Signal Trans. and Intestinal Colonization by V.Cholerae

信号传输

基本信息

批准号：
7011141
负责人：
Kenneth Milan Peterson
金额：
$ 24.78万
依托单位：
LOUISIANA STATE UNIV HSC SHREVEPORT
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2008-02-28
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：了解粘膜病原体（例如弧菌霍乱）在人类肠粘膜上定居是实时销售疫苗衍生物合理发展的关键能够诱导防止霍乱和其他肠子的保护性免疫腹泻疾病。这些目前的肠胃外疫苗接种策略感染在很大程度上无效。尽管需要许多霍乱基因为了确定肠道定殖，分子机制是它们编码的蛋白质促进了对宿主组织的纤维依从性理解不佳。这项研究建议中描述的研究代表了试图在分子水平上理解V。霍乱辅助定殖因子ACFB和ACFC蛋白到肠道这种新兴人类病原体的定殖特性。 ACFB是一个75 kDa的内部属于大型信号转导蛋白的膜蛋白参与细菌趋化性。 v.Cholerae ACFB突变体显示了变化使用群板运动/趋化性测定并产生的运动表型霍乱毒素和毒素调节的菌毛水平降低。 ACFC是26 kDa 与细菌硫酸盐结合蛋白相似的周质蛋白参与溶质转运和细菌趋化性。 ACFC内的突变特别干扰V.霍乱朝向A迁移的能力半乳糖-6-硫酸盐的梯度在标准趋化测定中。这个建议概述了一系列旨在以分子详细了解的实验 ACFB和ACFC蛋白对弧菌趋化性/肠的贡献殖民化。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在改进的发展中的特性治疗和预防霍乱/肠道感染的方法。我在那里本提案中的四个具体目标：（1）趋化性/肠道殖民/菌毛生产测定法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的功能半乳糖-6-硫酸盐和该过程对弧菌的贡献趋化性/肠定植；（3）霍乱的婴儿小鼠模型感染和切除的肠道组织将用于确定 ACFBC基因内携带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在促进最大水平的菌毛合成水平中的作用。