Regulation of Vibrio Cholerae Virulence by ToxR and TCPP

Vibrio cholerae is the causative agent of the diarrheal disease cholera, a debilitating disease affecting as many as ten million people annually. The disease is caused by cholera toxin, a protein whose expression is regulated by two membrane-localized transcription factors, ToxR and TcpP. <P> This application investigates how ToxR and TcpP combine to activate the toxT promoter in V. cholerae, thus triggering virulence gene expression. In addition, we will investigate how ToxR activates a second promoter, ompLJ, in a TcpP- independent fashion. Expression of the porin, OmpU, leads to bile resistance in V. cholerae. <P> We hypothesize that ToxR functions by different mechanisms at the ompU and toxT promoters. With ToxR directly activating ompU while serving to facilitate TcpP-mediated promoter activation at toxT. <P>Finally, we will also develop a system for observing the the interaction of ToxR and TcpP in the membrane of living V. cholerae cells using FRET. This will allow us to observe a critic step in induction of virulence in real time as well as allow us to observe any changes in ToxR/TcpP interaction in response to a variety of environmental conditions. <P> Specific Aims of this project are: I. Determine the mechanism of ompU and toxT activation by ToxR. II. Determine the mechanisms of DNA-binding, ToxR interaction and RNA polymerase stimulation by TcpP and the role of each event in toxT activation. III. Measure ToxR/TcpP protein-protein interactions in living V. cholerae cells after tcpP induction using FRET (fluorescence resonance energy transfer)