Vibrio cholerae colonization and transmission factors in aquatic hosts

PROJECT SUMMARY Vibrio cholerae is an aquatic bacterium that causes diarrheal diseases ranging from mild to deadly. Pandemic O1/O139 serogroup strains cause cholera, a potentially deadly disease, whereas non-O1/O139 strains cause diarrhea that ranges from mild to severe. Mammalian models have been useful in characterizing major human V. cholerae virulence factors, but rabbits and mice are not natural V. cholerae hosts and require absent or damaged microbiota to enable colonization. Vertebrate fish are natural V. cholerae hosts and zebrafish are a model that recapitulates the entire infectious cycle in the presence of mature intestinal microbiota and immune responses. Fish are rapidly colonized by exposure to V. cholerae in water, develop diarrhea, excrete large numbers of hyperinfectious V. cholerae, and transmit the infection to naïve fish. Because this is a model in a natural host, it presents new opportunities for V. cholerae study that are not possible with mammalian models. However, the V. cholerae genes that are required for fish colonization and transmission are unknown and a major gap in our knowledge. The process by which V. cholerae becomes hyperinfectious when passing through a host is also not well understood. This proposal will examine the infectious processes used by pandemic O1 V. cholerae and test the following two hypotheses: 1) V. cholerae produces specific colonization factors in fish that are also important in human infections. Preliminary experiments identified genes that may be important for colonization using a Tn-Seq screen. Selected genes of interest identified by Tn-Seq will be deleted to assess colonization defects. Genes whose deletion reduces or prevents colonization will be characterized to determine how they facilitate fitness in fish. The effects of gene deletions that impact fish colonization will also be tested in the infant mouse model to assess defects in mammalian colonization. 2) Motility and chemotaxis are crucial for the transient hyperinfectious V. cholerae phenotype. RNA-Seq was used to compare differences in gene expression between actively colonizing and excreted V. cholerae. Genes particularly related to chemotaxis and motility were found to be highly upregulated in excreted V. cholerae, and a mutant that cannot produce a flagellum was found to be highly defective in transmission/hyperinfectivity. To identify the mechanisms for this, other genes important for motility and chemotaxis will be deleted and tested for defects in transmission/hyperinfectivity. Completion of the proposed work will significantly advance our understanding of the requirements for V. cholerae colonization and transmission in the environment and likely in humans as well. The long term goals of this project are to use the zebrafish model to better understand the V. cholerae life cycle in the environment that contributes to human disease, and to identify new strategies to combat V. cholerae disease and transmission.