Mechanism of Virulence Regulation by Membrane Activators

描述（由申请人提供）：霍乱弧菌的毒力基因调控需要四种不同寻常的转录调控蛋白的作用，即一对膜定位转录激活剂/效应分子，称为ToxR/ToxS和TcpP/TcpH。这些蛋白作用机制的工作模型认为，ToxR和TcpP共同激活一种蛋白ToxT的表达，该蛋白可以激活编码霍乱毒素和毒素共调节菌毛的毒力因子的基因。相比之下，单独的ToxR -独立于TcpP -可以调节OmpU和OmpT两种外膜蛋白的表达。激活剂和效应器之间的相互作用预计发生在质周空间，尽管通常效应器的作用不太明显。TcpH的作用是阻断针对TcpP的质周结构域的蛋白水解机制。TcpH是否在TcpP的功能中发挥其他作用将在本研究中进行评估。FoxS可能为ToxR赋予了其功能所必需的高阶结构。从这些膜定位激活剂功能的机制模型中产生的特定假设将被测试。我们对这些蛋白质的了解大多来自遗传和生化研究，这些研究将在本文提出的工作中继续进行。随着原核细胞研究的进展和影像学和细胞学资源的可用性，研究转录复合物膜定位的试剂和实验方法也被提出。

DESCRIPTION (provided by applicant): Virulence gene regulation in Vibrio cholerae requires the action of four unusual transcription regulatory proteins, pairs of membrane localized transcription activator/effector molecules called ToxR/ToxS and TcpP/TcpH. Working models for the mechanisms of action of these proteins hold that ToxR and TcpP collaborate to activate expression of a protein ToxT, which activates genes encoding virulence factors cholera toxin and toxin co-regulated pilus. By contrast, ToxR alone - independently of TcpP - can regulate expression of OmpU and OmpT, two outer membrane proteins. Interactions between the activators and effectors are predicted to take place in the periplasmic space, although in general the roles of the effectors are less well characterized. TcpH acts to block a proteolytic mechanism that targets periplasmic domain of the TcpP. Whether TcpH plays any other role in the function of TcpP will be assessed in this study. FoxS may serve to confer higher order structure on ToxR essential for its function. Specific hypotheses generated from these mechanistic models of membrane-localized activator function will be tested. Much of what we understand about these proteins has come from genetic and biochemical studies, and these will continue in the work proposed herein. With advances and imaging and cytological resources available for studying the prokaryotic cell, studies aimed at developing reagents and experimental approaches for studying membrane localization of transcription complexes are also proposed. Specific Aims I. Determine the mechanism of ompU and toxT activation by ToxR. II. Determine the role of DNA binding and RNA polymerase interaction by TcpP for toxT activation. Ill. Define the mechanisms and consequences of activator/effector periplasmic interactions. IV. Develop cytological methods for analyzing membrane localized activator function.