Characterizing the VxrAB Two Component Signal Transduction System

Pathogenic strains of Vibrio cholerae cause the acute diarrheal disease cholera, which can result in hypotonic shock and death within 12 hours of the first symptoms. V. cholerae is found primarily in the aquatic environment but can be transmitted to a human host through the consumption of contaminated food or water. In order to survive in the aquatic environment and human host, V. cholerae must sense and respond to the fluctuating external conditions encountered in these varied environments. To do this, V. cholerae and other bacterial species utilize two-component signal transduction systems (TCSs), which employ a sensor histidine kinase (HK) to sense a cognate signal and activate its associated response regulator (RR) to initiate a cellular response.One such TCS is the V. cholerae VxrAB (Vibrio type six regulator) TCS, which has been shown to positively regulate a number of important cellular processes, including virulence, a bacterial defense system known as the Type Six Secretion System (T6SS), and cell wall homeostasis. In this work we further characterize the VxrAB system, identifying it as a positive regulator of biofilm formation, demonstrating a role for its activation of T6SS within biofilms, and further characterizing the role of its regulon members in virulence and cell wall homeostasis. First, we identified VxrB as a new regulator of biofilm formation through the systematic analysis of V. cholerae RRs (Chapter 2). Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. VxrB, regulates expression of key structural and regulatory biofilm-genes in V. cholerae. Additionally, vxrB is encoded as part of a 5-gene operon, which encodes the cognate HK vxrA, and three genes of unknown function. ΔvxrA and ΔvxrB are both deficient in biofilm formation, while ΔvxrC enhances biofilm formation in a vxrB dependent manner, indicating that VxrC may act as a repressor of this system. This work revealed a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels.Given that VxrB co-regulates T6SS genes and biofilm genes, I next investigated the role of the T6SS in biofilms (Chapter 3). The T6SS is a contractile nanomachine capable to injecting toxins into neighboring cells. Given the close proximity of cells to one another in a biofilm environment I demonstrated that the T6SS can actively fire and kill susceptible neighboring cells within the biofilm. This is the first evidence of T6SS activity within V. cholerae biofilms and suggests that VxrB’s co-regulation of biofilm formation and T6SS genes may contribute to the ability of V. cholerae to persist in intra- and inter-species biofilms. Finally, though our lab previously established that VxrB-mediated virulence is partially due to its activation of the T6SS, we also demonstrated that other factors contribute to VxrB-mediated intestinal colonization. Using RNA-seq analysis we identified a number of hypothetical genes regulated by VxrB and tested their contribution to VxrB-mediated phenotypes, including virulence, T6SS activity, and cell wall homeostasis (Chapter 4). We identified two operons that contribute to intestinal colonization, VC1162-60 and VC2548-47, and one operon that contributes to cell wall homeostasis, VC2520-16, all of which appear to converge around the cell envelope and are conserved systems in numerous other bacteria. This work enhances our understanding of hypothetical proteins and provides a more in depth understanding how VxrB-related phenotypes are mediated