Systematic analysis of two-component signal transduction systems in Vibrio cholerae biofilm formation and virulence

Vibrio cholerae, the causative agent of cholera, is a facultative human pathogen that can inhabit aquatic environments and human intestines. Its survival in aquatic habitats is critical for its transmission to humans. However, how V. cholerae senses and responds to fluctuating environmental factors experienced during aquatic and intestinal growth remains to be explained.One way in which V. cholerae could sense and respond to changes in the environment is through use of a two-component signal transduction systems (TCS). The TCS can regulate a wide variety of behaviors and processes, including virulence, biofilm formation, stress response, and motility. The prototypical TCS consists of a membrane-bound histidine (HK), which senses environmental signals and a corresponding response regulator (RR), which mediates a cellular response. I investigated the role of each TCS in V. cholerae infection and biofilm formation by focusing on RRs, the most downstream component. First, I performed mutational analysis of all (53) RRs. I generated a set of 53 isogenic RR mutants in V. cholerae by deletion analysis. To test whether V. cholerae these RRs are involved in virulence, I screened my library of 53 RR deletion mutants in a murine model of infection. In addition to the 13 previously identified RRs that control virulence, I identified an uncharacterized TCS named VxrB (Vibrio type six regulator) that is required for intestinal colonization. Transcriptome analysis revealed that VxrB activates type VI secretion system (T6SS) genes, which encode a complex molecular machine to inject effector proteins into target cells. Identification of a new TCS controlling virulence is significant because it fills gaps in our knowledge about V. cholerae pathogenesis, contributes to the general understanding of how TCS is regulated and suggests new methods for manipulating pathogenic behavior to improve human health. This could represent a target for developing anti-bacterial strategies. Regulation of biofilm formation in V. cholerae involves several transcriptional regulators and alternative sigma factors, such as RpoN. The exact mechanism by which RpoN impacts biofilm formation is yet to be determined. RpoN functions together with the NtrC family of RR, thus raising the possibility that biofilm formation requires both RpoN and an NtrC family RR. In this study, I analyzed the role of the eight NtrC family RRs in biofilm formation and identified four of these RR’s regulating biofilm formation. LuxO positively regulates biofilm formation. In contrast FlrC, FlrA and NtrC negatively regulate biofilm formation. Consistent with this observation, whole-genome expression profiling and transcriptional reporter assays revealed that expression of the Vibrio polysaccharide (vps) genes and genes encoding the two positive transcriptional regulators, VpsR and VpsT, is increased in an ntrC mutant. Epistasis analysis showed that NtrC acts in parallel with HapR and CRP-cAMP complex, the negative regulators of biofilm formation. This study underscores the importance of NtrC family of response regulators in the regulation of biofilm formation in V. cholerae. Furthermore, elucidation of the mechanism for regulation of biofilm formation will provide the foundation for developing novel treatments and prevention strategies against cholera, and will facilitate anti-biofilm interventions.To examine whether V. cholerae TCS signal pathways are involved in biofilm formation, I screened my 53 RR deletion mutants for the expression of Vibrio polysaccharide (vps) genes and analyzed the ability of these deletion mutants to form three dimensional biofilms in a flow-cell environment. These studies led to the identification of two (ntrC and vxrB) regulators of vps gene expression and five (ntrC, vxrB, VC1348, flrC, and flrA) regulators of biofilm formation. Since VxrB has previously been shown to regulate virulence, I wanted to further investigate its role in biofilm formation. VxrB positively regulates biofilm formation and expression of vps genes. This study revealed that expression of biofilm genes and T6SS genes are co-regulated by VxrB.This study identified critical TCSs regulating virulence and biofilm formation in V. cholerae and furthered our knowledge of regulation of environmental adaptation by an important human pathogen.