Arthropod saliva in vector-borne disease transmission: Functional analysis of novel domains in salivary secretions

National Institute Of Allergy And Infectious Diseases

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Abstract

The main accomplishments of the Molecular Entomology Unit are: 1. We described the function of sialokinin, a salivary peptide of the Yellow Fever mosquito, Aedes aegypti. Sialokinin modulates macrophages, alters endothelial permeability, promotes leukocyte recruitment, induces nitric oxide release, and interferes with hemostasis via NK1R signaling., promoting blood perfusion at the bite site. We generated sialokinin knocked out mosquitoes using CRISPR/Cas9 technology. Mosquitoes lacking sialokinin have longer probing times, and reduced ability to recruit leukocytes and activate macrophages, which has potential consequences on pathogen transmission. These findings demonstrate the antihemostatic and immunomodulator role of sialokinin in vivo. 2. Characterization of Anopheles gambiae anti-hemostatic proteins. We performed a comprehensive biochemical and functional study to uncover the binding capabilities of 3 members of long form D7 (AngaD7L1, 2, and 3) family of secreted proteins. We assessed the physiological functions of these proteins in vivo and ex vivo, and demonstrated their relevance in mosquito blood feeding. We showed that AngaD7L2 has anti-coagulant activity while AngaD7L3 exclusively binds serotonin (5HT), a mediator of platelet aggregation and vasoconstriction. These novel activities had never been found before in Anopheline D7 long forms. We showed how these salivary proteins evolved novel and distinct functions to modulate the vascular biology of vertebrate hosts, and show strong anti-hemostatic (platelet aggregation, vasoconstriction, and coagulation) functions that likely facilitate blood-feeding. This work highlights the complex yet highly specific biological activities of a family of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding. 3. Aedes aegypti salivary proteins bind directly to the envelope protein of Zika virus (ZIKV). We evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance, and found that three salivary proteins (AAEL000793, AAEL007420, and AAEL006347) bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. However, these interactions have no effect on viral replication in cultured endothelial cells and keratinocyte. We also found that mosquito salivary proteins interacting with ZIKV are immunogenic in patients infected with ZIKV and DENV. These results suggest that mosquitoes release viral particles coated with salivary gland proteins into the hosts skin. We also found that antibodies from patients infected with DENV and ZIKV can recognize the virussalivary protein complex. This work offers valuable insight into the possible interactions of the virus with mosquito salivary proteins. 4. An update insight into the sialotranscriptome Ochlerotatus (Aedes) triseriatus, the main vector of La Crosse virus. We revisited O. triseriatus salivary gland contents using an Illumina-based sequencing approach of both male and female tissues. This work also represents the first salivary transcriptome of male O. triseriatus mosquitoes. We reported 10,317 DNA coding sequences, classified into several functional classes. The translated transcripts also served as a reference database for proteomic analysis of O. triseriatus female saliva, in which unique peptides from 101 proteins were found. Finally, comparison of male and female libraries allowed for the identification of female-enriched transcripts that are potentially related to blood acquisition and virus transmission. The updated sialotranscriptome study reported here provided novel data and extended our knowledge of the salivary repertoire of this mosquito. The data presented here may serve as reference for further studies in the absence of an O. triseriatus genome.