Signal Transduction and Pilus/Toxin Regulation by Vibrio cholerae

描述（由申请方提供）：了解粘膜病原体（如霍乱弧菌）定植于人肠粘膜的机制是合理开发能够诱导针对霍乱和其他肠道传染病的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胃肠外疫苗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，它们编码的蛋白质促进弧菌粘附宿主组织的分子机制知之甚少。本研究提案中描述的研究代表了一种在分子水平上理解霍乱弧菌TcpI“趋化性相关”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是一种分子量为75 kDa的内膜蛋白，属于参与细菌趋化性的信号转导蛋白大家族。霍乱弧菌tcpI突变体显示毒素共调节菌毛（TCP）和霍乱毒素表达模式的改变.具体地说，tcpI突变体在通常与缺乏菌毛/毒素产生相关的环境条件下（培养基pH 7.0-8.4）产生TCP定殖菌毛和霍乱毒素。这些数据表明，TcpI是一个阻遏物的菌毛/毒素生产在Luria肉汤在中性至碱性pH值。该建议概述了一系列的实验，旨在了解TcpI弧菌菌毛/毒素生产的贡献。霍乱弧菌TCP和霍乱毒素是关键的毒力决定因子。由于TcpI有助于这些重要毒力因子的产生，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关于这种蛋白质性质的信息，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）使用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调节级联中的作用水平，以通过过度表达毒素/菌毛合成的相关调节因子来影响毒力基因表达。公共卫生相关性：霍乱弧菌是世界范围内发病和疾病的主要原因。该细菌具有复杂的调节级联，其在时间和空间上控制响应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调节霍乱毒素和毒素共调节菌毛合成响应pH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.