-wide levels of cross-over interference were also suggested to differ between individuals [26] , [27] , but corresponding genetic variants – if existing - have not been identified thus far . We herein describe our efforts to take advantage of ( i ) the large multigenerational half-sib pedigrees typifying dairy cattle population and ( ii ) the systematization of genome-wide SNP genotyping with ∼50 K medium density arrays for “genomic selection" purposes [28] , to quantify inter-individual variation in recombination phenotypes as well as to map contributing genetic loci . The bovine haploid genome is estimated at 2 . 87 Gbp distributed over 29 acrocentric chromosomes and a pair of metacentric sex chromosomes [29] . Total map length was previously estimated at ∼31M and shown ( contrary to most other mammals ) not to differ between sexes [30] . The potential correlation between recombination rate and fertility , as well as the hypothesized effect of domestication on recombination rates [31] adds to the interest of a detailed characterization of recombination phenotypes in livestock .
The dataset available for analysis comprised 10 , 192 bulls from the Netherlands ( H ) and 3 , 783 bulls from New-Zealand ( NZ ) , that were genotyped for marker panels comprising respectively 50 , 876 [32] and 51 , 456 [33] SNPs of which 19 , 487 in common . The 13 , 975 bulls assorted in 429 three-generational paternal half-sib pedigrees of the structure shown in Figure 1 . All Dutch bulls were from the Holstein-Friesian ( HF ) breed , while in NZ 61% of the bulls were HF and 39% Jerseys ( J ) . SNP genotypes were phased [34] , and CO events identified in the gametes transmitted by generation II ( GII ) bulls to their GIII sons . We identified 259 , 752 CO in 10 , 106 gametes , corresponding to an average genome size of 25 . 7 M ( organs ) . Average number of CO for each of the 29 acrocentric chromosomes was remarkably well predicted ( r2 = 0 . 96 ) by ( i ) size in bp ( 1 = 0 . 07CO/10 Mb ) and ( ii ) the requirement for at least one chiasma per meiosis ( 0 = 0 . 48 CO ) ( Figure S1A ) . Also in agreement with the obligate chiasma theory , the frequency distribution of gametes with 0 , 1 , 2 , … CO-events was best explained [35] assuming near absence of nullichiasmatic meioses for all autosomes . Moreover , under a truncated Poisson model forcing the proportion of nullichiasmatic meioses at zero [36] , the most likely frequency of meioses with one chiasma was considerably lower than expected , and this was largely due to an excess of meioses with two chiasmata . This supports the preferred occurrence of a second chiasma , particularly for the larger chromosomes ( Figure S1B ) . Recombination rate ( RR ) computed in 60-Kb windows averaged 0 . 00062 ( i . e . ∼1 cM/1 Mb ) , but was strongly over-dispersed with an excess of “hot" and “cold" windows ( defined as windows with RR>2 . 5 standard deviations from the mean ) ( Figure S2A–S2C ) . Note that hot windows as defined here ( 60 Kb ) cannot be compared with recombination hotspots as defined in human and mouse genetics ( ≤5 Kb ) [14] , [15] , [37] . On average , 34% of CO events could be assigned to hot windows representing 13% of the genome . Hot and cold windows differed in base pair composition and repeat content ( Table S1 ) . Hot windows tended to concentrate in sub-terminal ( proximal chromosome end ) and terminal regions ( distal chromosome end ) , while cold windows concentrated in the middle of the chromosome arms as well as in terminal regions ( proximal chromosome end ) coinciding with the centromeres ( Figure S2D ) . Hot and cold windows tended to cluster in what we refer to ( following Chowdhury et al . [13] ) as recombination “jungles" and “deserts" , respectively . We measured chromosome-specific levels of cross-over or chiasma interference using the shape parameter ( ) of a gamma distribution [26] , [38] . We used a maximum likelihood approach extracting information from the frequency distribution of ( i ) the number of CO per gamete , ( ii ) CO-position ( in centimorgan ( cM ) ) for gametes with one CO , ( iii ) inter-CO distance ( in cM ) for gametes with two CO , and ( iii ) inter-CO distance ( in cM ) for gametes with three CO . Positive interference was evident for all chromosomes , manifesting itself by ( i ) a paucity of gametes with zero CO , ( ii ) less uniform than expected distribution of single CO position , and ( iii ) inflated distance between CO for gametes with multiple CO . The value of that maximized the overall likelihood averaged 2 . 6 ( range: 1 . 5–3 . 1 ) across all chromosomes ( versus 4 . 5 in human [26] ) . It was primarily determined by the inter-CO distance for gametes with two recombination events . Values of maximizing the likelihood of the frequency distribution of number of CO events and of CO-position for gametes with one recombination tended to be larger that the value of maximizing the likelihood of the inter-CO distance for gametes with two recombinations , while values of maximizing the likelihood of the distance between CO for gametes with three recombinations tended to be smaller . Of note , the observed distribution of CO events per gamete and hence of chiasmata per meiosis , was remarkably well accounted for by positive interference . There was no evidence for an effect of chromosome length on , whether maximizing the overall likelihood or that of the constituent parameters ( Figure S3A–S3B ) . Average genome-wide recombination rate ( GRR ) ( corrected for family size - M&M ) differed significantly between GII sires ( p<0 . 0001; range: 18 . 7–32 . 1 ) ( Figure S4A ) . We took advantage of the fact that 72 of the GII bulls had non-overlapping sets of GIII sons in H and NZ , to estimate the repeatability of GRR as the correlation between these independent measurements , yielding a highly significant Spearman's correlation coefficient of 0 . 58 ( p<3 . 7×10−7 ) ( Figure S4B ) . We estimated the heritability ( h2 ) of GRR at 0 . 22 in the Dutch HF breed . We used a Hidden Markov Model-based approach that simultaneously exploits linkage and linkage disequilibrium [34] to scan the genome for QTL influencing GRR . At each SNP position , all chromosomes in the dataset ( i . e . 2n chromosomes for a data set with n animals ) were assigned to one of 20 hidden states corresponding to “ancestral haplotype states" . The effect of these hidden haplotype states ( HHS ) on the GRR was then estimated using a mixed model including a polygenic effect to correct for population stratification ( M&M ) . We only used HF animals ( from both H and NZ ) in these analyses . We identified two genome-wide significant QTL , respectively on BTA10 ( z = 5 . 8 ) and BTA19 ( z = 4 . 9 ) ( Figure 2A ) . The lod-2 drop-off confidence interval ( CI ) of the BTA10 QTL spanned ∼1 . 4 Mb encompassing 47 genes . Three of these are strongly expressed in testis: TBC1D21 , TSSK4 , and REC8 . REC8 is a particularly appealing positional candidate as it codes for a member of the kleisin family of SMC ( structural maintenance of chromosome ) proteins , which localizes to the axial elements of chromosomes during meiosis in both oocytes and spermatocytes . The mouse homologue is a key component of the meiotic cohesion complex , which regulates sister chromatid cohesion and recombination between homologous chromosomes [39] , [40] . We therefore re-sequenced 7 . 2 Kb encompassing the REC8 gene ( including 1 . 2 Kb upstream of the start codon and 0 . 9 Kb downstream of the polyadenylation site; Figure S5A and Table S2 ) for animals selected to obtain the sequence of three HHS associated with an increase in GRR and one associated with a decrease in GRR ( as HHS with divergent effect on GRR should differ at the causative variant positions ) ( Figure 2B ) . We identified five SNPs located respectively in the 5′UTR ( ss428897146 and ss418642851 ) , intron 5 ( ss418642852 ) , exon 10 ( ss418642853 = E287K ) and intron 12 ( ss418642854 ) . Of note , two of these ( ss418642852 and ss418642854 ) segregated perfectly between the high and low GRR haplotypes . We developed 5′exonuclease assays for ss418642851 , ss418642852 , ss418642853 and ss418642854 ( Table S3 ) , and genotyped the GI and GII sires . We performed single point association analysis using a mixed model including the ( random ) effect of the SNPs as well as a polygenic animal effect to correct for stratification . Ss418642854 yielded a lod score of 9 . 12 , i . e . 3 . 7 units higher than any other BTA10 SNP , and 3 . 3 units higher than the highest BTA10 haplotype-based signal . The difference in GRR between alternate homozygotes at the ss418642854 SNP was 1 . 8 CO/genome ( Figure 2B ) . To provide additional support for the causality of the REC8 gene , we took advantage of the fact that 121 HF GII sires had also been genotyped with a recently developed high-density Illumina 777K SNP array , including 45 SNPs spanning the QTL CI . When performing single point association analysis using the same mixed model for all SNPs in the CI , the lod score still clearly maximized on top of the REC8 gene and for SNP ss418642854 ( Figure S4D ) . Taken together , these results support the fact that variation in the REC8 gene indeed underlies the identified QTL . The CI of the BTA19 QTL spans ∼0 . 6 Mb encompassing two genes: KCNJ2 and KCNJ16 . Neither is knowingly related to recombination , yet both are expressed in testes ( data not shown ) . Preliminary sequence analysis of the KCNJ2 and KCNJ16 open reading frames ( ORF ) of animals carrying haplotypes with significantly different effect on GRR did not reveal obvious variants that might underlie the observed effects ( data not shown ) . In addition to these two significant QTL , we obtained a suggestive lod score of 3 . 2 on BTA6 that maximized at the exact position of the RNF212 gene ( Figure 2A ) . This suggests that variation in RNF212 affects GRR in cattle as it does in human [12] , [13] . Homologues of RNF212 in C . elegans ( ZHP3 ) and yeast ( ZIP3 ) are known to be involved in meiotic recombination [12] . We re-sequenced 10 amplicons encompassing the entire RNF212 ORF and intron-exon boundaries ( Figure S5B and Table S2 ) in animals selected to obtain the sequence of two haplotypes increasing and two decreasing GRR ( Figure 2C ) . We identified eight SNPs located respectively in the 5′UTR ( ss418642855 , ss418642856 and ss418642857 ) , intron 1 ( ss418642858 ) , exon 3 ( ss418642859 ) , intron 4 ( ss418642860 ) , intron 9 ( ss418642861 ) and exon 12 ( ss469104611 = P259S ) . Five of these ( ss418642855 , ss418642858 , ss418642860 , ss418642861 and P259S ) segregated perfectly between the high and low GRR haplotypes . We developed five 5′exonuclease assays ( Table S3 ) and genotyped the GI and GII sires . Ss469104611 ( = P259S ) yielded a lod score of 18 , i . e . 15 . 3 units higher than any other BTA6 SNP and 14 . 8 units higher than the highest BTA6 haplotype-based signal . The difference in GRR between alternate homozygotes at the P259S variant was 3 . 3 CO/genome ( Figure 2C ) . We took advantage of the same 121 GII sires genotyped with the high-density 777K Illumina array , including 27 SNPs in the ∼1 Mb CI of the BTA6 QTL . Lod scores clearly maximized on top of the RNF212 gene , at the position of the ss469104611 variant ( Figure S4E ) . Taken together , these results strongly supported the causality of the RNF212 gene . We then computed , for each GII bull , the proportion of CO falling in hot windows ( i . e . the genome-wide hot-window usage or GHU ) . GHU differed significantly between GII sires ( p<0 . 002; range: 4%–58% ) , was repeatable ( Spearman's correlation: 0 . 46; p<0 . 0008 ) and had a heritability of 0 . 21 in Dutch HF ( Figure S6 ) . We scanned the genome for QTL affecting GHU in HF , and identified three suggestive QTL , respectively on BTA3 ( z = 3 . 7 ) , BTA25 ( z = 4 . 1 ) and BTAX ( z = 2 . 8 ) ( Figure 3A ) . The CI of the BTA3 QTL spans ∼2 . 1 Mb and encompasses three genes ( LOC781798 , LOC522984 and OLFM3 ) not obviously related to recombination . The CI for the BTA25 QTL ( UMD3 31 . 29–33 . 62 Mb ) contains 25 genes of unknown function . Most interestingly , the lod score peak on the X chromosome coincided with the position of two adjacent gonosomal PRDM9 paralogues ( hereafter referred to as PRDM9-XA and –XB ) . In mice and human , genome-wide hotspot usage has been shown to be genetically controlled , with variation in the PRDM9 C-terminal tandem array of Cys2His2 zinc-finger ( ZF ) domains having a major effect [19]–[22] . We therefore designed amplicons allowing specific amplification and sequencing of the complete PRDM9-XA and -XB ZF arrays . The C-terminal ZF arrays of the PRDM9-XA and PRDM9-XB reference sequences ( UMD3 build ) contain respectively eight and 20 ZF domains in tandem ( Table S2 ) . Sequence analyses indicate that bovine PRDM9 ZF arrays are rapidly evolving ( as they are in human and rodents but not in dogs [41]–[43] ) , and this is predicted to increase allelic heterogeneity . We thus decided to determine the sequence of the PRDM9-XA and –XB ZF arrays for 80 individuals representing all 20 hidden haplotype states . Not a single polymorphism , whether synonymous or not , was observed for the PRDM9-XA array . For PRDM9-XB , however , we detected ( i ) a VNTR-like length polymorphism ( as we detected a common allele with 22 ZF ) , and ( ii ) nine SNPs ( Figure 3B ) . Notably , eight of the nine SNPs were non-synonymous . Two affected residues that are predicted to mediate DNA binding , located respectively in ZF 11 out of 22 ( 11/20 ) ( ss5 = I23K , position +6 ) and 16/22 ( ss7 = L17T; position -1 ) . Four corresponded to R<->Q amino-acid substitutions at position 13 of ZF 2/22 ( ss1 ) , 9/22 ( ss2 ) , 11/22 ( ss5 ) and 18/22 ( ss8 ) . Two corresponded to A<->Y amino-acid substitutions at position 7 of ZF domains 10/22 ( ss3 ) and 14/22 ( ss6 ) . Based on these results , we decided to sequence the PRDM9-XB ZF array for all GI and GII sires . The 10 polymorphisms assorted in eight haplotypes observed at least five times , jointly accounting for 98 . 6% of the sequenced chromosomes ( Figure 3B ) . We tested the effect of PRDM9-XB haplotype on GHU using the mixed model described above , and obtained a lod score of 7 . 3 , i . e . 4 . 5 units higher than in the initial scan , hence strongly supporting the causality of the PRDM9-XB paralogue . Analysis of the effects of individual haplotypes indicates that: ( i ) ss5 has a major effect , the K allele decreasing GHU ∼30-fold when compared to the I allele ( hap1-hap3 contrast ) , ( ii ) ss1 has no effect on GHU ( hap1-hap2 contrast ) , ( iii ) the VNTR affects GHU as the loss of two ZFs decreases GHU ∼6-fold ( hap2-hap6 contrast ) , ( iv ) ss2 , ss3 , ss4 , ss7 , ss8 and ss9 have no effect on GHU ( hap6- ( hap5 , hap7 , hap8 ) contrasts ) , ( v ) ss6 affects GHU , the Y allele increasing GHU ∼6-fold when compared to the A allele ( hap4-hap8 contrast ) ( Figure 3C ) . The major effect of the ss5 variant was also apparent from single-point analyses , yielding a lod score of 4 . 6 ( Figure 3D ) . Rapid PRDM9 evolution presupposes accelerated turn-over and hence high polymorphism of recombination hotspots [41] . To test this hypothesis , we scanned the genome for cis-acting haplotype effects on LRR ( in HF ) . We tested the effect of hidden haplotype state of the GII sires on the recombination rate in an 800-Kb window centered on the interrogated SNP position ( M&M ) . We obtained one genome-wide significant effect on BTA6 ( Figure 4A ) . The observed signal was primarily driven by two haplotype clusters ( HS2 and HS9 ) , increasing recombination ∼4 to 5-fold ( Figure 4A ) . The association signal maximized in the middle of a 840-Kb recombination jungle , for which the observed recombination rate exceeded expectation by up to ∼8 . 5 SD ( Figure 4B ) . LRR in the corresponding 800-Kb window was of the order of 8–9% for GII sires heterozygous for either the HS2 or HS9 haplotypes . Eight additional peaks exceeded the genome-wide suggestive threshold ( by definition , expected by chance only once per genome scan ) , supporting the common occurrence of cis-acting haplotype effects on local recombination rate , and presumably reflecting polymorphisms in cis-acting recombination-triggering motifs [18] . We finally evaluated inter-individual variation in genome-wide interference levels ( GIL ) . As interference levels were primarily determined by inter-CO distance for gametes with two CO ( cfr . above ) , we used this metric for QTL mapping . Distances between CO were measured both in centimorgan ( GILcM ) and base-pairs ( GILbp ) , and expressed in standardized deviations from the chromosome mean . Both measures proved to significantly differ between GII sires ( GILcM: p<0 . 002; GILbp: p<0 . 001 ) , to be repeatable ( GILcM: = 0 . 36 , p<0 . 03; GILbp: = 0 . 53 , p<0 . 00003 ) but to have low heritability ( GILcM: 0 . 045; GILbp: 0 . 052 ) in Dutch HF ( Figure S7 ) . We nevertheless scanned the genome for QTL affecting GIL in HF . We identified no QTL when using GILcM , yet one genome-wide suggestive QTL ( z = 4 . 1 ) on BTA25 when analyzing GILbp . The CI of the QTL encompassed four genes ( E-NPP7 , LOC100297064 , FOX1 and TMEM114 ) not knowingly involved in recombination ( Figure 5; Figure S7 ) .
We herein estimate the male map length in domestic cattle at 25 . 7 Morgan based on the analysis of CO events in >10 , 000 sperm cells . This is ∼5 M lower than previous estimates [30] , but in better agreement with the relationship between number of chromosome arms and map length observed in other species [3] . Our findings suggest re-evaluation of ( i ) the presumed equal male and female recombination rate in cattle , and ( ii ) the inflation of recombination as a result of domestication . We demonstrate that GRR is repeatable , that it differs between sires , and that ∼21% of the observed variation is inherited in the HF breed . We identify two significant and one suggestive QTL influencing GRR . We provide evidence that two strong positional candidate genes , namely REC8 and RNF212 , are very likely causative . We reach this conclusion by targeting resequencing efforts to haplotype clusters with significantly different effect on GRR , leading to the identification of SNPs that exhibited highly significant increases in association signal . While variation in RNF212 has been previously shown to affect GRR in human [12] , [13] , the implication of REC8 is novel . For RNF212 , the variant yielding the strongest association is a missense variant resulting in a proline to serine substitution . Despite the fact that the corresponding protein segment is poorly conserved , P259S is a strong candidate causative variant . However , we cannot exclude that the causative variant is regulatory , lying outside of the sequenced RNF212 segments and in LD with P259S , nor that additional causative RNF212 variants exist . For REC8 , the causative variants are most likely regulatory , as coding variants strongly associated with GRR could not be detected despite the sequencing of haplotypes with opposite effects . The most strongly associated SNP ( ss418642854 ) is potentially causal , although the affected sequence is not strongly conserved . Thus , it remains possible that other variants outside the sequenced regions will show equal or even stronger association with GRR . Further sequencing and functional studies are required to achieve complete molecular understanding of these two QTL . Confirming previous findings in human and mice , we observed an overdispersion of LRR , CO tending to preferentially occur in hot windows ( exhibiting sequence features reminiscent of human recombination hotspots ) , while avoiding cold windows . As expected from human and mouse , hot windows tended to concentrate in sub-terminal regions , while cold windows were enriched at centromeres and in the middle of chromosome arms . The propensity for CO to occur in hot windows ( GHU ) was shown to be a repeatable and heritable phenotype in HF ( h2≈21% ) . We identified three genomic loci with suggestive evidence for an effect on GHU . Strikingly , one of these co-localized with two X-linked PRDM9 paralogues . By resequencing bulls representing all hidden haplotype clusters , we identified nine SNPs and a VNTR-type polymorphism in the PRDM9-XB paralogue . Using a haplotype-based approach , we provide strong evidence that an I to K substitution at DNA binding position +6 of ZF 11 decreases GHU ∼30-fold , without affecting GRR . Moreover , we provide suggestive evidence that the VNTR-like polymorphism as well as an A to Y amino-acid substitution at position 7 of ZF domain 14 independently modulate GHU ∼6-fold . Surprisingly , four of the eight non-synonymous variants correspond to R<->Q substitutions at amino-acid position 13 of four distinct ZF domains . None of these variants appear to affect GHU . While this could indicate that the corresponding position is highly mutagenic , we believe that it is more likely that this finding reflects the spreading of a variant within the ZF array by a process of concerted evolution of tandem repeats [44] . Likewise , ss3 and ss6 both correspond to A<->Y substitutions at amino-acid position 7 . Surprisingly , no polymorphisms were observed in the equivalent ( although shorter ) PRDM9-XA array . The reason for this striking difference remains unknown , especially given the fact that both PRDM9-XA and PRDM9-XB appear to be expressed in bovine testes ( data not shown ) . In further support of the rapid coevolution of PRDM9 and recombination hotspots in the bovine , we identify haplotypes with significantly different propensity to engage in recombination at a specific BTA6 jungle . We hypothesize that this results from sequence differences at recombination triggering motifs . This model predicts epistatic interactions between PRDM9 variation and BTA6 haplotype , and analyses to uncover such effects are ongoing . We demonstrate that , as expected , all chromosomes are subject to positive interference , multiple CO being more distant that expected by chance alone . By applying a gamma-model to the distance between MLH1 foci , Lian et al . [27] observed that interference might increase with decreasing chromosome size . It was subsequently indicated , however , that the observed trend might be due to inappropriate modeling of finite chromosome size [45] . It has been suggested that crossovers might involve two pathways [f . i . 46]: ( i ) the pairing pathway not subject to interference , and ( ii ) the disjunction pathway undergoing interference . As the proportion of pairing over disjunction CO increases with decreasing chromosome size , the two-pathway model predicts a decrease in interference levels with decreasing chromosome size as observed in budding yeast [47] . However , we did not find evidence for an effect of chromosome size on levels of interference , in general agreement with Broman and Weber [26] for human . We devised a novel metric to quantify genome-wide interference , and showed that it is repeatable and differs significantly between individuals , yet modestly heritable . We obtain preliminary evidence for the existence of a QTL influencing this trait on BTA25 . The corresponding signal was observed when measuring inter-CO distance in base pairs but not when measured in centimorgan . Further studies will be required to verify the genuine nature of this QTL .
Marker phasing was conducted with the Phasebook software package [34] . We exploited Mendelian rules to phase SNP genotypes in sons ( GII and GIII ) , and linkage information to phase SNP genotypes in sires ( GI and GII ) . CO events were then identified as phase switches in the gametes transmitted by the GII sires to their GIII sons . Double-CO occurring in intervals that were separated by less than three informative markers were attributed to genotyping errors and ignored . CO in 2-Mb windows for which the recombination rate of the GII sire was significantly >5% were attributed to GII phasing errors and ignored . The distribution of CO-events was surveyed using a graphical interface to identify as many other artifacts as possible . Assuming absence of chromatid interference , the proportion of gametes with i CO from meioses with j chiasmata ( ) , follows the binomial distribution:As a consequence , the proportion of gametes with i CO from all meiosis ( ) equalsin which pj correspond to the proportion of meiosis with j chiasmata . The likelihood of a dataset with n0 gametes with 0 CO , n1 gametes with 1 CO , n2 gametes with 2 CO , etc . equalsL is a function of the unknown parameters pj . We determined the values of pj that maximized L . Values considered for j were limited to six . The recombination rate in a defined 60 Kb window was computed as where n is the total number of CO events identified on the corresponding chromosome in the analyzed population , xi is the size ( in bp ) of the marker interval to which CO i has been mapped , oi the overlap ( in bp ) between the 60-Kb window and CO interval i , and T is the total number of analyzed gametes . To normalize window-specific recombination rates for local marker density and informativeness , we simulated ( 1 , 000 times ) genotypes for the GIII sons by randomly “dropping" CO events on the phased GII chromosomes assuming a uniform distribution of CO events following a Poisson process ( with mean corresponding to the real data ) , randomly sampling one of the two paternal chromosomes , while keeping the original maternal chromosome intact . The entire phasing and CO mapping process was then reinitiated with these in silico generated SNP genotypes . The corresponding simulations yielded an average recombination rate with standard deviation for each window . This allowed us to express the actual recombination rate measured for a given window in standardized deviations from the mean ( across simulations ) . Chromosome-specific levels of CO interference were quantified using the shape parameter of a gamma distribution , following Broman and Weber [26] . We determined – for each chromosome – ( i ) the frequency distribution of CO events per gamete , ( ii ) the CO position ( in cM ) for gametes with one CO , ( iii ) the inter-CO distance for gametes with two CO , ( iv ) the inter-CO distance for gametes with three CO . We then compared these distributions with theoretical expectations under various levels of interference , accounting for chromosome size . To that end , we simulated series of “chiasmata" ( CH ) along four stranded bundles with gamma-distributed intervals . The shape parameter was varied from 1 ( no interference ) to 15 with 0 . 1 increments . The rate parameter was always said at 2 . The values of the gamma variables were multiplied by 25 , to obtain an average inter-CH distance of 50 . The CH-series were then converted to single chromatid CO-series , by retaining CH events with a probability of 0 . 5 . The average inter-CO distance was therefore 100 ( “cM" ) . We then randomly sampled at least 500 , 000 independent segments of n cM from these chains , where n corresponds to the actual size of the studied bovine chromosome in cM . For these 500 , 000 “gametes" , we computed the frequency distribution of ( i ) CO-events , ( ii ) CO-position for gametes with one CO ( 5 cM bins ) , ( iii ) inter-CO distance for gametes with two CO ( 5 cM bins ) , and ( iv ) inter-CO distance for gametes with three CO ( 5 cM bins ) . We then evaluated the goodness-of-fit between the real and simulated data by maximum likelihood . The likelihood of the data ( L ) was assumed to be:in which N is the total number of studied gametes , Nri is the number of CO-events characterizing gamete i , P ( Nri ) is the probability of having Nr CO-events ( which is determined by the value of ) , Di is the CO-position ( gametes with one CO ) or inter-CO distance ( s ) ( gametes with >1 CO ) , and is the probability of Di given Nri ( which is determined by the value of ) . was computed for gametes with 1 , 2 and 3 CO and set at 1 for the other gametes ( there is no additional information to be extracted from gametes with 0 CO; gametes with >3 CO are rare and their information likely to be less reliable ) . For simplicity , the probability of the two inter-CO distances for gametes with two CO were considered independent . Accordingly , this likelihood equation can be reformulated as:in which LNr is the likelihood of the observed frequency distribution of number of CO per gamete , and equals , where fi is the expected frequency ( given ) of gametes with i CO ( given ) and Ni is the observed number of gametes with i CO; is the likelihood of the observed frequency distribution of CO-positions for gametes with one CO , and equals , where fi is the expected frequency ( given ) of single-CO gametes with CO-position i ( 5 cM bin ) , N1i is the observed number of single-CO gametes with CO-position in bin i , and l is the length ( in cM ) of the considered chromosome; is the likelihood of the observed frequency distribution of inter-CO distance for gametes with two CO , and equals , where fi is the expected frequency ( given ) of inter-CO distance i ( 5 cM bin ) for double-CO gametes , N2i is the observed number of inter-CO distance in bin i for double-CO gametes , and l is the length ( in cM ) of the considered chromosome; is the likelihood of the observed frequency distribution of inter-CO distance for gametes with three CO , and equals , where fi is the expected frequency ( given ) of inter-CO distance i ( 5 cM bin ) for triple-CO gametes , N3i is the observed number of inter-CO distance in bin i for triple-CO gametes , and l is the length ( in cM ) of the considered chromosome . The values of fi needed to compute the corresponding likelihoods were obtained from the simulations performed under varying values of . The inter-CO distance for gametes with two CO events appeared to be the most influential parameter in determining ( Figure S3 ) . We therefore focused on this measure to perform genetic analysis and QTL mapping ( see also hereafter ) of crossover interference . Inter-CO distances for gametes with two CO were measured in centimorgan ( “GILcM" ) or in base-pairs ( “GILbp" ) , and were normalized by subtracting the mean inter-CO distance for that chromosomes and multiplying by the standard deviation . We noted that estimates of GRR decreased with increasing family size ( Figure S4C ) and attributed this to errors in determining the sire's phase . To correct GRR for this factor we used 10 paternal half-sib families with >100 G III sons . From these families we randomly sampled ( 1 , 000 times ) from 1 to 10 sons with corresponding SNP genotypes . Phasing of the GI , GII and GIII bulls was conducted with Phasebook on these purposely limited data-sets , including determination of CO events in the paternal gametes transmitted to GIII sons . For each of the 10 families we then compared average GRR estimated with 1 , 2 , … 10 sons ( over the 1 , 000 simulations ) with GRR estimated with all sons ( >100 ) , yielding a set of values where i corresponds to the number of used sons ( 1 to 10 ) for family j . These values were averaged across families to generate , i . e . an overall effect on GRR of family size i , used to correct the actual GRR estimates obtained from families with <10 half-sibs . Narrow sense heritabilities ( h2 ) of recombination phenotypes ( measured in the GIII sons ) were estimated using two mixed models [48] . The first modeled average phenotypes of GII sires , and included an overall mean , a random individual animal effect ( with variance-covariance structure proportionate to twice the coefficient of kinship between corresponding GII sires ) , and a random error proportionate to the inverse of the number GIII sons per GII sire . The second modeled the individual phenotypes of the gametes transmitted to GIII sons . It included an overall mean , a random individual animal effect ( with variance-covariance structure proportionate to twice the coefficient of kinship between corresponding GII sires ) , a random permanent GII sire effect , and a random error . Variance components were estimated by restricted maximum likelihood ( REML ) analysis [49] . QTL were mapped using a previously described mixed model approach that simultaneously exploits linkage and LD information [34] . At each SNP position , homologues in the data set were assigned to one of 20 hidden states corresponding to “ancestral haplotype clusters" . The utilized mixed models was the same as the first one used to estimate h2 ( i . e . modeling average phenotypes of the GII sires and adjusting the random error such that it would be proportionate to the inverse on the number of GIII observations per GII sire ) , with addition of a random “ancestral haplotype cluster" effect . The covariance between the effects of the 20 possible “ancestral haplotype clusters" was assumed to be zero . Significance thresholds were empirically determined by phenotype permutation [50] , following standard guidelines [51] . Phenotypic values were permuted amongst half-sibs , a genome-scan conducted , and the highest ( across the genome ) value of the likelihood ratio test ( LRT ) stored . QTL were considered significant if the corresponding LRT exceeded the 95% percentile of the LRT-values obtained by permutation ( i . e . if it exceeded the value of the LRT expected to occur by chance alone once every twenty genome scans ) . QTL were considered suggestive if the corresponding LRT exceeded the 63% percentile of the LRT-values obtained by permutation . To see the latter , a LRT that is not exceeded in 100−63 = 37% of genome scans is exceeded on average once per genome scan as 0 . 37 = e−1 ( assuming that such events are Poisson distributed ) . A linkage signal is defined as being suggestive if it is obtained by chance alone on average once per genome scan . To identify cis-acting haplotype effects on local recombination rate , we defined 800 Kb windows centered around the interrogated marker position . At that marker position , we selected the GII sires that were heterozygous for “ancestral haplotype clusters" [34] and tested the additive effect of “ancestral haplotype cluster" of the GII sires on the recombination phenotype of their GIII sons by ANOVA . The recombination phenotype of GIII sons was defined as the probability that a paternal CO event would have occurred in the interrogated window measured as the degree of overlap between CO encompassing marker intervals and interrogated window . We designed primer pairs to amplify and sequence either the entire gene ( REC8 ) , the ORF ( RNF212 , KCNJ2 , KCNJ16 ) , or the ZF array ( PRDM9-XA and PRDM9-XB ) ( Table S2 ) . Animals to re-sequence were selected based on the ancestral haplotype clusters they carried at the most likely position of the corresponding QTL . Amplifications , purification of the amplicons and direct sequencing of the amplicons were carried out using standard procedures . Genotyping of candidate QTN was conducted using 5′ exonuclease ( Taqman ) assays for REC8 and RNF212 ( Table S3 ) , or by amplicon sequencing for PRDM9-XB .
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Homologous recombination is an essential cellular process that determines proper chromosome segregation during meiosis , affects fertility , and influences evolvability . Nevertheless , the components of the recombination apparatus remain incompletely characterized in mammals . One approach to identify such components is to identify the genes that underlie inherited variation in recombination phenotypes . In addition to providing mechanistic insights , this would allow the study of the evolutionary forces that shape the recombination process . In this paper , we take advantage of genotypes for 50 , 000 genome-wide SNP markers to measure four recombination phenotypes ( genome-wide recombination rate , genome-wide hotspot usage , locus-specific recombination rate , genome-wide cross-over interference ) for >750 bulls on the basis of >250 , 000 cross-overs detected in sperm cells transmitted to >10 , 000 sons . We quantify the heritability and scan the genome for Quantitative Trait Loci ( QTL ) influencing each one of these recombination phenotypes . We perform a detailed genetic analysis of three such QTL , thereby providing evidence that genetic variants in REC8 and RNF212 influence genome-wide recombination rate , while genetic variants in an X-linked PRDM9 paralogue influence genome-wide hotspot usage .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"genetics",
"biology",
"genomics",
"genetics",
"and",
"genomics"
] |
2012
|
Genetic Variants in REC8, RNF212, and PRDM9 Influence Male Recombination in Cattle
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Serine/arginine-rich ( SR ) proteins are important splicing factors which play significant roles in spliceosome assembly and splicing regulation . However , little is known regarding their biological functions in plants . Here , we analyzed the phenotypes of mutants upon depleting different subfamilies of Arabidopsis SR proteins . We found that loss of the functions of SC35 and SC35-like ( SCL ) proteins cause pleiotropic changes in plant morphology and development , including serrated leaves , late flowering , shorter roots and abnormal silique phyllotaxy . Using RNA-seq , we found that SC35 and SCL proteins play roles in the pre-mRNA splicing . Motif analysis revealed that SC35 and SCL proteins preferentially bind to a specific RNA sequence containing the AGAAGA motif . In addition , the transcriptions of a subset of genes are affected by the deletion of SC35 and SCL proteins which interact with NRPB4 , a specific subunit of RNA polymerase II . The splicing of FLOWERING LOCUS C ( FLC ) intron1 and transcription of FLC were significantly regulated by SC35 and SCL proteins to control Arabidopsis flowering . Therefore , our findings provide mechanistic insight into the functions of plant SC35 and SCL proteins in the regulation of splicing and transcription in a direct or indirect manner to maintain the proper expression of genes and development .
Alternative splicing ( AS ) is an important mechanism in the regulation of gene expression by excising introns and ligating different exons to produce multiple mRNA isoforms from a single gene . This post-transcriptional process greatly enhances transcriptome and proteome complexity [1 , 2] . In humans , pre-mRNAs from >95% protein-coding genes are alternatively spliced to produce mature mRNAs [3 , 4] . Of the total intron-containing genes , >60% and >48% undergo AS in Arabidopsis and rice , respectively [5 , 6] . In addition , AS plays a key role in the life process by modulating the gene expression in development [7–11] . Mutations in AS may result in a wide range of diseases in humans [1 , 12] . In plants , aberrant AS may affect their growths and defense responses [13–16] . There are five different types of AS , including exon skipping , intron retaining , mutually exclusive exons , alternative 5' splice site and 3' splice site selection [3 , 6] . In vertebrates , exon skipping is the most frequent type , whereas intron retention is the most common event in plants [17] . Two elements are necessary for AS: 1 ) cis-acting elements , a specific RNA sequence often found in exons or introns ( ESE/ESS , exon splicing enhancer/silencer; ISE/ISS , intron splicing enhancer/silencer ) , and 2 ) the trans-acting elements [7 , 10] , proteins which promote the joining of exons . Pre-mRNA splicing takes place in a large RNA-protein complex known as spliceosome , composed of five small nuclear ribonucleoprote in particles ( U1 , U2 , U4/U6 , U5 snRNPs ) and a large number of non-snRNP proteins , including serine/arginine-rich ( SR ) proteins [18–20] . The interactions between SR proteins and snRNPs are important for splicing . In mammals , it is accepted that SC35 and SF2/ASF interact with both U1-70K and U2AF35 , the subunits of U1 and U2 snRNPs respectively , thereby playing a role in the selection of 5' and 3' splice sites [21] . In Arabidopsis , SRZ21 , SRZ22 and SCL33 were also found to interact with U1-70K [22 , 23] . SR proteins contain one or two RNA recognition motifs ( RRM ) at the N-terminal domains and serine/arginine-rich ( RS ) domain at the C-terminal [8 , 24–30] . The two different domains have disparate functions , with the RRM domain interacting with pre-mRNAs and the RS domain regulating the protein-protein interactions [24 , 29 , 31 , 32] . The RS domain can influence protein subcellular localization by modulating its phosphorylation [33] . The SR proteins are dynamic [34] , and they assemble in nuclear speckles at the subcellular level [34–38] . SR proteins participate in many processes , including mRNA export [39 , 40] , maintenance of genome stability [41 , 42] , microRNA processing [43] and transcription [44] . There are seven SR proteins in humans ( SF2/ASF , SC35 , SRp20 , SRp75 , SRp40 , SRp55 and 9G8 ) [27 , 45] . In vivo , transcription and RNA processing are coupled [46–49] , and SC35 was found to participate in transcription in animals , and influences transcriptional elongation by regulating the functions of RNA polymeraseII ( RNAPII ) [44 , 50 , 51] . Deletion of the ASF/SF2 or SRp20 resulted in cell and embryo lethality [41 , 52 , 53] . Mutation of ASF/SF2 may lead to cancer [54 , 55] , and disruption of SC35 leads to a heart disease [56] . Plants have a much higher number of SR proteins with 18 in Arabidopsis and 24 in rice [15 , 57] . In Arabidopsis , the total SR proteins are divided into six subfamilies: SR ( ASF/SF2-like , SR34 , SR34a , SR34b , and SR30 ) , RSZ ( 9G8-like , RSZ21 , RSZ22 , and RSZ22a ) , SC ( ortholog of SC35 ) , SCL ( SC35-like , SCL28 , SCL30 , SCL30a , and SCL33 ) , RS ( RSp31 , RSp31a , RSp40 , and RSp41 ) , and RS2Z ( RS2Z32 , and RS2Z33 ) . Among these six subfamilies , the latter three are specific to plants [15] . SR proteins play important roles in plant development . Overexpression of atSRp30 affects the splicing and growth of plants , resulting in late flowering , reduced apical dominance , and larger flowers and rosette leaves . Overexpression of atRS2Z33 leads to an increased number of embryos , thicker hypocotyl and cotyledons , altered shapes of root hairs and trichromes , and elevated cell size [58 , 59] . SR45 , which contains one RRM in the middle and two RS domains with each in the N-terminal and C-terminal respectively , is therefore not a classical SR protein . Sr45-1 , a transfer DNA ( T-DNA ) insertion mutant , exhibits abnormal phenotypes , including late flowering time , narrow leaves , reduced root growth and an altered number of petals and stamens [60] . Recently , SR45 was found to be involved in RNA-direct DNA methylation ( RdDM ) , however , the detailed mechanism is not clear [61] . Compared with the studies on SR proteins in vertebrates , the functions of SR proteins in plants remain poorly understood . Here we analyzed the functions of six subfamilies of Arabidopsis SR proteins using a genetic approach . We addressed the molecular basis for the functions of SC35 and SC35-like proteins . Arabidopsis SC35 is an ortholog of human SC35 splicing regulator , containing a RRM domain and a RS domain in its N- and C-terminal , respectively [62] . The four SCL proteins in Arabidopsis all contain a RRM domain and a RS domain in the N- and C-terminal , respectively . In addition , SCL proteins contain an N-terminal domain rich in arginine , proline , serine , glycine and tyrosine [15 , 62] . We identified polytropic defects of the sc35-scl mutant ( scl28 scl30 scl30a scl33 sc35 ) in plant development , including delayed flowering time , serrated leaves , shorter root length and abnormal silique phyllotaxy . In the sc35-scl mutant , 213 genes were found to show significant changes in AS , including all the common AS patterns . In addition , the expression levels of 1249 genes were altered in the sc35-scl mutant . Therefore , our findings demonstrated that SC35 and SCL proteins regulate plant development in a redundant manner by modulating splicing and transcription of a subset of genes .
To study the function of SR proteins , we identified single null mutants of 17 SR protein genes: scl28 , scl30 , scl30a , scl33 , sc35 , sr30 , sr34 , sr34a , sr34b , rs31 , rs31a , rs40 , rs41 , rsz21 , rsz22 , rs2z32 , and rs2z33 . Under a long-day condition , we observed no obvious morphological alterations of these single null mutants . To study the functional relationships between these SR proteins , we generated multiple mutants of different families of SR proteins according to their phylogenetics and structures by crossing the related single T-DNA insertion mutant in addition to clustered regularly interspaced short palindromic repeats/ CRISPR associated proteins 9 ( CRISPR/Cas9 ) -mediated mutagenesis [63] . We first generated sr ( sr34 sr34a sr34b sr30 ) and rs quadruple mutant ( rs31 rs31a rs40 rs41 ) . Given that RSZ and RS2Z subfamilies containing one or two Zn knuckles in the middle of the RS and RRM domains are evolutionarily related [25] , and that SCL proteins have a similar structure to that of SC35 [15 , 62] ( S1 Fig ) , we generated the rsz-rs2z quintuple mutant ( rsz21 rsz22 rsz22a rs2z32 rs2z33 ) and the sc35-scl quintuple mutant ( S2 Fig ) to analyze the functions of these subfamilies . Apart from the sc35-scl quintuple mutant ( scl28 scl30 scl30a scl33 sc35 ) , we observed no visible phenotypes of the sr quadruple mutant , rs quadruple mutant , and rsz-rs2z quintuple mutant ( S3 Fig ) . Compared with the wild-type ( Col-0; WT ) , the sc35-scl mutant is characterized by serrate rosette leaves , appearing as early as at the first euphylla ( Fig 1A–1C ) . In addition , several smaller rosette leaves were observed during the late vegetative stage . Under a long-day ( 16h: 8h light: dark ) condition , the sc35-scl mutant displayed a delayed flowering phenotype ( Fig 1E ) , with approximately four more rosette leaves than WT ( Fig 1C and 1D ) , and an altered phyllotaxis arrangement ( Fig 1F ) . The roots of the quintuple mutant are shorter in comparison to those of WT seedlings ( Fig 1G and 1H ) . To generate the sc35-scl mutant , we obtained several double , triple and quadruple mutants of the SC35 and SCL genes . These double ( scl28 scl30 , scl28 scl33 , scl28 sc35 , scl30 scl33 , scl30 sc35 , scl30a scl33 , scl30a sc35 , and scl33 sc35 ) and triple mutants ( scl28 scl30 scl30a and scl30 scl30a scl33 ) have no obvious phenotypic alteration , only the quadruple mutant ( scl28 scl30 scl30a scl33 ) mutant shows mild phenotypic changes , including serrated rosette leaves and late-flowering ( S4 Fig ) . Considered together , these results suggested that the SC35 and SCL proteins play a redundant role in plant development . To elucidate the expression patterns of SC35 and SCL proteins , we fused the upstream regulatory sequences of SC35 , SCL28 , SCL30 , SCL30a and SCL33 to the β-glucuronidase ( GUS ) reporter gene and transformed these fusions into Arabidopsis plants . For each fusion protein , three independent transgenic lines were selected to analyze the GUS staining in seedlings and different tissues of adult plants . We observed overlapping expressions of the five SR proteins , which widespread in seedlings , rosettes , stem leaves , siliques and flowers ( Fig 2 ) , implicating that the five SR proteins have ubiquitous functions in plant growth . To address the subcellular distributions of SC35 and SCL proteins , these proteins were fused to yellow fluorescent protein/cyan fluorescent protein ( YFP/CFP ) , and transiently expressed or coexpressed in tobacco leaves . SC35 and SCL proteins were observed to form nuclear speckles ( Fig 3A ) and colocalize in these subnuclear domains ( Fig 3B ) . To elucidate the relationships between U2 snRNP and SC35/SCL proteins , YFP-tagged U2AF65a and CFP-fused SC35/SCL proteins were transiently coexpressed in tobacco leaves . Colocalization between U2AF65a and SC35/SCL proteins was observed in nuclear speckles ( Fig 4A ) . In addition , yeast two-hybrid assays revealed the interactions between U2AF65a and SC35/SCL proteins ( Fig 4B ) . To study the relationships between U1 snRNP and SC35/SCL proteins , YFP-tagged U1-70K [64] and CFP-fused SC35/SCL proteins were transiently coexpressed in tobacco leaves . Colocalization between U1-70K and SC35/SCL proteins was observed in nuclear speckles ( Fig 4C ) . Similarly , we observed the interactions between U1-70K and SC35/SCL proteins as indicated by yeast two-hybrid assays ( Fig 4D ) . As SC35 and SCL proteins interact with subunits of U1 and U2 snRNPs , we then investigated the global AS of the sc35-scl quintuple mutant . To this end , we applied high-throughput sequencing to analyze mRNAs in 12 d old plants of the mutant using WT as a control . Two distinct groups of RNA-seq data from three biological repeats were formed in the hierarchical clustering ( S5A and S5B Fig ) . The reads mapped to the genome and aligned against splice junctions were summarized in S1 Table . Compared to WT , the 10114 splicing events were found to be changed in the sc35-scl quintuple mutant , including exon skipping , intron retained , alternative 3’ splice site , alternative 5’ splice site , alternative start , and alternative end ( S6B Fig ) . Among these genes with altered splicing , 213 genes have the significant alternation ( p<0 . 05 ) of the splicing pattern . Real-time reverse transcription polymerase chain reactions ( RT-PCRs ) of several genes ( Fig 5 ) were applied to validate sequencing data by comparing the exclusion/inclusion ratio ( the ratio of skipped events to unskipped events ) and splicing efficiencies between WT and sc35-scl mutant ( the ratios of spliced RNA to unspliced RNA ) [65 , 66] . For these genes , the splicing results revealed by RT-PCRs are consistent with those from RNA-sequencing data ( Fig 5; S7 Fig ) . The results of semi-quantitative reverse transcription and polymerase chain reaction ( qRT-PCR ) were observed to be consistent with the RNA-sequencing data ( S8 Fig ) . In addition , the alternative splicings of representative genes also change in double , triple , and quadruple mutants of SC35/SCL proteins , however , the changing rates increase from double , triple , quadruple to quintuple mutants of SC35 and SCL genes ( Fig 5; S7 Fig ) , supporting that the proper splicing of these genes depend on the interactive roles of the five SC35/SCL proteins . The different isoforms of a specific gene may play disparate functions in special development stages or different tissues [65 , 67–69] . We investigated the functions of different splicing isoforms of the PIF6 gene to further validate the RNA-sequencing data . PIF6 is a transcription factor containing an active photochromo-binding motif and a bHLH-heterodimerization domain , and plays a role in seed germination [67 , 70] . PIF6 has two distinct splicing isoforms: PIF6-α and PIF6-β . PIF6-β lacks the bHLH-heterodimerization domain , and therefore loses the ability to interact with DNA or other proteins . Overexpression of PIF6-β influences seed dormancy and hypocotyl length under red light , and results in an increased rate of seed germination and decreased hypocotyl growth under red light [67] . The real-time RT-PCR indicated that efficiency of the third exon skipping increases approximately 3 . 5 fold in the mutant compared with that of WT ( Fig 6A and 6B ) , resulting in the elevation of the PIF6-β isoform . Accordingly , the seed germination rate of the sc35-scl mutant is higher ( Fig 6C ) , and hypocotyl length of the quintuple mutant is shorter than that of WT under red light ( Fig 6D ) . To clarify the molecular mechanism for the role of SC35 and SCL proteins in the regulation of splicing , using the exhaustive evaluation of the matriX motifs ( XX motif ) method , we identified a specific SC35/SCL protein-binding sequence containing a short AGAAGA motif ( Fig 7A ) , and the splicings of several genes with this motif were confirmed by real-time RT-PCR , consistent to RNA-seq data ( S9 Fig ) . To investigate whether SC35 and SCL proteins directly interact with this motif , we used RNA electrophoretic mobility shift assays ( EMSAs ) to test the interaction between a purified SCL protein and the Biotin-labeled RNA motif . We examined the binding of SCL30 to the RNA fragment of AT1G53250 locus , which was randomly selected from genes containing this short AGAAGA motif ( Fig 7B ) . The AS pattern of AT1G53250 was confirmed by real-time RT-PCR ( Fig 7C; S9 Fig ) . As shown in Fig 7D , the amount of RNA-protein complex increases proportionally to the increase of purified SCL30 . In contrast , the amount of RNA-protein complex decreases as the unlabeled probe increases ( Fig 7D ) . Finally , the unlabeled probe completely abolished the binding of labeled probes ( Fig 7D , lane 9 ) , indicating that SCL30 binds directly to this specific RNA sequence . To further verify the specificity of the binding of SCL30 to this RNA sequence , we mutated the AGAAGA to UCUUCU ( S10A Fig ) , and performed a competition assay . The results showed that the mutated probe failed to compete the binding of SCL30 to the un-mutated probe ( S10B Fig ) , further supporting that the binding of SCL30 to the RNA sequence is specific . By comparing the RNA-seq data of WT and sc35-scl mutant , we noticed that there were 1249 differentially expressed genes ( fold change>1 . 5 , p<0 . 05 ) ; the expressions of 720 and 529 genes increased and decreased , respectively ( S2 Table ) . These genes of altered expression are involved in different biological processes , including genes involved in the ribosome , phenylalanine metabolism , plant hormone signal transduction , and spliceosome ( Fig 8A ) . We verified the expressions of key genes involved in the ribosome , phenylalanine metabolism , and plant hormone signal transduction by real-time RT-PCRs in seedlings of WT and sc35-scl mutant ( Fig 8B ) . Importantly , the expressions of a group of genes without intron also changed ( Fig 8C ) , raising a possibility that SC35 and SCL proteins might play a role in the regulation of transcription . As both transcription and degradation of mRNA affect the accumulation level of the mRNA , we compared the transcript decay rates of several genes without intron between WT and sc35-scl to uncover the role of SC35/SCL proteins in the transcriptional regulation . To this end , cordycepin , a drug which is structurally analogous to adenosine , was used to inhibit the transcription and followed by the evaluation of mRNA decay [71–73] using EIF-4A transcript , which is stable and has a prolonged half life , as a control [73 , 74] ( S11 Fig ) . The results showed that the mRNA decay efficiencies of some genes in sc35-scl were similar to those in WT ( S12 Fig ) , supporting a role of SC35 and SCL proteins in the transcriptional regulation of a population of genes in a direct or indirect manner . In animals , it was known that SR proteins interact with RNA polymerase II ( RNAP II ) [75–77] , and that deletion of the SR proteins attenuates the production of nascent RNAs [44] . Firefly luciferase complementation imaging assays were performed to address the potential interactions between plant SC35/SCL proteins and NRPB4 , a specific subunit of RNAP II [69] . NRPB4 was fused to CLUC , and the five SR proteins were fused to NLUC . CLUC/NLUC pairs of constructs were transiently coexpressed in tobacco epidermal leaf cells , and complemented luciferase signals were observed between NRPB4 and SC35/SCL proteins ( Fig 9A ) , indicating that SC35 and SCL proteins interact with NRPB4 . These interactions between NRPB4 and SC35/SCL proteins were further tested by the Co-IP assays . Flag-fused SCL28 and SCL30 were observed to co-immunoprecipate with YFP-fused NRPB4 ( Fig 9B ) , but not with SCL30a , SCL33 , and SC35 , possibly due to weak interactions between NRPB4 and them . An obvious phenotype of the sc35-scl quintuple mutant is late-flowering . Under the long-day condition , plants of the sc35-scl mutant bolt at approximately 30 d after sowing , whereas WT plants at approximately 23 d ( Fig 1E; Fig 10A ) . In the quintuple mutant from the RNA-seq data , we found that the expression of Flower Locus C ( FLC ) , which encodes a MADS-box DNA binding protein , a key regulator of flowering time in Arabidopsis [78 , 79] , increases significantly compared with that of the WT ( S2 Table ) . Real-time RT-PCR further confirmed that loss of functions of SC35 and SCL proteins resulted in a sharp increase of FLC ( Fig 10B ) . In Arabidopsis , there are four different splicing isoforms of FLC: FLC . 1 , FLC . 2 , FLC . 3 and FLC . 4 . Among them , the isoform of FLC . 1 encodes a functional FLC protein and has an abundant expression in Arabidopsis [66] . We first investigated the splicing of the last intron by real-time RT-PCR and found that the proportions of spliced to unspliced introns were identical in WT and sc35-scl mutant ( the splicing efficiencies are 0 . 917 and 0 . 973 in WT and sc35-scl , respectively ) ( Fig 10C and 10D ) , indicating that the SC35 and SCL proteins have no effect on AS of FLC transcripts , consistent with the RNA-seq data ( S3 Table ) . We then addressed whether the constitutive splicing of FLC has changed . To this end , we examined the splicing of the first intron of FLC by real-time RT-PCR and found that the proportion of spliced to unspliced transcript increases in the sc35-scl quintuple mutant compare with that in WT ( the splicing efficiency is 0 . 331 and 1 in WT and sc35-scl , respectively ) ( Fig 10E ) , suggesting that SC35 and SCL proteins regulate the splicing efficiency of the first intron of FLC . The level of unspliced FLC transcript is higher in sc35-scl mutant than in WT ( Fig 10D ) , we then asked if the elevated expression level of FLC is caused by not only the splicing , but also by the transcriptional regulation . To this end , we examined levels of Pol II of the FLC gene by chromatin immunoprecipitation ( ChIP ) . Eight primers on different regions of the FLC gene were designed for the real-time RT-PCR of the immunoprecipitated samples ( Fig 10F ) . The results showed that Pol II levels were higher in sc35-scl ( Fig 10G ) . We also tested the levels of the transcription-activating mark ( H3K4me3 ) and transcription-repressing mark ( H3K27me3 ) at FLC locus by ChIP-PCR assay , the results showed that the level of H3K4me3 increases ( Fig 10G ) , whereas the level of H3K27me3 decreases in FLC chromatin of the sc35-scl mutant compared with those in WT ( S13 Fig ) .
Splicing is an important mechanism in eukaryotes , and is thought to regulate gene expression at the co- and post-transcriptional levels [80 , 81] . Cis-acting elements and trans-acting elements interact to regulate this process . The SR proteins are important regulators belonging to the trans-acting elements [19] . Plants have a greater number of SR proteins than animals , with Arabidopsis encoding 18 SRs and rice encoding 22 SRs . However , compared to the extensive studies on SR proteins in animals , studies on SR proteins in plants were very limited . In previously studies , gain-of-function has been the main tool to study the functions of plant SR proteins [58 , 59] . However , due to the functional redundancy and dosage-dependence of SR proteins , the loss-of-function approach is necessary to address the roles of SR proteins in development and their molecular mechanisms . Here we systematically studied different subfamilies of the classical SR proteins in Arabidopsis using the genetic approach . The 18 classical SR proteins in Arabidopsis were crossed to generate four mutants , including sr quadruple mutant ( sr34 sr34a sr34b sr30 ) , rs quadruple mutant ( rs31 rs31a rs40 rs41 ) , rsz-rs2z quintuple mutant ( rsz21 rsz22a rsz22 rs2z32 rs2z33 ) , and sc35-scl quintuple mutant ( scl28 scl30 scl30a scl33 sc35 ) . Interestingly , no visible phenotypes were observed in the sr quadruple mutant , rs quadruple mutant , and rsz-rs2z quintuple mutant . These observations are different from those in animals in which deletion of ASF/SF2 arrests the cell growth [52] , and loss-of-function of SRp20 in the mouse leads to the embryonic lethality with the embryo failing to grow as early as in the morula stage [53] . Our results indicated that the functions of SR proteins are redundant in Arabidopsis . Importantly , the redundancy not only appears in the members of SR proteins of the same subfamily , but also to some degree among members from different subfamilies of SR proteins . The loss of functions of SC35 and SCL ( SC35-like , SCL28 , SCL30 , SCL30a and SCL33 ) results in pleiotropic changes in development , including serrate rosettes , late flowering , shorter roots , and anomalous phyllotaxis arrangement . At the subcellular level , SC35 and four SCL proteins co-localize in nuclear speckles . SC35 and SCL proteins were found to interact with the U1-70K and U2AF65a . In the sc35-scl quintuple mutant , pre-mRNA splicing of a population of genes are affected , including all events of AS with the alternative 3’ splice site the main target . In sc35-scl , the changed splicing isoform of PIF6 might affect the seeds dormancy and hypocotyl elongation as we have not detected obvious changes of the expression levels of several known genes related to seeds germination ( PIF1 , SPT , RBCS , CAB1 , APL3 , CH3 , ABI1 , ABI2 , ABI4 and ABI5 ) [82–84] and hypocotyl elongation ( PIF3 , PIF4 and PIF5 ) [85–88] ( S14 Fig ) , however we cannot rule out other unknown factors which may contribute to these phenotypes . It was known that the RRM domain ( s ) of SR proteins have the ability to bind to the target pre-mRNA , and preferentially to specific RNA sequences [87 , 89–91] . We found that SCL30 binds to the AGAAGA motif , in comparison to previous studies showed that the GAAG repeats function as the splicing enhancers [92–94] in animals , and RZ-1C and SR45 proteins bind to the AG-riched motif in plant [66 , 95] , suggesting that the AG-rich sequence might play conservative and important role in splicing both in plant and animals . It was known that most exons are bound by at least one SR protein [87 , 91 , 96 , 97] , we thought SR45 and SC35/SCL proteins may bind to specific motifs to play redundant and cooperative roles in splicing . RZ-1C , an RNA-binding protein , might also participate in this process together with these SR proteins . We also found that the AS patterns of a set of genes without the AGAAGA motif changed in the sc35-scl mutant . We speculated that the AS of different genes may depend on different motifs . Alternatively , the loss of SC35 and SCL proteins may influence the functions of other SR proteins . Among 1249 genes , of which the expression levels changed in the sc35-scl mutant , only 12 genes were found to have changed their splicing patterns as indicated in the sequencing data ( S4 Table ) . The expression levels of genes can be regulated by their constitutive splicing as previously reported [66 , 98]; however , we found that the expression levels of many genes without introns also change with the degradation of some transcripts not changed in sc35-scl , suggesting a direct or indirect role of SC35 and SCL proteins in the transcriptional regulation in addition to their functions in splicing . In mammals , many studies have proved that splicing factors interact with C-terminal domain ( CTD ) of NRPB1 in RNAP II , or other components of transcription machinery to influence the transcription process [99 , 100] . We tested the potential interactions between SC35/SCL proteins and CTD of NRPB1 , full-length of NRPB1 and NRPB4 , two RNAP II-specific subunits , luciferase complementary signals can be only detected between SC35/SCL proteins and NRPB4 ( Fig 9A ) . In addition , Co-IP also confirmed the interactions between NRPB4 and SC35/SCL , however no clear positive results between NRPB4 and SCL30a/SCL33/SC35 were observed in the experiment ( Fig 9B ) . We thought the interactions between them maybe too weak to be detected under the experimental condition . We also found that SC35/SCL co-localize partially with the biggest subunit RNAP II ( NRPB1 ) in nuclear speckles ( S15 Fig ) , implicating that these SR proteins may exist in the large complex containing RNAP II to regulate gene expression at transcriptional and post-transcriptional levels . The result that SC35/SCL proteins regulate both the splicing and transcription was further supported by our analysis of the expression level of FLC gene which increases significantly in the sc35-scl mutant with a later flowering phenotype . Several genes ( FPA , FVE , FY , VIL2 , VRN1 , VRN2 , and VRN5 ) involved in the regulation of FLC expression by the autonomous or vernalization pathway [101–104] have no obvious changes in their AS patterns , and only mild increases in their expression levels in the sc35-scl mutant ( S2 Table; S3 Table; S16 Fig ) . It was known that the increases of these regulators lead to earlier flowering . We suspected that the elevated expression of FLC due to losses of functions of SC35 and SCL proteins might play a predominant role in the control of the later flowering phenotype of sc35-scl mutant . The splicing efficiency of FLC . 1 did not change in the quintuple mutant ( Fig 10C and 10D ) . However , SC35 and SCL proteins suppress the constitutive splicing of the first intron of FLC ( Fig 10E ) , thereby resulting in a higher expression of FLC in sc35-scl . In addition , the result that the level of unspliced FLC increases in sc35-scl suggested the regulation may occur at the level of transcription . The repression of SC35 and SCL proteins on the FLC transcription was further supported by the chromatin status in this locus with a decrease in H3K27me3 and increases in Pol II and H3K4me3 occupancies in the sc35-scl mutant . It has been known that the FLC antisense RNA COOLAIR and chromatin modifications influence the FLC expression [105–107] . We found that SC35 and SCL splicing factors regulate the splicing efficiency of the first intron of FLC and histone methylation status at FLC locus , thus revealed a novel regulatory pathway in the control of expression level of FLC and flowering . In animals , it was found that SR proteins regulate the chromatin structure and dynamics through interaction with histones [108 , 109] , and yeast SR-like protein Npl3 was also found to play a role in chromatin remodeling and histone modification [110] . It is of interest to address if SC35 and SCL or other plant SR proteins have roles in histone modifications or not . Future studies will focus on investigating the potential interactions between histones and SR proteins , and uncovering more functions of SR proteins in plants .
Arabidopsis thaliana ( ecotype Col-0 ) , T-DNA insertion mutants scl28 ( CS853758 ) , scl30 ( CS805508 ) , scl30a ( Salk_056672 ) , scl33 ( Salk_058566 ) , sc35 ( Salk_033824C ) , sr30 ( Salk_116747C ) , sr34 ( CS878689 ) , sr34a ( Salk_087841C ) , sr34b ( Salk_055412 ) , rs31 ( Salk_014656C ) , rs31a ( CS834516 ) , rs40 ( Salk_118875 ) , rs41 ( CS803022 ) , rsz21 ( Salk_114234 ) , rsz22 ( CS809215 ) , rs2z32 ( Salk_031147C ) , rs2z33 ( Salk_051525 ) were obtained from TAIR . All the mutants were confirmed by PCR ( primers were listed in S5 Table ) . SR34 and RSZ22a were mutated by CRISPR/Cas9 [63] . T0 seeds were selected in Murashige and Skoog ( MS ) medium containing 50mg/L Hygromycin B . Homozygous transgenic lines were confirmed by PCR and sequencing . All seeds were germinated in MS medium ( 1% sucrose and 0 . 8% agar ) , after vernalizing at 4°C for 3 d or 4 d . Plants were grown in a green house under a 16 h light/8 h dark photoperiod . The multiple mutants including quintuple mutant ( scl28 scl30 scl30a scl33 sc35 ) and quadruple mutants ( sr34 sr34a sr34b sr30 , rsz21 rsz22 rs2z32 rs2z33 , and rs31 rs31a rs40 rs41 ) were generated by crossing between corresponding single mutants in combination with the CRISPR/Cas9 method . A seed germination assay was performed according to the previous report [67] . Freshly harvested seeds from brown siliques were sown on the 0 . 8% MS plates and placed in a growth chamber under a 12 h light/12 h dark photoperiod at different temperatures . The germination rates were scored after 7 d according to the standard [67] . For the measurement of flowering times , the seeds of Col-0 and sc35-scl were vernalized in 4°C for 4 d and sown in the soil under LD ( 16 h light/8 h dark ) . The bolting time and the numbers of rosette leaves were scored . The cDNAs of SC35 , SCL28 , SCL30 , SCL30a , SCL33 , U170K and U2AF65a were amplified by PCR from Col-0 cDNAs ( primers used were shown in the S5 Table ) , then subcloned into the plasmids pCambia1300-35S-N1-YFP and pCambia2300-35S-N1-CFP . The constructs were confirmed by sequencing and introduced into Agrobacterium tumefaciens ( GV3101 ) by electroporation . Transient expression and image processing were conducted according to the protocol [111] . For colocalization analysis , plasmid pairs SCL28-CFP/U170K-YFP , SCL28-CFP/U170K-YFP , SCL28-CFP-SC35-YFP , SCL30-CFP/U170K-YFP , SCL30-CFP/U2AF65a-YFP , SCL30-CFP/SC35-YFP , SCL30-CFP/SCL28-YFP , SCL30-YFP/SCL30a-CFP , SCL30-YFP/SCL33-CFP , SCL30a-CFP/U170K-YFP , SCL30a-CFP/U2AF65a-YFP , SCL30a-CFP/SC35-YFP , SCL30a-YFP/SCL33-CFP , SCL30a-CFP/SCL28-YFP , SCL33-CFP/U170K-YFP , SCL33-CFP/U2AF65a-YFP , SCL33-CFP/SC35-YFP , SCL33-CFP/SCL28-YFP , SC35-CFP/U170K-YFP and SC35-CFP/U2AF65a-YFP were co-expressed in tobacco leaves . After 48 h , inoculated leaf discs were visualized under a DeltaVision PersonalDV system ( Applied Precision ) using the Olympus UPLANAPO water immersion objective lens ( 60 ×/1 . 20 numerical apertures ) . The filters used for YFP were exciter ( 492/18 nm/nm ) , emitter ( 535/30 nm/nm ) , for CFP were exciter ( 430/24 nm/nm ) and emitter ( 470/24 nm/nm ) . For yeast two-hybrid assays , the coding regions of SCL28 , SCL30 , SCL30a , SCL33 and SC35 were cloned in pGBKT7 and pGADT7 plasmids ( primers used were shown in S5 Table ) . The coding regions of U1-70K and U2AF65a were cloned in pGADT7 plasmid ( primers used were shown in S5 Table ) . These constructs were confirmed by sequencing and cotransformed pairwisely into yeast strain AH109 according to the Pro-Quest Two-Hybrid System Manual ( Matchmaker user’s manual , Invitrogen ) . Transformants were cultivated on the SD-Leu-Trp medium at 30°C in an incubator for approximately 3 d , and tested in selection plates: SD-Leu-Trp-His-Ala medium and SD-Leu-Trp-His medium were supplemented with 1mM or 5mM 3-amino-1 , 2 , 4-triazole ( 3-AT ) , respectively . The results were tested after 3–6 d of growth at 30°C . Total RNAs were extracted from 12 d seedlings of WT and quintuple mutant ( scl28 scl30 scl30a scl33 sc35 ) using the RNeasy plant mini kit ( Qiagen ) . The total RNAs were treated with DNase I followed by mRNA isolation using magnetic beads with Oligo ( dT ) . Fragmented mRNAs were used as templates for PCR amplification and the construction of the RNA-seq library . Agilent 2100 Bioanalyzer and the ABI Step One Plus Real-time PCR System were used for the quantification and qualification of the sample library . Finally , the library was sequenced using Illumina HiSeq TM 2000 . The sequencing data termed raw reads were subjected to quality control ( QC ) . After QC , raw reads were filtered into clean reads , and aligned to the reference sequences with SOAP aligner/SOAP2 . The reads with strand direction were aligned to the TAIR10 genome using SOAP aligner/SOAP2 , allowing no more than five mismatches . The ASD ( AS detector ) software ( http://www . novelbio . com/asd/ASD/html ) was used for the detection of AS events . To calculate the p-value of AS events , first count the number of junction reads that align either to the inclusion or exclusion isoforms in both the WT and sc35-scl quintuple mutant , and calculate a p-value using junction read-counts between WT and sc35-scl quintuple mutant by Fisher exact test . Then calculate read coverage for the alternative exon and its corresponding gene in both WT and sc35-scl quintuple mutant , and calculate a second p-value by Fisher exact test according to the alternative exon read coverage relative to its gene reads coverage between WT and sc35-scl quintuple mutant . Finally combine the above two p-values to obtain an adjusted p-value using a weighted arithmetic equation for assessing the statistical difference of AS between WT and sc35-scl quintuple mutant [112 , 113] . The AS events with p-value<0 . 05 were considered as the significant change . Using the EB-seq algorithm to analyze the differential expression genes , the standard was a fold change>1 . 5 or<0 . 667 , p<0 . 05 . Pathway analysis was used to identify the significant pathway of the differential genes according to the Kyoto Encyclopedia of Genes and Genomes ( KEGG ) database . We used the Fisher’s exact test to select the significant pathway . The threshold of significance was defined by the p-value and false discovery rate ( FDR ) [114–117] . The promoters of SCL28 , SCL30 , SCL30a , SCL33 and SC35 were cloned into the pBI101 plasmid ( primers used were shown in S5 Table ) . The transgenic Arabidopsis plants generated using the flower dip method [118] were selected on hygromycin ( 50mg/L ) and confirmed by PCR . At least three T2 independent lines were analyzed . GUS staining was performed according to published literature [119] with a modified buffer ( 1mg/ml 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid cyclohexylammonium salt , 50mM Na3PO4 , pH 7 . 0 , 0 . 1% Triton X-100 , 2mM K4Fe ( CN ) 6·3H2O , 2mM K3Fe ( CN ) 6 , and 10mM EDTA ) . The seedings and plant tissues ( rosette , stem leaf , silique and inflorescence ) were immersed in the GUS buffer overnight at 37°C in dark , and then cleared with 75% ethanol [120] . The total RNAs were extracted from 12 d Arabidopsis seedlings using the RNeasy Plant Mini Kit ( Qiagen ) . RNAs were treated with master mix reagents ( Toyobo ) to remove the genomic DNA , and the reverse transcription was conducted using the master mix reagents to generate the cDNAs . The cDNAs were diluted approximately five-fold and used as a template for quantitative PCR using a SYBR Green Master Mix ( Takara ) . Quantitative real-time PCR was performed in a Bio-Rad CFX Real-time System ( primers used were shown in S5 Table ) . The ACTIN2 gene was used as an internal control and for data normalization . The data obtained were analyzed through a Bio-Rad iCycleriQ Real-time Detection System , and three biological repeats were performed . The splicing efficiency was measured as previously reported [66 , 112 , 121] . The splicing efficiency was calculated by determining the level of spliced RNA normalized to the level of unspliced RNA in the intron-retained splicing events , and in the exon-skipped splicing event , the exclusion/inclusion ratio was calculated by determining the level of skipped RNA normalized to the level of unskipped RNA [112] . The 3’ unspliced primers were designed crossing the intron-exon junction , and the 5’ primers were designed on the intron or the next exon . The 3’ spliced primers were designed to span the exon-exon junction . The primers related to unskipped RNA were designed to span the exon-exon junction , whereas the skipped primers were designed to span the exon with the exon after the next . These primers were then used for real-time RT-PCR . The data of the splicing efficiency were derived from three biological repeats . The seedings of WT and sc35-scl mutant were incubated in cordycepin , the samples were then collected at one hour intervals . Total RNA were extracted from the samples using RNeasy Plant Mini Kit ( Qiagen ) , then cDNAs were obtained by inverse transcription . Real-time PCR was used to test the expression levels of related genes which were normalized to the expression level of corresponding genes at 0 h to evaluate the mRNA decay efficiencies . The software of the XX motif was used to analyze the RNA motif [122] which is extracted from proximate the splice site of 100bp in the AS genes ( p<0 . 05 ) . The parameters used were described as follows: expected occurrences of motifs per sequence: zero , one or multiple occurrences; order of background model: 2; similarity threshold for merging motifs/PWMs: low; pseudocounts: 10%; number of gaps n fivemer seed: 0; start search with these seed patterns: includes 5-mer nucleotides , palindromes and tandem repeats . We selected one sequence of approximately 26 nt ( AGAUUAAAGAAGAGGAAAGGAGAAGG ) from AT1G53250 , containing the motif . The sequence was synthesized in vitro ( Takara ) , and the 5’ end was labeled with biotin . SCL30 was cloned in the plasmid of pET28a , fused with His-tag . The constructs were expressed in E . coli ( transetta ) and cultured at 37°C , and the expressions of SR proteins were induced by 0 . 4M isopropyl β-D-1-thiogalactopyranoside ( IPTG ) . The Ni-NTA agarose beads were used to purify SR proteins . The binding assays were performed according to the manual ( Light Shit Chemiluminescent RNA EMSA Kit , Thermo Pierce ) . The 20μl reaction system contained 2μg tRNA , 10nM of labeled RNA and the purified individual SR protein of different concentrations . The RNA-protein mixtures were incubated for approximately 30min at room temperature and fractioned on a 6% native polyacrylamide gel under 100V for approximately 60min in 0 . 5x TBE buffer , then transferred to a nylon membrane ( GE Healthcare ) . The biotin-labeled RNAs on the nylon membrane were detected using a chemiluminescent nucleic acid module ( Thermo Pierce ) [123] . For the competition assay , purified SR proteins , 2μg tRNA and the biotin-labeled 10nM specific RNA was added in all lanes , and unlabeled RNA was added to lanes 6–9 . Lane 5 , 0 . 4μg SR protein; lane 6 , 0 . 4μg SR protein+0 . 625μM unlabeled RNA; lane 7 , 0 . 4μg SR protein+1 . 25μM unlabeled RNA; lane 8 , 0 . 4μg SR protein+2 . 5μM unlabeled RNA; lane 9 , 0 . 4μg SR protein+5μM unlabeled RNA . The process was the same as that employed in the binding assay . The luciferase ( LUC ) complementation imaging assay was performed as previously described [124] . SR and NRPB4 proteins were fused to the C-terminal and N-terminal fragment of firefly luciferase , respectively . The NRPB4-NLUC and CLUC-SR were transferred into Agrobacteria strain GV3101 , and then co-infiltrated into the tobacco leaves using an injection syringe . At approximately 48 h , the leaves were injected with 100mM luciferin ( Sangon Biotech ) in 0 . 1% Triton X-100 , and fluorescence was quenched in the dark for several minutes , a Chemiluminescence Imaging System ( Tanon ) was then used to observe the luciferase signals . The plasmids pairs 35S::SC35/SCL-Flag and 35S::NRPB4-YFP were co-infiltrated into the tobacco leaves using an injection syringe . At approximately 48 h , the leaves were collected and ground in liquid nitrogen . The cell debris were treated with the three volumes of extraction buffer ( 50mM Tris-HCl at pH 8 . 0 , 150mM NaCl , 0 . 5% Triton X-100 , 0 . 2% 2-mercaptoethanol , 5% glycerol ) containing one proteinase inhibitor cocktail tablet/50 ml ( Roche ) , centrifuged for 20 min at 8 , 000g [125] . The total proteins incubated with the GFP agarose beads ( MBL ) about 3–4 h at 4°C . The columns were washed 5 times with washing buffer ( 50mM Tris-HCl at pH 7 . 5 , 100mM NaCl , 10% Glycerol , 0 . 05% Triton X-100 , 1mM EDTA ) and proteins were released by boiling the beads in SDS-PAGE loading buffer at 100°C for 10 min . The proteins were resolved by SDS/PAGE , and then the anti-Flag ( Sigma ) and anti-GFP ( Sigma ) antibodies were used to detect SC35/SCL-Flag and NRPB4-YFP , respectively . The ChIP assay was performed as previously described [126] using 12 d seedlings of WT and sc35-scl mutant . The seedlings of approximately 2 . 5g were harvested in cross-linking buffer ( 0 . 4M sucrose , 10mM Tris-HCl ( pH8 . 0 ) , 1mM PMSF , ImM EDTA , 1% formaldehyde ) for 10 min using vacuum infiltration and then halted in 2M glycine . After the addition of 5μg H3K27me3 , H3K4me3 ( Milipore ) and Pol II antibodies ( Abcam ) to the chromatin and incubation at 4°C overnight , the agarose beads of protein A and protein G were added and maintained at 4°C for approximately 2 h . After reverse cross-linking , DNA was purified and dissolved in 30μl water . The immunoprecipitated DNA was diluted and then quantified by real-time PCR . Real-time PCR data of Pol II were normalized to Actin , and H3K4me3 normalized to H3 . The Pol II enrichment at FLC was given as ratio of ( Pol II FLC/input FLC ) to ( Pol II Actin/input Actin ) and H3K4me3 enrichment as ratio of ( H3K4me3 FLC/input FLC ) [106] . Primers used for real-time PCR are listed in S5 Table .
|
SR proteins were identified to be important splicing factors . This work generated mutants of different subfamilies of the classic Arabidopsis SR proteins . Genetic analysis revealed that loss of the function of SC35/SCL proteins influences the plant development . This study revealed SC35/SCL proteins regulate alternative splicing , preferentially bind a specific RNA motif , interact with NRPB4 , and affect the transcription of a subset of genes . This study further revealed that SC35/SCL proteins control flowering by regulating the splicing and transcription of FLC . These results shed light on the functions of SR proteins in plants .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
[
"reverse",
"transcriptase-polymerase",
"chain",
"reaction",
"gene",
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"brassica",
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] |
2017
|
Depletion of Arabidopsis SC35 and SC35-like serine/arginine-rich proteins affects the transcription and splicing of a subset of genes
|
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