Vladimir Kokoza

Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity

Elucidation of molecular mechanisms underlying stage- and tissue-specific expression of genes activated by a blood meal is of great importance for current efforts directed towards utilizing molecular genetics to develop novel strategies of mosquito and pathogen control. Regulatory regions of such genes can be used to express anti-pathogen effector molecules in engineered vectors in a precise temporal and spatial manner, designed to maximally affect a pathogen. The fat body is a particularly important target for engineering anti-pathogen properties because in insects, it is a potent secretory tissue releasing its products to the hemolymph, an environment or a crossroad for most pathogens. Recently, we have provided proof of this concept by engineering stable transformant lines of Aedes aegypti mosquito, in which the regulatory region A. aegypti vitellogenin (Vg) gene activates high-level fat body-specific expression of a potent anti-bacterial factor, defensin, in response to a blood meal. Further study of the Vg gene utilizing Drosophila and Aedes transformation identified cis-regulatory sites responsible for state- and fat body-specific activation of this gene via a blood-meal-triggered cascade. These analyses revealed three regulatory regions in the 2.1-kb upstream portion of the Vg gene. The proximal region, containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh, is required for the correct tissue- and stage-specific expression at a low level. The median region, carrying sites for early ecdysone response factors E74 and E75, is responsible for a stage-specific hormonal enhancement of the Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic to the Vg gene. Furthermore, our study showed that several transcription factors involved in controlling the Vg gene expression, are themselves targets of the blood meal-mediated regulatory cascade, thus greatly amplifying the effect of this cascade on the Vg gene. This research serves as the foundation for the future design of mosquito-specific expression cassettes with predicted stage- and tissue specificity at the desired levels of transgene expression.

Transcriptional regulation of the mosquito vitellogenin gene via a blood meal-triggered cascade

In anautogenous mosquitoes, a blood meal is required for activation of genes encoding yolk protein precursors (YPP). Vitellogenin (Vg), the major YPP gene, is transcribed at a very high level following blood meal activation. It is expressed exclusively in the female fat body, the tissue producing most of mosquito hemolymph and immune proteins. In this paper, we analyzed the upstream region of the Aedes aegypti Vg gene in order to identify regulatory elements responsible for its unique expression pattern. To achieve this goal, we analyzed the gene using transgenic Drosophila and Aedes as well as DNA-binding assays. These analyses revealed three regulatory regions in the 2.1 kb upstream portion of the Vg gene. The proximal region containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh is required for the correct tissue- and stage-specific expression at a low level. The median region carrying sites for early ecdysone response factors E74 and E75 is responsible for hormonal enhancement of Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic of the Vg gene. The present work elucidates the molecular basis of blood meal-dependent expression of this mosquito gene, laying the foundation for mosquito-specific expression cassettes with predictable stage and tissue specificity.

Pathogenomics of Culex quinquefasciatus and Meta-Analysis of Infection Responses to Diverse Pathogens

Closing the Vector Circle The genome sequence of Culex quinquefasciatus offers a representative of the third major genus of mosquito disease vectors for comparative analysis. In a major international effort, Arensburger et al. (p. 86) uncovered divergences in the C. quinquefasciatus genome compared with the representatives of the other two genera Aedes aegypti and Anopheles gambiae. The main difference noted is the expansion of numbers of genes, particularly for immunity, oxidoreductive functions, and digestive enzymes, which may reflect specific aspects of the Culex life cycle. Bartholomay et al. (p. 88) explored infection-response genes in Culex in more depth and uncovered 500 immune response-related genes, similar to the numbers seen in Aedes, but fewer than seen in Anopheles or the fruit fly Drosophila melanogaster. The higher numbers of genes were attributed partly to expansions in those encoding serpins, C-type lectins, and fibrinogen-related proteins, consistent with greater immune surveillance and associated signaling needed to monitor the dangers of breeding in polluted, urbanized environments. Transcriptome analysis confirmed that inoculation with unfamiliar bacteria prompted strong immune responses in Culex. The worm and virus pathogens that the mosquitoes transmit naturally provoked little immune activation, however, suggesting that tolerance has evolved to any damage caused by replication of the pathogens in the insects. The genome of a third mosquito species reveals distinctions related to vector capacities and habitat preferences. The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.

Blocking of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes

To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and antiPlasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum, the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with antiPlasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite.

REL1, a Homologue of Drosophila Dorsal, Regulates Toll Antifungal Immune Pathway in the Female Mosquito Aedes aegypti

Signaling by Drosophila Toll pathway activates two Rel/NF-κB transcription factors, Dorsal (Dl) and Dorsal-related immune factor (Dif). Dl plays a central role in the establishment of dorsoventral polarity during early embryogenesis, whereas Dif mediates the Toll receptor-dependent antifungal immune response in adult Drosophila. The absence of a Dif ortholog in mosquito genomes suggests that Dl may play its functional role in the mosquito Toll-mediated innate immune responses. We have cloned and molecularly characterized the gene homologous to Drosophila Dl and to Anopheles gambiae REL1 (Gambif1) from the yellow fever mosquito Aedes aegypti, named AaREL1. AaREL1 alternative transcripts encode two isoforms, AaREL1-A and AaREL1-B. Both transcripts are enriched during embryogenesis and are inducible by septic injury in larval and female mosquitoes. AaREL1 and AaREL2 (Aedes Relish) selectively bind to different κB motifs from insect immune gene promoters. Ectopic expression of AaREL1-A in both Drosophila mbn-2 cells and transgenic flies specifically activates Drosomycin and results in increased resistance against the fungus Beauveria bassiana. AaREL1-B acted cooperatively with AaREL1-A to enhance the immune gene activation in Aag-2 cells. The RNA interference knock-outs revealed that AaREL1 affected the expression of Aedes homologue of Drosophila Serpin-27A and mediated specific antifungal immune response against B. bassiana. These results indicate that the homologue of Dl, but not that of Dif, is a key regulator of the Toll antifungal immune pathway in A. aegypti female mosquitoes.

Distinct Melanization Pathways in the Mosquito Aedes aegypti

Serine protease cascades are involved in blood coagulation and immunity. In arthropods, they regulate melanization, which plays an important role in immune defense and wound healing. However, the mechanisms underlying melanization pathways are not completely characterized. We found that in the mosquito Aedes aegypti, there are two distinct melanization activation pathways carried out by different modules of serine proteases and their specific inhibitors serpins. Immune melanization proteases (IMP-1 and IMP-2) and Serpin-1 mediate hemolymph prophenoloxidase cleavage and immune response against the malaria parasite. Tissue melanization, exemplified by the formation of melanotic tumors, is controlled by tissue melanization protease (CLIPB8), IMP-1, and Serpin-2. In addition, serine proteases CLIPB5 and CLIPB29 are involved in activation of Toll pathway by fungal infection or by infection-independent manner, respectively. Serpin-2 is implicated in the latter activation of Toll pathway. This study revealed the complexity underlying melanization and Toll pathway in mosquitoes.

Relish-mediated immune deficiency in the transgenic mosquito Aedes aegypti

The lack of genetic means has been a serious limitation in studying mosquito immunity. We generated Relish-mediated immune deficiency (RMID) by transforming Aedes aegypti with the ΔRel transgene driven by the vitellogenin (Vg) promoter using the pBac[3xP3-EGFP, afm] vector. A stable transformed line had a single copy of the Vg-ΔRel transgene. The Vg-ΔRel transgene expression was highly activated by blood feeding, and transgenic mosquitoes were extremely susceptible to the infection by Gram-negative bacteria. This RMID phenotype was characterized by severely reduced postinfection levels of antimicrobial peptides genes, defensin and cecropin. Crossing the RMID line with the wild-type strain produced the same RMID phenotype, indicating its dominant nature, whereas crossing with the Vg-def transgenic line, in which Defensin A was activated by blood feeding, restored the immunity to Enterobacter cloacae.

The Role of NF-κB Factor REL2 in the Aedes aegypti Immune Response

Mosquitoes transmit numerous diseases that continue to be an enormous burden on public health worldwide. Transgenic mosquitoes impervious to vector-borne pathogens, in concert with vector control and drug and vaccine development, comprise an arsenal of means anticipated to defeat mosquito-spread diseases in the future. Mosquito transgenesis allows tissue-specific manipulation of their major immune pathways and enhances the ability to study mosquito-pathogen interactions. Here, we report the generation of two independent transgenic strains of Aedes aegypti overexpressing the NF-?B transcriptional factor REL2, a homologue of Drosophila Relish, which is shown to be under the control of the vitellogenin promoter in the mosquito fat body after a blood meal. We show that this REL2 overexpression in the fat body results in transcriptional activation of Defensins A, C, and D, and Cecropins A and N, as well as translation and secretion of Defensin A protein into the hemolymph. We also demonstrate that induction of REL2 results in the increased resistance of the mosquito to tested Gram-negative and Gram-positive bacteria. Importantly, induction of transgenic REL2 leads to the significant decrease in susceptibility of A. aegypti to Plasmodium gallinaceum infection. Consistently, RNAi knockdown of REL2 in wild-type mosquitoes results in a delay in Defensin A and Cecropin A expression in response to infection and in increased susceptibility to both bacteria and P. gallinaceum. Moreover, our transgenic assays demonstrate that the N-terminus of the mosquito REL2, which includes the His/Gln-rich and serine-rich regions, plays a role in its transactivation properties.

Characterization of three alternatively spliced isoforms of the Rel/NF-κB transcription factor Relish from the mosquito Aedes aegypti

The Rel/NF-κB transcription factor Relish performs a central role in the acute-phase response to microbial challenge by activating immune antibacterial peptides. We cloned and molecularly characterized the gene homologous to Drosophila Relish from the mosquito Aedes aegypti. Unlike Drosophila Relish, Aedes Relish has three alternatively spliced transcripts encoding different proteins. First, the predominant Aedes Relish transcript of 3.9 kb contains both the Rel-homology domains and the inhibitor κB (IκB)-like domain, which is similar to Drosophila Relish and to the mammalian p105 and p100 Rel/NF-κB transcription factors. Second, Aedes Relish transcript contains Rel-homology domains identical to those of the major transcript but it completely lacks the IκB-like domain-coding region, which has been replaced by a unique 3′-untranslated region sequence. In the third transcript, a deletion replaces most of the N-terminal sequence and Rel-homology domains; however, the IκB-like domain is intact. All three Aedes Relish transcripts were induced by bacterial injection but not by blood feeding. In vitro-translated protein from the Rel-only construct specifically binds to the κB motif from Drosophila cecropin A1 and Aedes defensin genes. PCR and Southern blot hybridization analyses show that these three transcripts originated from the same large inducible mRNA encoded by a single Relish gene.

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.