Alberto Rascon

Proteases play essential roles in many important biological processes. Specifically, in the Aedes aegypti mosquito, proteases are important in the midgut (for blood meal protein digestion), the salivary gland (possible enhancement in viral infectivity and dissemination), and for egg/embryo survival (eggshell formation and melanization). However, the exact roles of Ae. aegypti proteases in these processes are either limited or still relatively unknown, which reveal vulnerabilities that can be exploited to attenuate transmission of human viral pathogens. Full understanding of protease function is needed for the development of novel vector control strategies and to better understand viral pathogen/host protease interactions. The work in the Rascón lab lies at the interface of both Chemistry and mosquito Biology, especially in the development of small molecule mosquito-specific inhibitors.

1) Determine the Function of ‘Orphan’ Midgut Proteases in the Blood Meal Digestion Process. At SJSU, the initial focus was on the four most abundant midgut proteases, which revealed the importance of three late phase proteases on fecundity (AaSPVI, AaSPVII, AaLT). However, no synergistic effect was observed when all three late phase proteases were knocked down at once. No other midgut proteases had been identified until 2010, when five new serine proteases (AaSPI-AaSPV) were discovered. These ‘orphan’ proteases lack targets and functions as they have yet to be characterized in vivo or in vitro. Additionally, two chymotrypsin-like enzymes (AaCHYMO and AaJHA15) have been identified and have been minimally studied in vitro, but again their exact role in digesting blood meal proteins are relatively unknown. Therefore, work has recently expanded to focus on the newly identified serine proteases (AaSPI-AaSPV) and the two chymotrypsin-like proteases.

2) Determine the Role of Midgut and Salivary Gland Proteases in Viral Pathogen Enhancement/Disruption. An uninfected Ae. aegypti mosquito becomes a vector upon imbibing a viremic blood meal. In the first 1-2 days after infection, the viruses invade the midgut epithelial cells through endocytosis, and replication is initiated thereafter. After ~3 days post infection, the viruses spread from the midgut, then throughout the mosquito body, ending in the salivary glands, with maximal viral titers observed 12-18 days post infection. Interestingly, work focusing on Dengue virus serotype 2 (DENV-2) in Ae. aegypti in vivo, resulted in a decrease in midgut DENV-2 RNA copies when feeding mosquitoes an infectious blood meal in the presence of a soybean trypsin inhibitor (STI). However, gene expression studies of Ae. aegypti non-infected and infected (DENV-2) mosquitoes revealed the downregulation of AaET in infected mosquitoes, indicating a possible immune role against the virus, and providing evidence that midgut proteases may be playing an immune role against DENV-2. However, more work needs to be conducted to conclusively state this. No other research labs have followed up on this idea. In addition, changes in salivary gland protease gene expression were observed to be up-regulated when the mosquito is infected with the DENV-2 serotype. Two main questions arise: Do these proteases aid in viral replication? Or are these salivary gland proteases involved in liquefying dermal tissues by specifically digesting the extracellular matrix components, enhancing infectivity and dissemination in the human host. The answer to these questions may lead to a new inhibitor target and affect viral pathogen transmission.

3) Determine the Role of Proteases in Eggshell Formation and Melanization. The Ae. aegypti mosquito lays her eggs in an aquatic environment, which allows water to freely pass through the transparent eggshell, but after two hours the eggshell darkens via melanization. The melanized eggshell serves as a protective layer for embryonic development, as well as preventing egg shrinkage caused by desiccation. Although the melanization process and the different protein types involved in this process are known, the role of proteases in the Ae. aegypti mosquito melanization process is not. Affecting the protective layer needed for embryonic development could serve as a new and different vector control strategy.