Research

The group’s ongoing and future research program broadly focuses on the innate immune systems and microbiomes of mosquito disease vectors, and comprises several independent but synergistically interacting projects. VIDEO PRESENTATION.

CURRENT DIMOPOULOS GROUP PROJECTS

Introduction
The group’s ongoing and future research program broadly focuses on the innate immune systems of mosquito disease vectors, and comprises several independent but synergistically interacting projects. We are interested in understanding the role and mechanisms of the mosquito’s innate immune system in the defense against human pathogens such as Plasmodium and the Dengue virus. A major focus is concentrated on Anopheles gambiae anti-Plasmodium defense systems that. Towards this, we have contributed with several pioneering discoveries. Our mission is to characterize the defense mechanisms employed by mosquito vectors against human pathogens. Our competitive advantage derives from a unique blend of core competencies in molecular entomology, innate immunity and functional genomics, as well as the access to state-of-the-art research infrastructure at the Johns Hopkins Malaria Research Institute. The long-term goal of our research program is to broaden the basic knowledge of this field and provide new tools for the development of vector borne disease control strategies.

 Development of mosquito gene expression analysis tools
The majority of our research projects involve high throughput transcription analyses for the study of infection responsive processes and immune response regulation. My group has acquired extensive expertise on microarray-based gene expression methodology, and developed the first full genome microarrays for Anopheles gambiae, Aedes aegypti, Culex pipiens and Plasmodium berghei. These genomics tools have been instrumental in a variety of project including the dissection of A. gambiae responses to Plasmodium infection, the annotation and basic characterization of the Ae. aegypti and P. pipiens transcriptomes, the study of C. pipiens responses to West Niles virus infection, and Ae. aegypti responses to dengue virus infection. We have also performed studies on Plasmodium gene regulation. We have also contributed towards the development of novel bioinformatics tools for the analysis of microarray data.
FUTURE DIRECTIONS: My group is continuously monitoring the development and evaluating the use of new functional genomics / post genomics technologies for our studies. Our Agilent Technologies-based mosquito microarrays provide cost effective and competitive performance for the next 2-3 years. High throughput sequencing-based gene expression assays are then expected to become a more powerful approach and will obviously be adopted for our studies. The current mosquito microarrays will continually be updated when the mosquito genome sequences are re-annotated in order to ensure representation of the most current transcript annotations.

The malaria parasite Plasmodium has a complex life cycle in the vector mosquito that involve several developmental transitions, interactions and invasion of epithelial tissues and evasion of the mosquitos' immune system. Gametocytes (GC) are ingested with an infectious blood meal and fertilize in the mosquito midgut to develop into a zygote which later transforms into a motile ookinetes (OK). The ookinetes will invade the midgut epithelium through a specific mechanism to reach the basal side where they develop into oocysts (OC). Thousands of sporozoites (S) will develop within the oocysts and disperse throughout the mosquito’s' hemolymph. From there the sporozoites will invade the salivary glands in which they can remain for weeks, prior to transmission to a new host. Salivary gland invasion also appears to be mediated by specific interaction.



 Relationships between An. gambiae immune defenses against different Plasmodium species.
Most studies addressing An. gambiae responses to Plasmodium infection have utilized the rodent Plasmodium berghei model system, which is more amenable to experimental manipulation than is the human parasite Plasmodium falciparum. However, An. gambiae is not the natural vector of the P. berghei parasite that differ significantly from the human pathogen with regard to a variety of biological processes. In order to address potential differences in mosquito defense responses to the two parasite species, and thereby validate the usefulness of the rodent model, we conducted a study were the transcriptional immune responses to infection of the An. gambiae gut was compared between the two parasite species. The results showed remarkably diverse responses to infection with the two parasite species that can be attributed to differences in infection level and in the biology of interaction between the two parasite species and the mosquito. The smaller number of induced putative immune genes upon P. berghei infection may indicate that the mosquito’s immune surveillance system is more capable of sensing P. falciparum, or P. berghei may in some way suppress the mosquito’s immune response, and that could partly explain the significantly higher infection levels of the rodent parasite in An. gambiae. Assays have also been conducted to address the mosquito responses to malaria infected blood in absence of parasite stages that infect mosquito tissues, and showed broad effects on gene regulation, that indicates the extensive qualitative differences between infected and non-infected blood. The capacity to mount an immune response to infected blood, in the absence of Plasmodium invasion of the mosquito gut, is likely beneficial in controlling Plasmodium infection since it would allow for enrichment of anti-Plasmodium factors prior to epithelial invasion. These experiments generated a wealth of information on novel putative anti-Plasmodium factors that can be pursued for functional dissection. This analysis was also extended to the level of gene function for twelve selected immune genes that were assessed for effect on infection with both parasite species through RNAi gene silencing methodology. Seven of these genes were found to influence mosquito resistance to both parasite species while two genes were specifically involved in defense against P. falciparum and another two were more specific for P. berghei. This study showed that the mosquito is utilizing both parasite species-specific and universal defense mechanisms to combat Plasmodium. Our recent studies using gene silencing of the negative regulator of Rel2, Caspar (Kim et al., 2006), which allows the transient stimulation of the IMD pathway, resulted in almost complete refractoriness of three major malaria vectors, A. gambiae, A. stephensi, and A. albimanus to the human P. falciparum (Garver et al., 2009). We have generated transgenic A. stephensi mosquitoes with overexpression of Rel2 in both midgut and fatbody tissues upon bloodmeal which suggests that Rel2-mediated immune responses are more specific in defending against human malaria parasites (see the section of Molecular characterization of the Imd pathway mediated resistance to P. falciparum).
FUTURE DIRECTIONS: The discovery of significant differences between infection responses to the two parasite species have prompted us to exclusively use the human pathogen P. falciparum in our ongoing and future studies. We are now addressing the immune system and anti-Plasmodium responses of natural field mosquitoes and have established collaboration with a research group in Cameroon to initially study the differences in immune gene expression between lab and field mosquitoes, and between different field mosquito populations.

                  

Tripartite interactions between bacteria – mosquito – pathogen, and anti-pathogen defenses.
The mosquito’s immune system is predominantly devoted to combating the bacterial and fungal pathogens that are present in its external environment and intestinal flora, and it was until recently unclear whether defense mechanisms have evolved that are specific for Plasmodium. One can argue that the mosquito’s immune surveillance system and antimicrobial effectors have to cope with a particularly broad spectrum of pathogens, including the Plasmodium parasite, as a result of its complex life style and diverse ecological niches. To address the relationships between the transcriptional immune responses to Plasmodium and bacterial immune challenges, we implemented a study that compared the gene expression in mosquitoes injected with Escherichia coli and Staphylococcus aureus to Plasmodium-infected mosquitoes. This study showed a significant overlap between the mosquito’s response to bacteria and Plasmodium infection and the bacteria infection-responsive transcripts included eight anti-Plasmodium genes. This observation supported the hypothesis that the mosquito is utilizing some of the same immune pathways and mechanisms for defense against these two classes of pathogen. RNAi gene silencing assays were also conducted to address potential overlaps in anti-Plasmodium and antibacterial gene function; this study showed that the genes displaying anti-Plasmodium activity also influenced the mosquito’s resistance to bacterial infection, while several genes with an effect on resistance to bacterial infection could not influence Plasmodium development. These findings suggest the mosquito is mainly employing its antimicrobial defense system in the fight against the malaria parasite. Although certain immune gene allele frequencies have been correlated with Plasmodium exposure in the field, there is little reason to believe that the mosquito would have undergone major adaptations to the malaria parasite and evolved highly specific anti-Plasmodium defense mechanisms.

                 
                                (Mosquito breeding site in the field)

Similarly to humans, the mosquito intestine harbors a natural microbiota which is necessary for maintaining normal physiological functions including host metabolism and immune homeostasis. In order to further address the role of the mosquito’s endogenous bacterial flora in a potential priming of the immune system, we used antibiotic-treatment to generate “bacteria-free” mosquitoes and tested these for permissiveness to Plasmodium infection. P. falciparum infection was 4- to 5-fold higher in bacteria-free compared to septic An. gambiae, suggesting that the bacteria flora of mosquitoes can in some way influence its susceptibility to Plasmodium and hence its vectorial capacity. We found that the mosquito’s natural bacterial flora show great variability between mosquitoes originating from the same colony and that it is an important regulator of mosquito permissiveness to Plasmodium. The mosquito’s natural microbiota and artificially introduced non-natural bacteria negatively affected malaria parasite development through a mechanism that appears to implicate in the innate immune system, and not a direct killing of Plasmodia by the bacteria. The natural bacterial flora is essential in inducing a basal level immunity that in turn enhances the mosquito’s ability in defending against the infection from the malaria parasites (Frolet et al., 2006). We then have undertaken a comprehensive functional genomics approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito’s microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes’ antimicrobial immune responses, plausibly through activation of basal immunity. In sum, the microbiota plays an essential role in modulating the mosquito’s capacity to sustain Plasmodium infection (Dong et al., 2009). An interesting observation here is that, the effect of certain immune genes on Plasmodium infection is dependent on the presence of the microbial flora, suggesting that their mode of action is complex. This finding suggests that future studies on gene specific anti-Plasmodium action should also consider the complex interplay between the microbiota and the mosquito’s immune defenses against the Plasmodium parasite. This relationship is further corroborated by observations from Dr. Barillas-Mury’s group, where RNAi gene silencing of one immune gene facilitated the proliferation of microbial flora but reduced the Plasmodium infection. The natural bacterial flora has also been shown to be involved in the suppression of other pathogenic organisms in other mosquito species (reviewed in Cirimotich et al., 2010). Tetracycline treatment of Culex bitaeniorhynchus rendered this mosquito more susceptible to the Japanese encephalitis virus and the Aedes aegypti mosquito microbial flora has been shown to stimulate a basal-level immunity which suppresses dengue virus infection.
We know from studies in Drosophila that the insect innate immune responses to bacterial infection involve the activation of the intracellular immune signaling pathways Toll and Imd, that are highly conserved throughout insect families. In order to address the implication of these pathways in the defense against P. falciparum an RNAi gene silencing strategy was employed that targeted the negative regulators Cactus and Caspar to activate the Toll and Imd pathways, respectively, upon parasite infection. Both pathways were capable of controlling susceptibility to infection and activation of the Imd pathway had the strongest effect with a ~90% reduction in P. falciparum infection level. This finding further supports the extensive overlaps between the mosquito’s antibacterial and anti-Plasmodium defense systems. We pursued the analysis further to address other immune defense reactions such as melanization, which is a major Plasmodium killing mechanism in refractory mosquitoes. We used a Sephadex bead melanization assay to test the implication of various immune genes in the melanization mechanism. These experiments showed that the RNAi –mediated silencing of the TEP1 and LRIM1 genes, which encode proteins known to mediate Plasmodium killing in malaria-susceptible mosquitoes, significantly compromised the ability of mosquitoes to melanize the beads. In contrast, silencing of two Plasmodium protective c-type lectins, CTL4 and CTLMA2, did not affect bead melanization. These data suggest that the anti-Plasmodium factors have multiple functions, as determinants of both Plasmodium and bacteria killing as well as melanization of the parasite and other foreign bodies, while the Plasmodium protective factors are specifically utilized by the parasite for evasion of mosquito defense mechanisms.
Recently, we have begun to investigate how bacteria in the mosquito midgut may directly impact Plasmodium development. Using bacteria isolated from mosquitoes collected in Zambia, southern Africa, we have supported previous analyses with laboratory bacteria that show Gram-negative bacteria are able to inhibit P. falciparum oocyst formation when bacteria and parasites are introduced in the same blood feed (Pumpuni et al., 1993; 1996; Gonzalez-Ceron et al., 2003). One bacterium in particular had a potent effect on ookinete formation and, through RNAi gene silencing, it was shown that the Imd pathway was not responsible for this inhibition. Further analyses with this bacterium showed that inhibition could occur in culture, suggesting that a mosquito response is not necessary. The exhibited mechanism of inhibition has been linked to the production of anti-Plasmodium molecules by the bacterium. These experiments suggest that the microbiota in the mosquito midgut, along with the immune response of the mosquito mounted against these bacteria, are responsible for protecting Anopheles mosquitoes from Plasmodium infection. It may be possible to drive these or other inhibitory bacteria into wild mosquito populations to increase resistance to malaria parasite infection, and studies to address these ideas are ongoing.
Mosquito – gut bacteria-dengue virus interactions
Similar to the data gathered from the mosquito-bacteria-Plasmodium interactions, we have found strong evidence that the mosquito’s natural microbial flora plays a role in limiting dengue virus infection in the mosquito midgut (Xi et al. 2008). Such experimental assays showed that mosquitoes that were rendered aseptic by way of antibiotic treatment had significantly higher dengue viral loads compared to mosquitoes that have its intact midgut microbial flora (Xi et al. 2008). Furthermore, the Toll and Jak-STAT pathway have been identified as been important pathways in the response against dengue virus infection. Although, an anti-dengue virus effector has yet to be described, quantitative PCR assays looking at the mRNA abundance of Toll pathway downstream effectors show an up-regulation of those genes upon exposure to bacteria via a sugar meal (GD group, unpublished data).
A survey on the microbiota of field-collected Ae. aegypti mosquitoes has yielded a panel of different bacterial isolates belonging to six bacterial phylogenetic classes; with the most dominant being the Gammaproteobacteria, Alphaproteobacteria and Betaproteobacteria. Reintroduction of some of the bacterial isolates into the mosquito resulted in a significant decrease on dengue viral loads compared to mosquitoes that did not harbor these bacteria. The phenotype observed could be the result of a direct effect of the bacteria on dengue virus (through secondary metabolites) or indirect effects (through elicitation of the immune system and/or steric hindrance). Currently we are working at identifying the mechanisms of antiviral activity of these bacterial strains.
FUTURE DIRECTIONS: Our working hypothesis is that the presence of bacteria and fungi in the mosquito primes or activates immune activity against Plasmodium in addition to having a direct effect on parasite development in the mosquito gut. Consequently, the mosquito’s anti-Plasmodium defense system is largely shaped by its microbial exposure in nature, which is continuous, in contrast to the relatively low exposure rate to Plasmodium. Current and future projects aim at the identification of other mechanism(s) and stage(s) at which the presence of bacteria negatively impacts upon the development of malaria parasites. We have initiated the functional characterization of some of the bacteria induced immune genes using RNAi gene silencing and other assays to address their specific role in defense. We will also continue to address the mosquito’s microbial exposure in nature since it is likely to vary significantly between different ecological niches and thereby be responsible for some of the differences in vectorial capacity between different mosquito populations.


Molecular characterization of the hyper-variable pattern recognition receptor AgDscam function in anti-Plasmodium defense
The innate immune system of the mosquito, unlike that of vertebrates, appears to lack the capacity to transiently adapt its pattern recognition repertoire toward the recognition of specific pathogens or to produce immunological memory. Instead, it relies on the limited diversity of its repertoire of ~130 germ line-encoded pattern recognition receptors. Malaria transmission in nature involves a variety of mosquito populations and parasite strains that differ in their genetic makeup. This variability complicates the development of malaria control strategies based on transgenic expression of anti-Plasmodium factors in vector mosquitoes, since their parasite-killing activity may vary depending on parasite strain- and species-specific differences. A broad-spectrum anti-Plasmodium factor would therefore be preferred for use in such control strategies. One of these recently identified anti-Plasmodium factors is the Down syndrome cell adhesion molecule (Dscam). Like its D. melanogaster ortholog, Dscam in Anopheles gambiae (AgDscam) possesses 82 alternatively spliced Ig (immunoglobulin) domain exons, which have the potential for generating 31,920 alternative splice forms with different interaction specificities. We have shown that AgDscam is an essential hypervariable receptor of the Anopheles gambiae immune surveillance system, demonstrating phenotypic plasticity in response to different spectra of pathogen exposure. We have shown that AgDscam produces splice form repertoires that are pathogen challenge-specific in the sense that they are enriched with receptor molecules having increased affinity and defense specificity for the eliciting pathogen. In order to investigate whether AgDscam anti-Plasmodium responses are in a splice-form specific manner, we have used siRNA gene silencing approach to target corresponding isoforms of AgDscam, our results have shown that Pf eliciting isoform has specificity against human malaria parasites, while Pb induced isoforms are more relevant in defending again rodent malaria parasites. To take a step further, we investigated this specificity by generating transgenic A. stephensi mosquitoes which are overexpressing either Pf or Pb specific isoform upon blood meal (carboxypeptidase promoter driven). Interestingly, transgenic mosquitoes overexpressing Pf specific isoform showed significant resistance to human malaria parasites, while no resistance to rodent malaria parasites, vice versa, transgenic mosquitoes with overexpression Pb specific isoform is refractoriness to rodent malaria parasites. Transgenic mosquitoes also showed specificities in defending the microbial proliferation in their midguts. At cellular level, we used confocal microscopy to visualize the co-localization of AgDscam with both P. falciparum and P. berghei which suggests that AgDscam is directly associated with the recognition of the parasites, we then focused on the splice form specificity of this association by in vivo overexpression these specific isoforms throught transgenesis approach. Transgenic mosquitoes with Pf specific isoform has more protein co-localized with the P. falciparum, while the control transgenic mosquitoes showed decreased binding of the protein to the parasites, suggesting that the recognition of AgDscam splicing forms have specificities against different species of parasites. Then we asked whether the major immune pathways are playing roles in the regulation of AgDscam splicing, as a high through-put strategy we used CombiMatrix microarray which have unique probes for each individual exons for the total 101 exons, our results have shown that AgDscam splicing is regulated by the Toll and Imd immune pathways (Dimopoulos group, unpublished data). It has been proposed that for eukaryotes alternative splicing was a very important step towards higher efficiency, because information can be stored much more economically. AgDscam’s broad range of anti-pathogen activity renders it a particularly interesting candidate for the development of anti-Plasmodium control strategies based on transgenic expression in mosquitoes; it has a high probability to display activity against a variety of Plasmodium strains and species, and thereby overcome the constraints of the more “specific” anti-Plasmodium factors in the field where diversity is large.

A schematic diagram of A. gambaie Dscam


Splicing of mRNA is performed by an RNA and protein complex known as the spliceosome and mechanisms of alternative splicing are highly variable. A regulator of Drosophila Dscam’s mutually exclusive splicing, the heterogeneous nuclear ribo-nucleoprotein hrp36, has been shown to act specifically within the exon 6 cassette to prevent the inclusion of multiple exons or multiple exon 6 variants (Olson et al., 2007). We are focusing on identification of alternative splicing regulators of AgDscam and elucidation whether these putative splicing factors are regulated by major immune pathways. To identify the candidate putative splicing factors that control AgDscam alternative splicing, we have blast searched Drosophila putative splicing factors (Mount and Salz, 2000; Park et al., 2004) against A. gambiae full genome and selected a panel of putative splicing factor genes and pre-messenger RNA processing factors for the further analysis. We performed expression analysis with these putative factors in the mosquito Sua5B cell lines which have been challenged with LPS, PGN and heat inactivated bacteria. Splicing factors that had a detectable effect on AgDscam mRNA isoform levels were reanalyzed. We have identified a couple of putative splicing factors which showed the implication of regulation of AgDscam exon 4 alternative splicing. We are now focusing on more thorough studies to see how these splicing factors are regulated by the immune pathways.
FUTURE DIRECTIONS: A better understanding of AgDscam’s anti-Plasmodium activity with regard to its infection-stage specificity, splice form specificity, and immune-related regulation are the first essential steps towards the complete dissection of its biology, which can permit the development of control strategies based on this gene. We have pursued several studies that aim at a detailed understanding of AgDscam function in anti-Plasmodium defense by use RNAi silencing, immunohistochemical staining, microarray analyses, and interaction analyses. We have also conducted the studies focusing on the regulation of AgDscam splicing and identified several splicing factors which play important roles in the AgDscam alternative splcing. We also have tested the anti-Plasmodium defense specificity of AgDscam specific isoform and for the first time we have assessed the efficacy of AgDscam in genetically modified mosquito lines that over-express pathogen specific splice forms. For the future more thoroughly analysis we want to use in vitro recombinant protein approach to investigate the specificity of association between pathogens and pathogen specific splice form repertoires in much greater detail. We also want to elucidate the mechanism of alternative splicing of AgDscam, more specifically, the mechanism of immune pathways in the regulation of AgDscam alternative splicing. These analyses will elucidate one of the most remarkable players of the insect innate immune system, which allows it to cope with a broad spectrum of microbes through the microbe challenge specific production of pattern recognition receptors. The ultimate goal of this study is to develop a malaria control strategy based on the knowledge we gain here.

Molecular characterization of the Imd pathway mediated resistance to P. falciparum
The innate immune system of Anopheles, the malaria vector’s main line of defense against Plasmodium parasites, is engaged at multiple stages of parasite infection. Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Through the microarray based expression analyses, in conjunction with the RNAi-based depletion of the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel1 activation reduced infection levels. RNAi –based depletion of the negative regulator of Rel2, Caspar, which allows the transient stimulation of the IMD pathway, results in almost complete refractoriness of the three major malaria vectors, A. gambiae, A. stephensi, and A. albimanus to the human P. falciparum hints that the Imd pathway is a major player in the defense against P. falciparum (Garver et al., 2009). More interestingly, we could also show that among the most potent anti-Plasmodium immune factors identified to date are TEP1, APL1, LRRD7, and FBN9, all controlled by the IMD pathway through its transcription factor Rel2. The Rel2 gene will through alternative splicing produce a full-length form (Rel2-F) which includes the carboxyl-terminal ANK and death domains, and a shorter form (Rel2-S), lacking these inhibition domains, which can constitutively translocate to nucleus where it actives transcription of anti-Plasmodium effector genes (Meister et al., 2005). Interestingly, transient activation of Rel2 through gene silencing of Caspar resulted in a minimal fitness cost of mosquitoes, in terms of longevity and fecundity at laboratory conditions. These properties of the IMD pathway and its downstream transcription factor Rel2 suggests that, if appropriately manipulated, it could be used for the development of malaria control strategies based on Plasmodium falciparum-resistant genetically modified mosquitoes.
To test this hypothesis we explored this approach through the development of genetically modified super-immune Anopheles mosquitoes that express the active form of the Rel2 (Rel2S) transcription factor under the control of the blood meal-inducible carboxylpeptidase (Cp) or vitellogenin (Vg) promoter, in order to target the malaria parasite at its early stages of development that are associated with the lumen and basal side of the mosquito’s midgut tissue. We generated Cp-Rel2 and Vg-Rel2, and a third hybrid super-immune transgenic line with blood meal-inducible expression of Rel2 in both tissue compartments. These transgenic mosquito lines displayed potent anti-P. falciparum activity and provided a unique opportunity to gain insight into the spatial and temporal specificities of the mosquito’s Rel2-mediated anti-Plasmodium defenses. Longevity and fecundity studies of these super-immune transgenic mosquito lines suggested a modest impact of transgene expression on these fitness determinants. As a proof of principle, we show for the first time that the mosquito's innate immune system can be used in a genetic engineering approach to develop a control strategy for human malaria (Dimopoulos group, unpublished data). .
FUTURE DIRECTIONS: We have shown that a transgenic Rel2-mediated innate immune response fulfills several criteria required for an anti-Plasmodium effector system that could be used for malaria control. First, we have shown that genetically modified super-immune mosquitoes activate multiple anti-Plasmodium factors that are likely to act independently against Plasmodium, thereby decreasing the possibility for the development of resistance by the parasite. Second, Rel2 confers resistance to P. falciparum in three independent and evolutionary divergent malaria vector species, potentially rendering this approach feasible in the 40 different Anopheline vectors of malaria. Third, transient Rel2 activation in the midgut tissue had no significant effect on fitness as measured by longevity or fecundity in laboratory conditions. Fourth, our approach did not involve the introduction of a foreign recombinant gene, but only the enhancement of the mosquito’s innate immune system through overexpression of its own Rel2 transcription factor, thereby decreasing the possibility of unexpected adverse effects relating to expression of a heterologous protein.
For the future more thorough analysis we want to investigate whether this approach will provide protection in the field which is characterized by an enormous genetic diversity of wild Plasmodium populations. We also want to assess the fitness of transgenic mosquitoes in the field conditions. A plausible future scenario could involve the spread of a Rel2 transgene through a powerful genetic drive system that can overcome the fitness cost of transgene expression, thereby providing super-immune properties to existing natural malaria vector populations. This approach has the advantage of being logistically simple and self-propagating as well as environmentally friendly, since it does not eliminate the mosquito from its ecologic niche or involve chemical insecticide or drug treatments.


The Aedes aegypti anti viral defense system
JAK-STAT pathway
The Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway is an evolutionary conserved immune signaling cascade that was originally identified and characterized in mammals because of its ability to mediate cytokine signaling (Schindler et al., 1992; Fu et al., 1992; Shuai et al., 1993). Activation of the canonical D. melanogaster JAK-STAT pathway is initiated by the extracellular binding of the unpaired ligand Upd to the transmembrane receptor Domeless (Dome), a distant homolog of the vertebrate type I cytokine receptors, which will undergo a conformational transformation leading to the self-phosphorylation of the associated JAKs (Hop). The activated Hop will in turn phosphorylate Dome, resulting in the formation of docking sites for the cytoplasmic STATs. The recruitment of STATs by the Dome/Hop complex will induce STATs phosphorylation and dimerisation that ultimately leads to its translocation into the nucleus where it will activate transcription of specific target genes. Two negative regulators, PIAS (protein inhibitor of activated STAT) and SOCS (suppressor of cytokine signaling), have been shown to suppress the JAK-STAT pathway in D. melanogaster.
The participation of the JAK-STAT pathway in anti-viral responses has been demonstrated in both mice and humans with deficient STAT-1. Recent studies have shown implication of the JAK-STAT pathway in the control of DCV infection in D. melanogaster. In A. aegypti, the dengue virus infection-responsive nature of two JAK-STAT pathway genes, dome and SOCS, also suggested the involvement of this pathway in the mosquito’s responses against dengue virus. To investigate this hypothesis, we have employed a combination of reverse genetic and transcriptomic analyses, and found that the RNAi-mediated suppression of the JAK-STAT pathway renders mosquitoes approximately 5-fold more susceptible to dengue virus infection, clearly showing that this pathway is also an important component of the mosquito’s anti-dengue defense (Souza-Neto et al., 2009). To understand how the JAK-STAT pathway transcriptionally orchestrates an anti-dengue response, we analyzed the transcript abundance of PIAS-silenced mosquitoes using whole genome microarrays. Comparative analysis identified 18 genes whose transcription is responsive to dengue and controlled by the JAK-STAT pathway. Further evaluation revealed that the RNAi silencing of at least two of these genes [dengue virus restriction factor (DVRF) 1 and DVRF2] results in mosquitoes more susceptible to dengue virus, suggesting that they are part of the JAK-STAT-mediated mechanism that impairs dengue infection in mosquitoes.

JAK-STAT Genetically Modified mosquito
Previous work from our group has established the JAK-STAT pathway as a key player in the A. aegypti anti-DENV immune response. However, since relatively little is known about the molecular mechanisms by which JAK-STAT acts to control virus replication, we are undertaking a transgenic approach to further characterize the biology of this pathway. The generation of transgenic A. aegypti lines that over-express JAK-STAT pathway components in response to blood-feeding will enable us to study the spatio-temporal pattern of JAK-STAT pathway-mediated DENV resistance, and to functionally test candidate effector genes by RNAi-mediated knockdown in a JAK-STAT-activated background.

Analysis of transcriptome divergence between geographically diverse Aedes aegypti field strains, and correlation with susceptibility to dengue virus infection
Most studies of vector immunity have been performed with laboratory mosquito strains which have been maintained under insectory conditions for decades. As compared to natural mosquito populations, laboratory mosquito strains are exposed to lower doses and a much narrower range of microbes; this together with the genetic bottleneck of a small initial parental population size often results in a loss of genetic variability. In contrast, sequence diversity of immunity-related genes is predicted to be higher in field mosquitoes to allow them to cope with the broad range of microbes that they come into contact with. Although most field studies have focused on genetic polymorphisms, natural and laboratory mosquito populations are also likely to differ in their transcriptomic responses to pathogen infection.
FUTURE DIRECTIONS: Together with collaborators, we have collected a panel of field-derived A. aegypti populations from distinct DENV-endemic geographical locations. We plan to compare the transcriptomes and DENV susceptibilities of these populations with our laboratory strain. These data would not only provide valuable information about immune gene regulation and usage in natural mosquito populations, but may also allow us to identify novel immune genes that control DENV in field mosquitoes.
Aedes aegypti salivary gland transcriptome responses to dengue virus infection

DENV infection of the mosquito salivary gland is essential for vertical pathogen transmission to vertebrates. In addition, mosquito saliva contains many vasodilatory and anti-coagulant compounds that facilitate bloodmeal acquisition. Despite the important role of the salivary gland in pathogen transmission, the anti-DENV immune response in this organ has not been well-characterized. We have undertaken a microarray gene expression approach to characterize this response and to identify candidate genes that modulate virus replication in this organ.
FUTURE DIRECTIONS: We have identified two categories of gene candidates that have possible roles in DENV transmission: genes that could directly modulate virus replication in the salivary gland, and genes that potentially affect bloodmeal acquisition by regulating mosquito host-seeking or probing behavior. These candidates are now being evaluated through RNAi-mediated gene knockdown assays. We hope that this transcriptomic and functional analysis of DENV infection in the salivary gland will further our understanding of the host-pathogen interaction in this organ.

The molecular biology of Dengue virus – mosquito interaction is largely unknown. We have made use of our A. aegypti genome microarrays in conjunction with other functional genomics tools to study the mosquito’s responses to infection with the Dengue virus. A comparison of gene expression between non-infected and Dengue (DENV-2) virus infected mosquitoes, at the stage when the virus spreads from the mosquito gut to other tissues, identified 240 up-regulated and 192 down-regulated genes in the non-gut tissues and 28 up-regulated and 35 down-regulated genes in the gut tissue. A significant proportion of these genes have predicted functions in the mosquito’s innate immune system with a particular bias towards components of the Toll immune signaling pathway. The negative regulator Cactus was down-regulated while several pattern recognition receptors, signaling modulation and transduction factors and effector molecules were up-regulated. This suggested implication of the Toll pathway in the anti-Dengue defense. To address this hypothesis at the functional level, we tested the impact of Toll pathway activation, through RNAi silencing of the Cactus gene, on virus infection in the gut according to Frolet et al., 2006. This lead to an approximately four-fold decrease in infection level compare to the GFP dsRNA treated mosquitoes. Hence, the A. aegypti Toll pathway appears to play a major role in defending the mosquito against Dengue.

Hemocyte responses to dengue virus infection.
Hemocytes are important immune effector cells that are thought to participate in pattern recognition, to mediate fat body production of immune peptides; and are considered the main immune factor that determines the systemic immune response in the mosquito.
To assess the effects of dengue virus infection on hemocytes, a microarray assay was conducted at 7d post-infection from the hemolymph collected from infected mosquitoes. The microarray analysis shows a differentially expressed set of genes belonging to several functional classes. Interestingly, several immune-related genes were down-regulated, indicating a modulation of the hemocyte transcriptome by dengue virus.
MD2-like proteins in dengue virus infection
As mention above, TLRs have been suggested to be involved in dengue virus infection in mosquito. TLRs are important PRRs able to recognize wide range of PAMPs. The diversity of pathogen recognition assists by various co-receptors. MD2 is a co-receptor assist TLR4 recognizing LPS in vertebrate system. Our microarray data revealed up-regulation of MD2-like proteins upon dengue virus infection; however, the function(s) and molecular mechanisms of these MD2-like proteins have not been elucidated. The role of these proteins on dengue virus infection is being investigated by using the RNAi-mediated gene knock-down and the knowledge might help better understanding in mosquito-virus interactions. FUTURE DIRECTIONS: Current and future experiments focus on the dissection of this anti-Dengue immune response through gene silencing of other genes that show regulation upon infection and a determination of the temporal and spatial specificity of virus clearance in the mosquito. Insect immunity to viruses is poorly described and these studies will therefore contribute significantly to the field and also provide potential tools for the development of dengue control strategies.


Molecular analysis of photic inhibition of blood-feeding in Anopheles gambiae
Anopheles gambiae mosquitoes exhibit an endophilic, nocturnal blood feeding behavior. Despite the importance of light as a regulator of malaria transmission, our knowledge on the molecular interactions between environmental cues, the circadian oscillators and the host seeking and feeding systems of the Anopheles mosquitoes is limited. We have shown that the blood feeding behavior of mosquitoes is under circadian control and can be modulated by light pulses, both in a clock dependent and in an independent manner. Short light pulses (~2-5 min) in the dark phase can inhibit the blood-feeding propensity of mosquitoes momentarily in a clock independent manner, while longer durations of light stimulation (~1-2 h) can induce a phase advance in blood-feeding propensity in a clock dependent manner. The temporary feeding inhibition after short light pulses may reflect a masking effect of light, an unknown mechanism which is known to superimpose on the true circadian regulation. Nonetheless, the shorter light pulses resulted in the differential regulation of a variety of genes including those implicated in the circadian control, suggesting that light induced masking effects also involve clock components. Light pulses (both short and long) also regulated genes implicated in feeding as well as different physiological processes like metabolism, transport, immunity and protease digestions. RNAi-mediated gene silencing assays of the light pulse regulated circadian factors timeless, cryptochrome and three takeout homologues significantly up-regulated the mosquito's blood-feeding propensity. In contrast, gene silencing of light pulse regulated olfactory factors down-regulated the mosquito's propensity to feed on blood. Our study show that the mosquito’s feeding behavior is under circadian control. Long and short light pulses can induce inhibition of blood-feeding through circadian and unknown mechanisms, respectively, that involve the chemosensory system.
FUTURE DIRECTIONS: We are interested in gaining a better understanding of how environmental cues can influence mosquito behavioral attributes that are important for disease transmission.


COLLABORATIONS: Dr. Roberto Barrera (CDC, Puerto Rico), Dr. Bruce Christensen (University of Wisconsin), Dr. George Christophides (Imperial College London), Dr. Mariano Garcia-Blanco (Duke University), Dr. Marc Muskavitch (Boston College), Dr. Clara Ocampo (CIDEIM, Cali, Colombia), Dr. Pedro Oliviera (Universidade Federal do Rio de Janeiro, Brasil), Dr. Alexander Raikhel (University of California, Riverside), Dr. Claudia Romero (Universidad del Norte, Barranquilla, Colombia), Dr. Mike Strand (University of Georgia), Dr. Marcos Sorgine (Universidade Federal do Rio de Janeiro, Brasil), Dr. Giuliano Gasperi and Dr. Ludvik Gumulski (University of Pavia, Italy), Dr. Juan Pascale (Gorgas Institute, Panama). Dr. Andy Waters (University of Glasgow, UK)



FUNDING: The group and its members have received funding from: National Institutes of Health, National Science Foundation, Ellison Medical Foundation, American Society of Microbiology, A STAR, Johns Hopkins School of Public Health, Lang Family, Bloomberg Family Foundation.

 

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