Activation mechanisms of the transcriptional regulator complex ToxRS in Vibrio cholerae

Cholera is a life-threatening diarrhoeal disease caused by the human pathogenic bacterium Vibrio cholerae. Responsible for the disease are specific virulence genes, which belong to the ToxR- regulon and are encoding for cholera toxins and pili for adherence. A hypothesis in the field suggests, that modulation of regulation is correlating with increased adaptation of pathogens to the corresponding host systems. For example, as for V. cholerae the virulence gene regulation seems more complex for the current pandemic strain biotype O1 El Tor as compared with its antecessor biotype O1 classical, although both encode similar virulence factors. This may have developed into a competitive situation for the O1 El Tor, leading to a replacement of the O1 classical, by showing less mortality, better adaptation and perhaps improved host persistence. With this project, the molecular function and interplay of the key-regulatory and cytoplasm membrane located complex ToxRS will be characterized. So far no study exists which describes the molecular principle of ToxR activation, respective ToxRS interactions. Based on our preliminary and interesting data, we will continue with important projects to resolve ToxRS interactions and functions. Therefore we will focus on issues such as: i) ToxRS interaction, ii) Proteolysis control of ToxR, and iii) Mechanisms of ToxR activity. The inhere used techniques will comprise in vitro and in vivo analyses of ToxR stability, cell localisation, activation assays, NMR protein structural analysis and fluorescence microscopy on single cells for determination and visibility of ToxRS complex formation and operator localisation. For the herein obtained data, we hope to contribute to alternative approaches for anti-cholerae therapy, since the understanding of the molecular function of the ToxRS activation complex will provide the opportunity to screen for putative inhibitors. Such agents could be used in cholera patients to produce termination of virulence gene expression and would subsequently lead to abrogation of V. cholerae colonization in the gut.

Cholera, caused by the bacterium Vibrio cholerae, is an acute diarrheal infection which remains a major global threat due to the high burden of morbidity and mortality. Derived from the aquatic environment the bacteria enter the human host. The master regulator for virulence genes and cholera toxin expression is ToxR, which is depending on environmental signaling. In this project we identified cysteine residues of ToxR as a potential redox switch that regulates its stability and activity. Environmental stimuli, such as nutrient availability, pH and bile salts, influence ToxR transcriptional activity, its proteolysis and structural interaction and hence indicated a mechanism for rapid adaptation to different habitats and stress conditions. Our results indicate that transcriptional activity of ToxR is dependent on a variety of factors, such as the presence of operators binding sites, the co-activator ToxS and Dsb proteins, all of which enhance the dimerization and thus activity of ToxR. Detailed elucidation of virulence gene regulation in V. cholerae at each level is of great value in order to find new affordable therapeutic approaches that interfere with regulatory circuits. Here, our findings contribute significantly to the understanding of the V. cholerae infection cycle by providing a new picture of the regulatory flexibility of the ToxRS regulation system.