Gene Regulation by ToxR, TcpP and ToxT in V. Cholerae

霍乱弧菌中ToxR、TcpP和ToxT的基因调控霍乱弧菌、霍乱毒素和毒素协同调节菌毛（分别为CT和TCP）的主要毒力因子由一系列调控因子控制，最终导致ToxT被两种膜定位激活剂ToxR和TcpP激活。弓形虫是霍乱毒素和毒素协同调节菌毛基因表达的直接激活剂，也是其他在霍乱弧菌生物学和致病性中作用尚不明确的基因的表达。膜激活因子，ToxR和TcpP，各自依赖于同源效应蛋白，ToxS和TcpH，尽管这种依赖的机制可能对每对蛋白不同。先前资助期的一个主要发现是TcpP受TcpH拮抗的膜内蛋白水解调节。这个过程需要YaeL蛋白酶作为从细胞中消除TcpP的两种蛋白酶中的第二种。TcpP是激活弓形虫基因所必需的，最近NIH支持这项工作的另一个重大发现是鉴定了一个受弓形虫直接转录控制的小RNA分子（tarA）。基于重要的初步数据和已发表的手稿，目前的建议有三个目标：1。全面分析TcpP和TcpH的相互作用，以及调控细胞内TcpP水平的膜内蛋白水解途径，包括鉴定尚不为人知的I位点蛋白酶；2. ToxR/TcpP调控通路小分子抑制剂的筛选、靶点表征及作用机制研究；3、tarA的作用机制。

DESCRIPTION (provided by applicant): Gene regulation by ToxR, TcpP and ToxT in V. cholerae The major virulence factors of Vibrio cholerae, cholera toxin and toxin-coregulated pilus (CT and TCP, respectively) are controlled by a cascade of regulators that ultimately leads to activation of ToxT by two membrane-localized activators, ToxR and TcpP. ToxT is the direct activator of cholera toxin and toxin-coregulated pilus gene expression, as well as expression of other genes whose roles in the biology and pathogenicity of V. cholerae are less well defined. The membrane activators, ToxR and TcpP, each depend for their activity on a cognate effector protein, ToxS and TcpH, although the mechanism of this dependence may be different for each pair of proteins. A major discovery of the prior funding period is that TcpP is subjected to regulate intramembrane proteolysis that is antagonized by TcpH. This process requires the YaeL protease as the second of two proteases that eliminate TcpP from the cell. TcpP is necessary to activate the toxT gene, and another major discovery from the most recent period of NIH support for this work was identification of a small RNA molecule (tarA) under direct transcription control of ToxT. Based on significant preliminary data and published manuscripts, the current proposal has three aims: 1. Comprehensive analysis of TcpP and TcpH interaction and the regulated intramembrane proteolysis pathway that governs TcpP levels in the cell, including the identification of the still unknown site I protease; 2. Screens for small molecule inhibitors of the ToxR/TcpP arm of the regulatory pathway and characterization of the targets of those inhibitors as well as the mechanism of inhibitor; 3, the tarA mechanism of action. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a bacterium that causes the human diarrheal disease cholera. The proposed studies are designed to uncover new knowledge mechanisms used by V. cholerae to regulate its virulence traits. One of the aims is intended to identify small molecules that interfere with key steps in the regulation pathway leading to disease. Such molecules might represent new classes of drugs that target virulence.