Signal Trans. and Intestinal Colonization by V.Cholerae

描述（由申请人提供）：了解 粘膜病原体如霍乱弧菌定殖于人肠粘膜 是合理开发减毒活疫苗衍生物的关键 能够诱导针对霍乱和其他肠道疾病的保护性免疫力， 妇科疾病。目前的胃肠外疫苗接种策略， 感染基本上是无效的。尽管许多霍乱弧菌基因需要 已经确定了肠道定植的分子机制， 它们编码的蛋白质促进弧菌粘附到宿主组织， 不太了解。本研究提案中描述的研究代表了 试图在分子水平上理解V的贡献。 肠内胆固醇辅助定植因子AcfB和AcfC蛋白 这种新兴的人类病原体的殖民特性。AcfB是一个75 kDa的内 属于信号转导蛋白大家族的膜蛋白 参与细菌的趋化性。霍乱弧菌acfB突变体显示出改变的 使用群体平板运动性/趋化性测定法测定运动性表型，并产生 降低霍乱毒素和毒素共调节菌毛的水平。AcfC是一个26 kDa 一种与细菌硫酸盐结合蛋白非常相似的周质蛋白 参与溶质运输和细菌趋化性。acfC内的突变 特异性地干扰霍乱弧菌迁移到 半乳糖-6-硫酸盐的梯度。这项建议 概述了一系列旨在了解分子细节的实验， AcfB和AcfC蛋白对弧菌趋化性/肠道的贡献 殖民化这些研究的长期目标是了解 AcfB和AcfC的结构和功能，以便我们可以使用信息 关于这两种蛋白质的性质， 治疗和预防霍乱/肠道感染的方法。我来了 本建议中的四个具体目标：（1）趋化性/肠道 定殖/菌毛产生测定将用于确定 促进趋化性和/或菌毛合成的AcfB;（2）体外/体内模型 系统将定义绑定所需的AcfC功能 半乳糖-6-硫酸及其对弧菌的作用 趋化性/肠道定植;（3）幼鼠霍乱模型 感染和切除的肠组织将用于确定 携带acfBC基因内突变的霍乱弧菌的定殖缺陷; (4)基于重组酶的体外表达技术（RIVET）将阐明 AcfB在促进最大水平的菌毛合成中的作用。

DESCRIPTION (provided by the 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 is poorly understood. The studies described in this research proposal represent an attempt to understand at the molecular level, the contribution of the V. cholerae accessory colonization factor AcfB and AcfC proteins to the intestinal colonization properties of this emerging human pathogen. AcfB is a 75 kDa inner membrane protein that belongs to a large family of signal transducing proteins involved in bacterial chemotaxis. V.cholerae acfB mutants display an altered motility phenotype using a swarm plate motility/chemotaxis assay and produce reduced levels of cholera toxin and toxin-coregulated pilus. AcfC is a 26 kDa periplasmic protein that closely resembles bacterial sulfate binding proteins involved in solute transport and bacterial chemotaxis. Mutations within acfC specifically interfere with the ability of V. cholerae to migrate toward a gradient of galactose-6-sulfate in a standard chemotaxis assay. This proposal outlines a series of experiments aimed at understanding in molecular detail the contributions of the AcfB and AcfC proteins to vibrio chemotaxis/intestinal colonization. The long-term goal of these studies is to understand the structure and function of AcfB and AcfC so that we can use the information regarding the properties of these two proteins in the development of improved methods for treating and preventing cholera/enteric infections. There I are four specific aims in the present proposal: (1) chemotaxis/intestinal colonization/pilus production assays will be used to determine the features of AcfB that promote chemotaxis and/or pilus synthesis; (2) in vitro/in vivo model systems will define the features of AcfC required for binding galactose-6-sulfate and the contribution of this process to vibrio chemotaxis/intestinal colonization; (3) the infant mouse model of cholera infection and excised intestinal tissue will be used to determine the nature of the colonization defect in V. cholerae carrying mutations within acfBC genes; (4) recombinase-based in vitro expression technology (RIVET) will elucidate the role of AcfB in promoting maximal levels of pilus synthesis.