Biotype-specific evolution

PROJECT SUMMARY Cholera is presently caused by O1 serogroup, El Tor biotype V. cholerae, which emerged in 1961 to initiate the ongoing seventh pandemic. The prior 6 cholera pandemics were caused by O1 serogroup classical biotype V. cholerae. Over the past 60 years, classical biotype has essentially disappeared from the aquatic environment and as a cause of cholera. However, the reasons for this are unknown. V. cholerae in the environment is found in association with numerous vertebrate fish species. The proposed work will use a zebrafish model for V. cholerae that can investigate interactions between V. cholerae and natural aquatic hosts covering the entire infectious cycle. Previous work found dramatic differences in the timeline of zebrafish colonization by classical and El Tor biotypes, with classical being cleared within 72 h and El Tor able to colonize for up to 14 days with high levels of bacterial replication. We hypothesize that this prolonged fish colonization and replication provided a strong selective advantage to El Tor, allowing it to replace classical in environmental niches. V. cholerae El Tor biotype has 2 pathogenicity islands termed VSP-1 and VSP-2 that classical lacks. Data from preliminary experiments indicate an El Tor strain deleted for VSP-1 has a normal fish colonization phenotype, whereas a strain deleted for VSP-2 is defective in prolonged colonization. Therefore, it is likely that gene(s) within VSP-2 are essential for prolonged colonization. Aim 1 of this proposal will use a progressive deletion strategy to identify and characterize gene(s) within VSP-2 that are important for prolonged colonization and assess whether such genes are sufficient to prolong classical colonization. On the host side, innate and adaptive immunity provide protection from invading pathogens. Fish have innate immune responses very similar to mammals, as well as adaptive immune responses that develop over the first 4-6 weeks of life. We hypothesize that classical biotype is rapidly cleared by a strictly innate response, whereas El Tor can only be cleared by an adaptive immune response. Aim 2-1 of this proposal will test these hypotheses using larval zebrafish, which have a fully functioning innate response but an undeveloped adaptive response. Aim 2-2 will further test the hypothesis that El Tor clearance requires adaptive immunity by using zebrafish mutants that are defective in adaptive immunity. Completion of the proposed work, using zebrafish as an environmental V. cholerae host model, will significantly advance our understanding of V. cholerae evolution and selective pressures in a natural reservoir. The long term goal of this work is to better understand the V. cholerae life cycle, how it contributes to pathogenesis in humans, and identify new strategies to combat V. cholerae disease and transmission. .