Signal Transduction and Pilus/Toxin Regulation by Vibrio cholerae

描述（由申请人提供）：了解诸如霍乱弧菌等粘膜病原体在人类肠道粘膜定殖的机制是合理开发能够诱导对霍乱和其他肠道腹泻疾病产生保护性免疫的减毒活疫苗衍生物的关键。目前针对这些感染的肠外接种策略在很大程度上是无效的。虽然许多霍乱弧菌肠道定植所需的基因已被确定，但它们编码的蛋白质促进弧菌粘附宿主组织的分子机制尚不清楚。本研究计划中描述的研究代表了一种从分子水平上理解霍乱弧菌TcpI“趋化相关”蛋白对这一重要人类病原体肠道定植特性的贡献的方法。TcpI是一种75 kda的内膜蛋白，属于参与细菌趋化的信号转导蛋白大家族。霍乱弧菌tcpI突变体显示毒素协同调节菌毛（TCP）和霍乱毒素表达模式的改变。具体而言，tcpI突变体在通常缺乏菌毛/毒素产生的环境条件下（培养基pH为7.0-8.4）产生TCP定植菌毛和霍乱毒素。这些数据表明，在中性至碱性ph下，TcpI是Luria肉汤中菌毛毒素产生的抑制因子。本文提出了一系列旨在了解TcpI对菌毛毒素产生的贡献的实验。霍乱弧菌TCP和霍乱毒素是关键的毒力决定因素。由于TcpI有助于产生这些必需的毒力因子，因此了解TcpI的结构和功能非常重要。我们的长期目标是利用有关这种蛋白质特性的信息来开发治疗和预防霍乱肠道感染的改进方法。本提案有两个具体目的：(1)利用ELISA/弧菌凝集试验确定TcpI促进毒素/菌毛合成的特征；(2)确定TcpI在ToxR/TcpP/ToxT调控级联中的作用水平，通过过表达毒素/菌毛合成相关调控因子来影响毒力基因的表达。公共卫生相关性：霍乱弧菌是世界范围内发病率和疾病的主要原因。这种细菌具有复杂的调控级联反应，在时间和空间上控制肠内信号的毒力因子合成。拟议的研究将确定霍乱弧菌趋化蛋白在ph响应下调节霍乱毒素和毒素共同调节菌毛合成的机制。这可能允许制定控制这种可怕的人类疾病传播的策略。

DESCRIPTION (provided by applicant): Understanding the mechanisms by which mucosal pathogens such as Vibrio cholerae colonize the human intestinal mucosa is key to the rational development of live-attenuated vaccine derivatives capable of inducing protective immunity against cholera and other enteric diarrheal diseases. Current parenteral vaccination strategies for these infections are largely ineffective. Although many V. cholerae genes required for intestinal colonization have been identified, the molecular mechanisms by which the proteins they encode promote vibrio adherence to host tissue are poorly understood. The studies described in this research proposal represent an approach to understand at the molecular level, the contribution of the V. cholerae TcpI "chemotaxis related" protein to the intestinal colonization properties of this important human pathogen. TcpI is a 75-kDa inner membrane protein that belongs to a large family of signal transducing proteins involved in bacterial chemotaxis. V. cholerae tcpI mutants display an altered pattern of toxin- coregulated pilus (TCP) and cholera toxin expression. Specifically, tcpI mutants produce the TCP colonization pilus and cholera toxin under environmental conditions normally associated with a lack of pilus/toxin production (media pH of 7.0-8.4). These data show that TcpI is a repressor of pilus/toxin production in Luria Broth at neutral to basic pH. This proposal outlines a series of experiments aimed at understanding the contributions of TcpI to vibrio pilus/toxin production. V. cholerae TCP and cholera toxin are critical virulence determinants. Since TcpI contributes to the production of these essential virulence factors, it is important to understand the structure and function of TcpI. Our long-term goal is to use the information regarding the properties of this protein in the development of improved methods for treating and preventing cholera enteric infections. There are two SPECIFIC AIMS in this proposal: (1) Define the features of TcpI that promote the synthesis of toxin/pilus using ELISA/vibrio agglutination assays; (2) Determine at what level TcpI acts within the ToxR/TcpP/ToxT regulatory cascade to influence virulence gene expression by over-expressing relevant regulators of toxin/pilus synthesis. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a major cause of morbidity and disease worldwide. This bacterium has a complex regulatory cascade that temporally and spatially controls virulence factor synthesis in response to intraintestinal signals. The proposed studies will determine the mechanism by which a V. cholerae chemotaxis protein regulates cholera toxin and toxin co-regulated pilus synthesis in response to pH. The may allow the development of strategies to control the spread of this dread human disease.