PROJECT ABSTRACT/SUMMARY There is a gap in our understanding of how specific lineages of Vibrio cholerae cause outbreaks of cholera - an infection of the small intestine that triggers severe watery diarrhea, dehydration, and, too often, death. Cholera is endemic in many regions of the world, causing over 120,000 deaths every year. Cholera is also one of the first diseases to emerge when the health care system and sanitation infrastructure break down during an economic or civil crisis, as witnessed just recently in Yemen with 500,000 cases and 2,000 deaths. The scientific premise for this proposal is a longstanding appreciation that cholera outbreaks emerge from single clonal lineages – strains that are closely related genetically. Although its natural aquatic environment harbors a diverse V. cholerae population, with both toxigenic (producing the main virulence factors of cholera toxin and toxin-coregulated pilus) and non-toxigenic strains, cholera outbreaks are caused by single clonal lineages. We propose that toxigenic strains use the type VI secretion system (T6SS) as an active competition mechanism to prevent other strains from colonizing the small intestine. The T6SS structurally resembles the injection apparatus of T4 bacteriophage and delivers toxic T6SS effectors into adjacent bacteria. Delivery of effectors is lethal unless the receiving cell produces immunity proteins that sequester the incoming toxins. The T6SS is encoded in three distinct loci on the chromosome. Each locus hosts a horizontally acquired genetic element with a distinct toxin–immunity pair called a module. Together, the three effector modules in a strain comprise its effector module set (e.g., AAA-module set for toxigenic strains). We discovered that strains with identical modules are compatible and co-exist, while strains with different modules compete on contact. Our central hypothesis is that toxigenic V. cholerae acquired the most competitive T6SS module set (AAA) to exclude incompatible cheater strains. To test our hypothesis for the funded parent grant, we will resolve how the AAA module set is acquired (Aim I), how it is used in vivo (Aim II) and how it contributes to clonal dominance (Aim III). In this revised proposal, we are adding a sub-aim (Aim IIIb) to test when and where AAA strains become dominant in human populations in Bangladesh exposed to cholera. This proposal investigates how bacterial competition mechanisms contribute to the clonal nature of outbreaks, allowing us to exploit strategies that interfere with the ability of toxigenic lineages to expand and cause cholera.