Identification of antimicrobial activities in the saliva of the malaria mosquito Anopheles gambiae.

Introduction. Female mosquitoes feed on blood to acquire nutrients to develop eggs. Both male and female mosquitoes need plant sugar to survive, to fly and to enhance reproduction. To obtain blood, hematophagous insects necessarily have to break their host’s skin, which is a barrier to pathogen infection: skin surface microbes might contaminate host circulation, and therefore the ingested blood meal. On the other hand, sugar sources are similarly often contaminated by several microbial pathogens naturally living on nectar and honeydew. Perhaps for this reason, a common finding in sialotranscriptomes of blood feeding arthropods is an overabundance of proteins known to have antimicrobial activity and a large number of still unknown antimicrobial families. The aim of the work is the identification and characterization of novel antimicrobial activities in the salivary glands of the malaria vector Anopheles gambiae. Materials and methods. To express recombinant proteins in a eukaryotic system, we have employed an in vitro wheat germ transcription/translation procedure. cDNAs encoding for 16 putative antimicrobial peptides were cloned in an expression vector designed to add an His-tag at the C-terminus of the recombinant proteins. To verify the correct expression and abundance of recombinant proteins (range of protein concentrations between 5 to 20 micromolar), Western blot and Coomassie Staining (or Silver Staining) were performed. Bacterial growth inhibition assays were performed using a 96-well plate platform and a microplate reader to monitor bacterial growth in the presence of recombinant peptides. Quantitative Real time RT-PCR was also performed to investigate the response of candidate genes to immune challenge triggered by sugar meals infected with Gram positive, Gram negative bacteria and yeast. Results. From the An. gambiae sialome, we have compiled a short sub-catalogue of putative antimicrobials salivary candidates (16 genes). Selection criteria were the following: i) sequence features (length, biochemical properties, similarities with members of antimicrobials families) and ii) expression profiles. Three categories of genes were indeed considered: i) genes expressed specifically or principally in the females glands (possibly involved in the defence against pathogens on human skin); ii) genes expressed in both sexes (encoding for factors possibly playing a protective role during sugar feeding) and iii) genes expressed in the salivary glands and in other organs. We have successfully synthesized fourteen recombinant proteins using the in vitro wheat germ transcription/translation system. Preliminary results indicate that some of the recombinant proteins exert antimicrobial activities as observed by bacterial growth inhibition assays. Moreover, transcriptional modulation was evidenced for some of the candidates upon bacterial challenge. Conclusions. Preliminary results of this work show that antimicrobials activities are encoded by short peptides expressed in the mosquito salivary glands. Even though most of these peptides do not show any sequence similarity with proteins in databases, novel antimicrobial features were revealed and further studies are required to confirm our data and to understand likely ways of action.