Discovery of novel Antimicrobial Peptides in the salivary glands of the malaria mosquito Anopheles gambiae

INTRODUCTION. Mosquito females need a blood meal to attain nutrients for egg development. A successful blood meal relies mainly on the activity of anti-haemostatic factors in the mosquito saliva, that is also the carrier of several pathogens transmitted by mosquitoes such as Plasmodium. Mosquitoes initially probe under the host skin and salivate, searching for a blood vessel to pierce and to start sucking the blood. This process exposes mosquito mouthparts to microbes on host skin surface that might contaminate the ingested blood meal. Moreover, both male and female mosquitoes need plant sugar to survive, to fly and to enhance reproduction: these food sources are stored for several hours in the crop and could be similarly contaminated by microbes living on nectar and honeydew. Salivary gland transcriptomes of haematophagous arthropods proved the occurrence of salivary peptides with unknown function showing possible antimicrobial sequence features. Aim of this work is the identification of novel antimicrobial peptides (AMPs) in the salivary glands of the malaria vector An. gambiae. MATERIALS AND METHODS. From salivary transcriptome we selected a list of 16 putative AMPs, based on sequence properties and expression profiles (enrichment in female glands or female/male glands). Recombinant peptides were obtained by chemical synthesis (feasible for 4 protein < 60 aminoacids) or using a cell-free in vitro transcription/translation system (successful for 10 out of 12 peptides) and employed in bacteria growth inhibition assays. Transcriptional regulation was also investigated by RTqPCR, challenging mosquitoes with Gram - (Escherichia coli) and Gram + (Staphylococcus aureus) bacteria. Systemic infection was induced by bacteria injection in thoraxes, while local infection was stimulated by infected sugar meals. RESULTS AND CONCLUSIONS. Functional studies were performed employing in vitro growth inhibition assays and revealed antimicrobial activity for some of the recombinant peptides. Indeed, preliminary data indicate that hyp15 inhibits the growth of E. coli bacteria, while hyp10, hyp6.3 and hyp14.5 inhibit the growth of S. aureus and, finally, hyp6.2 and hyp12 reduce the growth of both strain. Transcriptional profiles of candidate genes showed a balance between systemic and local innate immune responses. Indeed, 4 genes (hyp14.5, hyp14.5-1, hyp55.3 and hyp15) were induced by bacteria injection, while 4 genes (hyp10, hyp12, sg2 and sg2a) resulted activated by bacteria feeding. Overall, this study describes the identification of novel antimicrobial activities in the salivary glands of the malaria mosquito An. gambiae.