Environmental Modulation of Toxt-Dependent Transcription

University Of Texas San Antonio, San Antonio TX

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Abstract

Cholera is an often-fatal diarrheal disease caused by the bacterium Vibrio cholerae. This disease remains a health threat for the majority of the world, causing thousands of deaths every year. ToxT is the primary transcriptional activator of virulence genes in V. cholerae, and strains lacking toxT do not express the critical factors cholera toxin and toxin-coregulated pilus, and are unable to colonize the intestine and cause disease. ToxT transcriptional activity is regulated by certain environmental signals, and specifically by the presence of bile. Given the essential role of ToxT in cholera pathogenesis, a deeper understanding of this protein is critical for the development of new therapeutics and vaccines against this disease. Our studies will focus on characterizing the structure and function of ToxT, and determining the molecular mechanism(s) of environmental modulation of ToxT transcriptional activity. We have created a scanning alanine mutant library of ToxT in which every amino acid has been replaced with Alanine. We will utilize this library to examine the roles of individual amino acids in the function of ToxT. Our approach first involves characterizing the residues that are essential for ToxT activity, determining which amino acids are required for i). DNA binding, ii). dimerization, and iii). contact with RNA Polymerase. We will determine the structure of ToxT utilizing X-ray crystallography, and integrate the information into the functional studies to refine our understanding of ToxT transcriptional activity. We will determine the mechanism of environmental modulation of ToxT transcriptional activity by environmental signals, by i). characterizing differential transcriptional activation of ToxT-dependent promoters, ii). identifying ToxT amino acids involved in environmental modulation, iii). identifying additional V. cholerae genes involved in environmental modulation of ToxT activity, and iv). determining the effects of environmental modulation on ToxT dimerization. Finally, the relevance of the critical ToxT residues involved in function and environmental modulation will be assessed by testing the virulent properties of V. cholerae strains containing these mutant toxT alleles. Our ultimate goal is to learn how ToxT functions, in order to utilize this information to design new therapeutics and/or rationally designed live attenuated vaccines to combat cholera.