Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Jun Zhu

Abstract

Vibrio cholerae causes human infection through ingestion of contaminated food and water, leading to the diarrheal disease, cholera. In aquatic environments this bacterium displays an expression profile that is distinct from that observed during infection. It can also form matrix-encased aggregates known as biofilms, typically on chitinous surfaces, which can be important for transmission and infectivity. Upon entry into the host, a tightly regulated circuit coordinates induction of two major virulence factors: cholera toxin (CT) and a toxin co-regulated pilus (TCP). This study finds that bile components present in the host intestine can affect both of these processes. Certain bile salts, including taurocholate (TC), serve as host signals to activate V. cholerae virulence through inducing the activity of a transmembrane virulence regulator TcpP. In this study we show that Ca2+, an abundant metal ion in the gut, enhances bile salt-dependent virulence activation. Induction of TCP by murine intestinal contents is counteracted when Ca2+ is depleted by the high affinity calcium chelator EGTA, suggesting that calcium present in the gut is a relevant signal for V. cholerae virulence induction in vivo. We further show that in conjunction with TC, Ca2+ affects dimerization and membrane diffusion of TcpP as analyzed by bacterial two-hybrid and fluorescence recovery following photobleaching assays. When applied to mature biofilms, TC induces an increase in number of detached cells with a concomitant decrease in biofilm mass. Inhibition of protein synthesis did not alter rates of detachment, suggesting that V. cholerae undergoes a passive dispersal. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix and cell-free media contains a higher amount of free polysaccharide with TC, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, V. cholerae induces virulence in response to taurocholate only after exiting from the biofilm. Our data suggests a model in which V. cholerae ingested as a biofilm has co-opted the host-derived bile salt signal to sequentially detach from the biofilm and go on to activate virulence, which is further promoted by the synergistic effects of physiological levels of Ca2+.

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