Regulation of Vibrio cholerae virulence by ToxR and TcpP

ToxR 和 TcpP 对霍乱弧菌毒力的调节

• 批准号: 7651339
• 负责人: ERIC S KRUKONIS
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7651339
• 关键词: Affect; Bile fluid; Binding; Binding Sites; Biochemical; Cells; Cessation of life; Cholera; Cholera Toxin; Cues; DNA Binding; DNA-Directed RNA Polymerase; Dehydration; Diarrhea; Disease; Elements; Ensure; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic; Goals; Growth; In Vitro; Infection; Life; Measures; Mediating; Membrane; Oral Rehydration Therapy; Pilum; Proteins; Regulation; Resistance; Role; System; Time; Toxin; Vibrio cholerae; Virulence; Virulence Factors; mutant; pandemic disease; porin; promoter; protein protein interaction; reconstitution; response; transcription factor

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)

描述（由申请人提供）：霍乱弧菌是腹泻病霍乱的病原体，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转录因子ToxR和TcpP的调节。本应用研究了ToxR和TcpP如何结合激活霍乱弧菌中的toxT启动子，从而触发毒力基因表达。此外，我们将研究ToxR如何以不依赖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的胆汁耐药。我们假设在ompU和toxT启动子上，ToxR通过不同的机制起作用。ToxR可以直接激活ompU，同时促进tp介导的toxT启动子激活。”最后，我们还将开发一个系统来观察霍乱弧菌活细胞膜上ToxR和TcpP的相互作用。这将使我们能够实时观察到诱导毒力的关键步骤，并使我们能够观察到ToxR/TcpP相互作用在各种环境条件下的任何变化。本课题的具体目的是：1 .确定ToxR活化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各事件在toxT激活中的作用III。荧光共振能量转移法测定TcpP诱导霍乱弧菌活细胞中ToxR/TcpP蛋白的相互作用