Membrane stress and ToxR-mediated virulence of Vibrio cholerae

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The Gram-negative bacterium Vibrio cholerae, which is a natural inhabitant of the coastal aquatic and estuarine environment, is the causative agent of the gastrointestinal disease cholera. Virulence gene regulation in V. cholerae is highly complex with the master regulator components ToxR/ToxS acting at the top of a regulation cascade, which is responsible for the control of virulence as well as housekeeping/fitness genes. Despite its importance, signal input and in particular the function of ToxS in this master regulatory system have remained unknown. In this project we will investigate a new mode of activation of this system, as we found that the activity of the membran-bound transcriptional regulator ToxR is influenced by a protease (DegS), which is also part of the sigmaE membrane-stress response pathway. In addition, the ToxR response is influenced by another signal transduction system, which we identified as a two-component regulatory system, termed OsmRK. We also observed, that the expression of the ToxR-regulated porins ompU and ompT varies significantly, depending on environmental stress conditions and the allelic status of toxS, osmRK and degS. First analyses indicate, that these regulatory effects are ToxR-mediated, and that ToxR may be activated by proteolytic modification caused by DegS, and may then be further degraded by yet unknown proteases. In addition, ToxS seems responsible for stabilizing ToxR and thereby maintaining its activity in the membrane. Thus, our data indicate that ToxR activity is conrtolled by a novel and more complex variant of regulated intramembrane proteolysis (RIP). The proposed project will provide additional experimental evidence for this just emerging new regulatory pathway and will address further questions such as: (i) what is changed or modified within ToxR while receiving activating signals? (ii) Is ToxR a direct target for DegS? (iii) How is ToxR activity influenced by the OsmRK system? And finally, (iv) what is the nature of the signals recognized by DegS and OsmRK and do these systems cooperate in a single pathway? The expected results will significantly contribute to our understanding of ToxR-mediated regulation, which is not only crucial for virulence gene regulation in V. cholerae and other pivotal cell functions in Vibrio spp., but may also reveal a novel regulatory potential of the RIP pathway.