Vibrio cholerae is the causative agent of the severe diarrheal disease, cholera, that is endemic in much of Asia, Africa, and South America. The species is quite diverse, although only strains of the O1 or O139 serogroup cause epidemic disease. However, a significant amount of disease occurs globally in sporadic episodes and is caused by strains belonging to non-O1/non-O139 serogroups. Unlike pathogenic O1 and O139 strains, the vast majority of pathogenic non-O1/non-O139 strains do not carry the well characterized virulence factors for colonization (TCP) and toxin production (CT), and presumably cause disease by an unknown mechanism(s). Whole genome sequencing of a clinically isolated non-O1/non-O139 strain, AM-19226, has revealed the presence of open reading frames (ORFs) having significant similarity to genes encoding a Type Three Secretion System (TTSS). These ORFs appear to be conserved among a subset of non-O1/non-O139 strains, as well as pandemic strains of V. parahaemolyticus. Many pathogenic bacteria use TTSSs to translocate virulence factors into the cytosol of host cells, and we hypothesize that the presence of similar ORFs in V. cholerae represents a newly identified mechanism for host cell interaction and virulence acquired by these strains. Experiments suggest that the TTSS is indeed functional and has a role in pathogenesis. Deletion of a critical component of the TTSS severely attenuates the ability of strain AM-19226 to colonize the infant mouse model. Furthermore, an effector protein (whose secretion is TTSS dependent) has been identified, and it appears to have a role in the reorganization of host cell actin. We propose to use three complimentary in vitro approaches to begin to understand TTSS mediated pathogenesis in V. cholerae. The first approach is to identify additional effector proteins that promote virulence, using complimentary in vitro and in vivo model systems. The second approach is to dissect how effector proteins interact with host cells at the molecular level to cause disease, and the third is to use genomic approaches to begin to understand the regulatory network governing expression of the genes encoding effector proteins and the TTSS structural apparatus. Project Narrative: Today cholera is considered a health threat mainly in developing nations, regions lacking modern sanitation facilities, and in countries experiencing disrupted civil infrastructure due to war or environmental crises. The disease poses a threat to individuals traveling to endemic areas, and the United States CDC considers some strains a Category B level BioTerrorism threat because of the potential for spread via contaminated food or water. While O1 and O139 epidemic causing strains are well studied, this proposal seeks to understand the virulence mechanisms employed by strains of other serogroups that represent an emerging threat.