Signal Trans. and Intestinal Colonization by V.Cholerae

描述(由申请人提供)：了解通过哪些机制 霍乱弧菌等粘膜病原体定植于人的肠粘膜 是合理开发减毒活疫苗衍生品的关键 能够诱导对霍乱和其他肠道的保护性免疫 腹泻病。目前针对这些疾病的非肠道疫苗接种策略 感染基本上是无效的。尽管许多霍乱弧菌需要基因 肠道定植的分子机制已被确定 它们编码的促进弧菌与宿主组织黏附的蛋白质是 人们对此知之甚少。本研究提案中描述的研究代表了一种 试图在分子水平上理解V。 霍乱辅助定植因子ACFB和ACFC蛋白对肠道的影响 这种新出现的人类病原体的定植特性。ACFB是一个75 kDa的内部 属于信号转导蛋白大家族的膜蛋白 参与细菌趋化性的。霍乱弧菌ACFB突变株显示一种改变的 利用蜂群平板运动/趋化试验的运动表型和产物 降低霍乱毒素和毒素共调节菌毛的水平。ACFC是一个26 kDa的 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作用。ACFC内部的突变 特别干扰霍乱弧菌向 标准趋化试验中半乳糖-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蛋白在弧菌趋化/肠道中的作用 殖民主义。这些研究的长期目标是理解 ACFB和ACFC的结构和功能，以便我们可以利用这些信息 关于这两种蛋白质的性质在发展中的改进 治疗和预防霍乱/肠道感染的方法。我在这呢 本建议的四个具体目标：(1)趋化性/肠道 将使用定植/菌毛生产分析来确定 促进趋化和/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-硫酸盐及其对弧菌的贡献 趋化/肠道定植；(3)霍乱幼鼠模型 感染和切除的肠道组织将被用来确定 携带acfbc基因突变的霍乱弧菌的定植缺陷； (4)基于重组酶的体外表达技术(RIVET)将阐明 ACFB在促进菌毛合成最大水平中的作用。

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.