Regulation of Vibrio cholerae virulence by ToxR and TcpP

描述(申请人提供)：霍乱弧菌是腹泻疾病霍乱的病原体，霍乱是一种使人衰弱的疾病，每年影响多达1000万人。这种疾病是由霍乱毒素引起的，霍乱毒素是一种蛋白质，其表达受到两种膜定位转录因子ToxR和TCPP的调节。这项应用研究了ToxR和TCPP如何结合激活霍乱弧菌中的toxT启动子，从而触发毒力基因的表达。此外，我们还将研究ToxR如何以不依赖于TCPP的方式激活第二个启动子ompLJ。孔蛋白OmpU的表达导致霍乱弧菌对胆汁产生耐药性。我们推测ToxR在ompU和toxT启动子上通过不同的机制发挥作用。ToxR在促进TCPP介导的启动子激活的同时，直接激活ompU。‘最后，我们还将开发一个利用FRET观察霍乱弧菌活细胞膜上ToxR和TCPP相互作用的系统。这将使我们能够实时观察毒力诱导的关键步骤，并允许我们观察ToxR/TCPP相互作用在各种环境条件下的任何变化。本项目的具体目标是：1.确定ToxR II激活ompU和toxT的机制；3.确定TCPP刺激DNA结合、ToxR相互作用和RNA聚合酶激活的机制以及各事件在毒素T激活中的作用；3.利用荧光共振能量转移技术(FRET)检测TCPP诱导的霍乱弧菌活细胞中ToxR/TCPP蛋白-蛋白质的相互作用

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)