UC Santa Cruz

V. cholerae the causative agent of the diarrheal disease cholera is able to thrive within the small intestines of human hosts, however, between epidemics is found as an autochthonous member of aquatic microbial communities throughout the world (Kaper, 1979; Gil, 2004; Huq, 2005). This pathogen must endure changes in environmental parameters such as temperature, salinity, pH, and iron availability between their human hosts and aquatic reservoirs as well as within the environment. Temperature is an important parameter that governs the prevalence and distribution of V. cholerae in the environment, impacts association between V. cholerae and zooplankton, and is a strong predictor of cholera outbreaks. Despite the influence that temperature has on the ecology of V. cholerae, the molecular details of how V. cholerae adapts to temperature change and the cellular processes that are influenced by temperature in order to facilitate temperature adaptation in V. cholerae remain unknown.

To gain a better understanding of how temperature impacts V. cholerae, we examined biofilm formation at various temperatures. In V. cholerae, biofilm formation is important for persistence in the environment and survival within the human host. We found that low-temperature growth enhances biofilm formation in V. cholerae through an increase in levels of the intracellular signaling molecule cyclic diguanylate (c-di-GMP). Furthermore, we identified six diguanylate cyclases, cdgA, cdgH, cdgK cdgL, cdgM, and vpvC, which are responsible for increasing c-di-GMP levels at low temperatures that impact biofilm formation and motility in V. cholerae.

Next we analyzed the genome-wide transcriptional profile of V. cholerae grown at different temperatures and determined that genes involved in virulence, biofilm formation, type VI secretion (T6SS), and cold shock were modulated by temperature. Mutational analysis and phenotypic characterization of the most highly induced gene at low temperatures, cspV (coding for cold shock protein V) revealed that this cold shock gene is important for biofilm formation and T6SS-mediated interspecies killing. In order to examine the effect of CspV in a more biologically relevant system, we tested the ability of CspV to colonize live zooplankton and perform T6SS-killing of bacteria on the these plankton. The strain lacking cspV had a severe attachment defect on the plankton surface, as well as a T6SS-killing defect of E. coli and Aeromonas sp., which suggests that this cold shock gene is important for environmental survival and transmission of V. cholerae to the human host.

Finally we begin to characterize a putative c-di-GMP effector/ protease system in V. cholerae that is predicted to be functionally homologous to the inside-out signal transduction system LapD/G in Pseudomonas fluorescens. We determined that temperature is a key signal in regulating this system likely through low-temperature modulation of cellular c-di-GMP levels and via temperature modulation of genes that encode two putative large adhesins, VCA0849 and VC1620. Based on our results we predict that at low temperatures c-di-GMP levels are increased, causing the putative receptor VCA1082-3 to bind c-di-GMP and inhibit degradation of VCA0849 by a predicted periplasmic protease VCA1081, thereby increasing biofilm compactness and thickness at low temperatures.

This dissertation describes mechanisms that promote the survival of V. cholerae in the environment and facilitate the transmission of this pathogen to human hosts. This research provides important insight into how environmental pathogens adapt to changes in temperature and persist in the environment and by this means perpetuate their infectious cycle