Characterization of Type VI Secretion System in Edwardsiella Ictaluri
CommitteeLawrence, Mark L.
Pinchuk, Lesya M.
Peterson, Daniel G.
Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is one of the most important bacterial diseases causing significant economic losses in the US catfish industry. Understanding the virulence mechanisms of E. ictaluri plays a vital role to develop preventives, such as vaccines for the disease. Therefore, further research is necessary to discover the new virulence mechanisms of this pathogen. The long-term goal of our group is to determine the mechanism of E. ictaluri pathogenesis and to develop effective live attenuated vaccines against ESC. The overall goal of this project is to understand the role of Type 6 secretion system (T6SS) in E. ictaluri virulence and determine the safety and efficacy of T6SS mutants in the catfish host. The central hypothesis is that T6SS in E. ictaluri provide an ability to invade the host cells and survive inside of the channel catfish neutrophils and macrophages, and mutation of T6SS genes will cause attenuation of the bacterial virulence. The rationale for the proposed research is that characterization of the T6SS in E. ictaluri will enlighten its role in E. ictaluri virulence, and T6SS genes can be targeted to develop live attenuated vaccines. In this study, we first constructed mutants of individual T6SS genes and a double mutant. The persistence, virulence, and vaccine efficacy of T6SS mutants were determined in the catfish fingerlings and fry infection model. The T6SS mutants Ei?evpC, Ei?evpC?hcp2, Ei?evpD, Ei?evpE, Ei?evpG, Ei?evpJ, and Ei?evpK were significantly attenuated and provided better protection against E. ictaluri 93-146 in channel catfish fingerlings. The role of T6SS mutants in adhesion and invasion of in vitro catfish epithelial indicated that Ei?evpN, Ei?evpO, and Ei?evpP significantly were less adherent and invasive. The survival and replication of T6SS mutants in in vitro catfish peritoneal macrophages cell line showed that T6SS mutants could survive up to 6 hours after phagocyted by catfish macrophages. The survival and resistance of T6SS mutants to stress conditions present in macrophages phagosome showed that hydrogen peroxide could limit the growth of T6SS mutants in BHI and minimal medium. Ei?evpA, Ei?evpH, Ei?evpM, Ei?evpN, and Ei?evpO exhibited a significant growth decrease.