Rewiring networks for a pathogenic lifestyle

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Project Summary Vibrio cholerae causes the severe diarrheal disease cholera that is endemic in much of Asia, Africa, and South America, and has recently reemerged in Haiti, Syria, and Ukraine. The species is highly diverse, although only O1 or O139 serogroup strains cause epidemic disease. However, increasing sporadic disease has been reported globally, and is caused by strains belonging to non-O1/non-O139 serogroups that present a public health threat both in developed and industrialized nations, including the United States. 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 the virulence mechanisms used by these strains are not well understood. Our study of pathogenic non-O1/non-O139 serogroup strains began with genomic sequencing of the clinically isolated O39 serogroup strain, AM-19226, which revealed a Type Three Secretion System (T3SS) that is conserved among other V. cholerae isolates. Like TCP and CT, the T3SS is acquired by horizontal gene transfer (HGT), and integrated into the ancestral chromosome. Our subsequent experiments identified two membrane localized transcriptional activators (MLTAs) encoded within the T3SS genomic pathogenicity island (PAI), which are essential for T3SS function in vivo and in vitro. We also found that ToxR, an ancestral MLTA required for TCP and CT expression, is important for T3SS regulation. ToxR is encoded by all strains of V. cholerae where it regulates core chromosomal functions, and is well-studied as an MLTA that cross-regulates PAI and ancestral gene expression. Interestingly, we found that the T3SS encoded MLTAs influence ancestral gene expression and phenotypes such as motility and biofilm formation. We hypothesize that in order to survive in aquatic environments and also cause disease, T3SS-positive V. cholerae must integrate virulence gene regulation with transcriptional circuits outside of the PAI, leveraging the activities of both ancestral and newly acquired, PAI encoded transcriptional regulatory factors. We propose to use complementary genetic and biochemical approaches to identify and characterize the mechanisms used by T3SS PAIs to coordinate motility, biofilm formation, and T3SS gene expression in response to environmental cues. Initial studies will define the regulons for T3SS MLTAs, and the conditions promoting regulation. We will investigate protein-protein interactions and MLTA transcriptional domain sequence specificity. Initial studies will focus on VttRA as the protein at the top of the regulatory hierarchy. Our overall goal is to discover how PAI encoded MLTA activity regulates virulence phenotypes and ancestral physiological traits that are necessary to maintain dual lifestyles of newly evolved pathogens.