Project Summary Cholera is a severe dehydrating diarrheal disease caused by Vibrio cholerae. This Gram-negative rod has the unusual capacity to colonize the small intestine and to cause explosive epidemics. Here we will address fundamental questions in V. cholerae-host interactions, leveraging many of the approaches and tools we have created in the past decades. V. cholerae O1, the cause of pandemic cholera, is divided into Ogawa and Inaba serotypes, which differ only by the presence or absence of methylation of the terminal O-antigen sugar respectively. Switching of the Ogawa and Inaba serotypes during cholera epidemics has been recognized for over a century, but the consequences of serotype conversion on pathogen fitness are not clear. We discovered that the Ogawa serotype has greater in vivo fitness than the Inaba serotype and that the two serotypes rely on distinct metabolic process for growth in vivo. Thus, there are unexpected direct or indirect phenotypic and physiological consequences of O-antigen methylation on V. cholerae growth in vivo. The consequences and mechanisms that underlie the in vivo fitness differences of the V. cholerae serotypes will be determined in Aim 1. Cholera toxin (CT) triggers the intestinal fluid secretion that largely accounts for choleric diarrhea. We found that CT also leads to the secretion of hundreds of host proteins identified in diarrheal fluid and re-models the intestinal epithelial transcriptional response to V. cholerae. Many of these proteins and transcripts are linked to innate immune responses and we found that one of these proteins, surfactant protein D (SP-D), restricts V. cholerae growth in the intestine. In Aim 2, we will analyze the protective mechanisms mediated by SP-D and investigate the roles of additional V. cholerae-induced secreted host proteins in impeding the pathogen’s colonization, to uncover host innate axes that protect against V. cholerae infection. Cholera epidemics often spread extremely rapidly, and host passaging increases V. cholerae infectivity, but the V. cholerae and host genes that govern cholera transmission are largely unknown. In Aim 3, we will leverage our experience with pathogen barcoding and a new computational framework to extend analyses developed in Aims 1 and 2, examining the roles of serotype and CT in modulating host-priming of infectivity. Additional pathogen pathways and host processes that control V. cholerae infectivity will also be elucidated, to deepen understanding of cholera transmission. Collectively, the proposed research will yield new understanding of the interconnected processes that govern V. cholerae intestinal colonization and infectivity as well as the host factors and mechanisms that limit colonization and control transmission. This work will provide new perspectives on the biology of the two V. cholerae serotypes, and on the actions of cholera toxin in stimulating innate host defense responses against the pathogen. Our findings will also hav...