Jayme A. Souza-Neto

Discovery of Insect and Human Dengue Virus Host Factors

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world’s population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1–4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and α-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense

Here, we show that the major mosquito vector for dengue virus uses the JAK-STAT pathway to control virus infection. Dengue virus infection in Aedes aegypti mosquitoes activates the JAK-STAT immune signaling pathway. The mosquitos susceptibility to dengue virus infection increases when the JAK-STAT pathway is suppressed through RNAi depletion of its receptor Domeless (Dome) and the Janus kinase (Hop), whereas mosquitoes become more resistant to the virus when the negative regulator of the JAK-STAT pathway, PIAS, is silenced. The JAK-STAT pathway exerts its anti-dengue activity presumably through one or several STAT-regulated effectors. We have identified, and partially characterized, two JAK-STAT pathway-regulated and infection-responsive dengue virus restriction factors (DVRFs) that contain putative STAT-binding sites in their promoter regions. Our data suggest that the JAK-STAT pathway is part of the A. aegypti mosquitos anti-dengue defense and may act independently of the Toll pathway and the RNAi-mediated antiviral defenses.

Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae.

Insect midgut-dwelling bacteria generate reactive oxygen species that inhibit malaria parasite development. Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development before invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

Reciprocal Tripartite Interactions between the Aedes aegypti Midgut Microbiota, Innate Immune System and Dengue Virus Influences Vector Competence

Dengue virus is one of the most important arboviral pathogens and the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It is transmitted between humans by the mosquitoes Aedes aegypti and Aedes albopictus, and at least 2.5 billion people are at daily risk of infection. During their lifecycle, mosquitoes are exposed to a variety of microbes, some of which are needed for their successful development into adulthood. However, recent studies have suggested that the adult mosquitos midgut microflora is critical in influencing the transmission of human pathogens. In this study we assessed the reciprocal interactions between the mosquitos midgut microbiota and dengue virus infection that are, to a large extent, mediated by the mosquitos innate immune system. We observed a marked decrease in susceptibility to dengue virus infection when mosquitoes harbored certain field-derived bacterial isolates in their midgut. Transcript abundance analysis of selected antimicrobial peptide genes suggested that the mosquitos microbiota elicits a basal immune activity that appears to act against dengue virus infection. Conversely, the elicitation of the mosquito immune response by dengue virus infection itself influences the microbial load of the mosquito midgut. In sum, we show that the mosquitos microbiota influences dengue virus infection of the mosquito, which in turn activates its antibacterial responses.

Engineered anopheles immunity to Plasmodium infection.

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquitos innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control.

Anopheles Imd pathway factors and effectors in infection intensity-dependent anti-Plasmodium action.

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasites ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.

Transcriptome Analysis of Aedes aegypti Transgenic Mosquitoes with Altered Immunity

The mosquito immune system is involved in pathogen-elicited defense responses. The NF-κB factors REL1 and REL2 are downstream transcription activators of Toll and IMD immune pathways, respectively. We have used genome-wide microarray analyses to characterize fat-body-specific gene transcript repertoires activated by either REL1 or REL2 in two transgenic strains of the mosquito Aedes aegypti. Vitellogenin gene promoter was used in each transgenic strain to ectopically express either REL1 (REL1+) or REL2 (REL2+) in a sex, tissue, and stage specific manner. There was a significant change in the transcript abundance of 297 (79 up- and 218 down-regulated) and 299 (123 up- and 176 down-regulated) genes in fat bodies of REL1+ and REL2+, respectively. Over half of the induced genes had predicted functions in immunity, and a large group of these was co-regulated by REL1 and REL2. By generating a hybrid transgenic strain, which ectopically expresses both REL1 and REL2, we have shown a synergistic action of these NF-κB factors in activating immune genes. The REL1+ immune transcriptome showed a significant overlap with that of cactus (RNAi)-depleted mosquitoes (50%). In contrast, the REL2+ -regulated transcriptome differed from the relatively small group of gene transcripts regulated by RNAi depletion of a putative inhibitor of the IMD pathway, caspar (35 up- and 140 down-regulated), suggesting that caspar contributes to regulation of a subset of IMD-pathway controlled genes. Infections of the wild type Ae. aegypti with Plasmodium gallinaceum elicited the transcription of a distinct subset of immune genes (76 up- and 25 down-regulated) relative to that observed in REL1+ and REL2+ mosquitoes. Considerable overlap was observed between the fat body transcriptome of Plasmodium-infected mosquitoes and that of mosquitoes with transiently depleted PIAS, an inhibitor of the JAK-STAT pathway. PIAS gene silencing reduced Plasmodium proliferation in Ae. aegypti, indicating the involvement of the JAK-STAT pathway in anti-Plasmodium defense in this infection model.

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.

Heparin-binding proteins of seminal plasma in Nellore bulls

The aim of this study was to identify heparin-binding proteins (HBPs) in seminal plasma of Nellore (Bos taurus indicus) bulls. Bulls (n=4), 30-36 months old, 500-550kg with satisfactory seminal quality were selected. After the centrifugation, samples of the seminal plasma were pooled and the HBPs were isolated by heparin-affinity chromatography. The recovered HBPs fractions were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) 12.5% was performed in vertical minigels. Eight bands with molecular weights ranging from 15 to 63kDa were observed. Two proteins were identified (22 and 25kDa), similar to those previously described in Bos taurus taurus bulls. Other bands identified in this study (39, 53, 58 and 63kDa) have not been previously observed and possibly they are specific to Nellore semen.

An Anopheles aquasalis GATA factor Serpent is required for immunity against Plasmodium and bacteria

Innate immunity is an ancient and conserved defense system that provides an early effective response against invaders. Many immune genes of Anopheles mosquitoes have been implicated in defense against a variety of pathogens, including plasmodia. Nevertheless, only recent work identified some immune genes of Anopheles aquasalis mosquitoes upon P. vivax infection. Among these was a GATA transcription factor gene, which is described here. This is an ortholog of GATA factor Serpent genes described in Drosophila melanogaster and Anopheles gambiae. Gene expression analyses showed an increase of GATA-Serpent mRNA in P. vivax-infected A. aquasalis and functional RNAi experiments identified this transcription factor as an important immune gene of A. aquasalis against both bacteria and P. vivax. Besides, we were able to identify an effect of GATA-Serpent knockdown on A. aquasalis hemocyte proliferation and differentiation. These findings expand our understanding of the poorly studied A. aquasalis-P. vivax interactions and uncover GATA-Serpent as a key player of the mosquito innate immune response.