Recombinant Expression and Potential Autocatalysis of Aedes aegypti Trypsin-like Serine Proteases (AaSPII and AaSPIV)

Abstract

Aedes aegypti mosquitoes can be found globally in tropical and subtropical urban areas and spread Zika, Dengue fever, yellow fever, and Chikungunya viruses. Current vector control methods are limited and nonspecific. The female Ae. aegypti mosquito uses blood meal proteins to obtain nutrients required for oogenesis; inhibition of the midgut trypsin-like serine proteases responsible for blood meal digestion may provide a novel method of vector control. Ae. aegypti blood meal digestion is complex and the role of uncharacterized serine proteases in blood digestion is unclear; specifically, a group of trypsin-like serine proteases (AaSPII-V) is expressed at constant levels before and following Ae. aegypti blood meal acquisition. This research focuses on the in vitro biochemical study of two specific Ae. aegypti trypsin-like serine proteases (AaSPII and AaSPIV) in order to gain further understanding of their role in blood meal digestion. The approach involved the successful cloning and bacterial expression of these soluble, recombinant proteases. Results from attempts to purify these proteases were unsuccessful but indicative of potential autocatalytic and autodigestive behavior. Future studies will focus on obtaining purified recombinant proteases for further study. The study of AaSPII and AaSPIV, as well as other midgut Ae. aegypti proteases, will aid in understanding the overall role proteases play in blood meal digestion and may eventually allow for the development of mosquito-specific enzyme inhibitors. v ACKNOWLEDGMENTS First and foremost, I would like to thank Prof. Alberto A. Rascón Jr., my research advisor, for the opportunity to perform this research in his laboratory and for the expert mentorship and guidance I received throughout my research endeavors. While working with Prof. Rascón, I gained invaluable experience and learned both technical and practical skills to help me succeed in the future as a research scientist. I would also like to extend my gratitude to the members of my graduate committee, Prof. Laura Miller Conrad and Prof. Marc d'Alarcao, for their support and assistance in developing this thesis. I also thank my friends and colleagues at San Jose State University and in the Rascón Lab for the scientific discourse, encouragement, and laughter we've shared. For their constant love and support, I want to thank my family and friends. My mother and father, Valerie and Gregg Eilerts, have provided a lifetime of support and encouragement and prepared me for a successful future. I thank my brother, Tom Eilerts, for always making me laugh and for his endearing attempts to help me overcome research struggles via jelly donut metaphors. I thank my sister, Renee Eilerts, for always welcoming me in her home and making sure I remembered to occasionally have fun outside of the lab (Go Sharks!). I thank my best friend, Chelsea Carman, for listening and supporting me through both my undergraduate and graduate education. Lastly, I want to extend my deepest gratitude to my partner, Morgan Hoffman, for his unwaver...