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 accomplishments of the Molecular Entomology Unit are: 1. Characterization of Ae. aegypti and Ae. albopictus anti-hemostatic and anti-inflammatory proteins. Ae. aegypti and Ae. albopictus mosquitoes are vectors of several arboviruses such as dengue, Zika and yellow fever that cause extensive human morbidity and mortality. We performed a comprehensive comparative biochemical and structural study to uncover the binding capabilities of Ae. aegypti D7L2 (AeD7L2) and Ae. albopictus D7L1 (AlboD7) and their relevance in mosquito blood feeding. We assessed the physiological functions of both salivary proteins in vivo and ex vivo. The physiological function(s) of both proteins was demonstrated by ex vivo platelet aggregation and in vivo leukocyte recruitment experiments. Pressure myography experiments showed AeD7L2 reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U46619, the stable analog of thromboxane A2. Our work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as yet another example of the sophisticated biology underlying arthropod blood feeding. 2. We solved the native crystal structures of two major salivary proteins (CqDVP-2 and CqDVP-4) from Cx. quinquefasciatus, an important, yet understudied disease vector. We showed that both structures have a -trefoil fold, a structural motif shared with the B-chain of the Ricin toxin, which is the portion of the toxin complex that binds to carbohydrates, including galactose, within the plasma membrane of host cells. While the physiological functions of the CqDVP proteins remain unknown, we have presented evidence that both CqDVP proteins bind to carbohydrates. These findings suggest that CqDVP proteins modulate host immune responses to mosquito bites, likely to facilitate blood meal acquisition. Through interactions with carbohydrates present in the plasma membranes of mast cells or other immune cells, these proteins may limit host inflammation while a mosquito feeds, which would reduce itch or other disturbing sensations and increase the mosquitos odds of survival. 3. We showed that SGS1, an Ae. aegypti salivary protein, is essential for Plasmodium gallinaceum infection of the mosquito midgut and salivary glands. RNA interference and CRISPR/Cas9 were used to evaluate the role of Ae. aegypti SGS1 in the P. gallinaceum life cycle. We found that SGS1 facilitates, but is not essential for, invasion of Ae. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could therefore be targeted in the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite, in a strategy to decrease malaria transmission. 4. We proposed the use of salivary gland proteins as biomarkers for malaria risk in South America. We detected a positive correlation among the antibodies against an An. darlingi salivary gland apyrase and antibodies against P. vivax and P. falciparum antigens in patients infected with malaria. Individuals with high IgG levels against the An. darlingi salivary apyrase were five times more likely to have malaria infection than uninfected persons. This is the first study describing salivary peptides of An. darlingi, the main vector of malaria in South America, as a potential tool to evaluate immune responses against mosquito saliva. With this work, we aim to improve estimation of malaria transmission intensity in South America. This study provides strong evidence to use salivary proteins as a tool for monitoring the human-mosquito exposure and biomarkers for malaria risk. 5. An insight into the sialotranscriptome and virome of Amazonian anopheline mosquitoes. The sialotranscriptome study reported here provided novel data on four New World anopheline species (An. braziliensis, An. marajorara, An. nuneztovari and An. triannulatus) and extended our knowledge of the salivary repertoire of An. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard with future RNAseq studies. A total of 60,016 sequences were submitted to GenBank, which will help in the discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure in South America. Additionally, we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of An. triannulatus, An. marajorara and An. darlingi species.