Regulation of Vibrio cholerae virulence by ToxR and TcpP

描述（由申请人提供）：霍乱弧菌是霍乱弧菌的病原体，霍乱是一种使人衰弱的疾病，每年影响多达一千万人。这种疾病是由霍乱毒素引起的，霍乱毒素是一种蛋白质，其表达受两种膜定位的转录因子ToxR和TcpP调节。本申请研究了ToxR和TcpP如何联合收割机激活霍乱弧菌中的toxT启动子，从而触发毒力基因表达。此外，我们将研究ToxR如何以TcpP非依赖性方式激活第二个启动子ompLJ.孔蛋白OmpU的表达导致霍乱弧菌的胆汁抗性。我们假设ToxR在ompU和toxT启动子处通过不同的机制起作用。ToxR直接激活ompU，同时促进toxT处TcpP介导的启动子激活。“最后，我们还将开发一个系统，用于使用FRET观察活的V. cholesterol细胞膜中ToxR和TcpP的相互作用。这将使我们能够在真实的时间内观察毒性诱导的关键步骤，并使我们能够观察ToxR/TcpP相互作用响应于各种环境条件的任何变化。本项目的具体目标是：一。确定ToxR II激活ompU和toxT的机制。确定TcpP对DNA结合、ToxR相互作用和RNA聚合酶刺激的机制，以及每个事件在toxT激活中的作用III。使用FRET（荧光共振能量转移）测量tcpP诱导后活的霍乱弧菌细胞中ToxR/TcpP蛋白质-蛋白质相互作用

DESCRIPTION (provided by applicant): 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. 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. 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. ' 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. 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)