Role of flagellins in motility and virulence of Vibrio human pathogens - PROJECT SUMMARY Annually ~4,500,000 people worldwide suffer infections from pathogenic Vibrio spp., 200,000 of which result in death. V. cholerae (Vc) and V. vulnificus (Vv), the etiological agents of cholera and non-cholera vibriosis respectively, inflict life-threatening gastroenteritis, and, in susceptible individuals infected with Vv, septicemia and necrotizing fasciitis. To effectively colonize the small intestine, Vc and Vv rely on motility conferred through a sheathed polar flagellum. The polar flagellum also contributes to biofilm formation by facilitating Vc and Vv attachment and microcolony formation, enabling Vc and Vv to persist in the environment and be transmitted as biofilms. Despite this, the mechanisms governing polar flagellar synthesis and function remain uncharacterized. Composed of 5-6 unique, differentially regulated protein subunits called flagellins and enclosed by outer membrane, the Vibrio polar flagellar filament is a complex and understudied feature of Vibrio flagellar synthesis and pathogenesis. To gain a better understanding of the contributions of the flagellins toward polar flagellar synthesis and flagellar-mediated motility and virulence, the proposed study will involve (1) high-content single- cell video tracking to analyze the effects of different flagellin compositions on Vc and Vv swimming characteristics (speed, directional change rate, and angular displacement). Additionally, we will (2) identify the effects of different flagellin compositions on Vc and Vv virulence using the suckling mouse model of cholera and Galleria mellonella larvae, a novel invertebrate animal model. Lastly, we will (3) determine the effects of flagellin composition on Vc and Vv biofilm formation by performing confocal laser scanning microscopy (CLSM) imaging of Vc and Vv biofilms at different timepoints, allowing dynamic analysis of biofilm formation over time in. Together, these studies will shed light on the individual contributions of the flagellins toward Vibrio flagellar synthesis and flagellar- mediated disease, addressing a long-standing gap in knowledge regarding Vibrio pathogenesis. Combined with my proposal’s training plan which is precisely designed to expand my skills in high-content microscopy and computational biology, this proposal will deepen my understanding of cutting-edge computational techniques involving computing platforms like MATLAB, BiofilmQ and Python programming. Additionally, with my sponsor’s guidance, I will receive training in bacteriology, genetics, animal infection studies and molecular biology which will be further enriched by my collaboration with the supporters of my proposal. My participation in training programs such as the Center for the Integration of Research, Teaching and Learning (CIRTL) and professional development opportunities including colloquia, grant writing workshops, conferences and organizing our department’s predoctoral seminar series, will allow me to develop my teaching and mentoring acumen, ensuring my training not only in research, but in training and outreach as well.
