Structure-function analysis of H. pylori VacA

幽门螺杆菌 VacA 的结构功能分析

• 批准号: 6749490
• 负责人: TIMOTHY L COVER
• 金额: $ 27.05万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6749490
• 关键词: Helicobacter; atomic force microscopy; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; bacterial toxins; conformation; cytotoxicity; disease /disorder model; epitope mapping; gastrointestinal disorder; gene expression; host organism interaction; immunoaffinity chromatography; intracellular transport; laboratory mouse; molecular pathology; molecular site; polymerization; protein reconstitution; protein structure function; site directed mutagenesis; tissue /cell culture; vesicle /vacuole; virulence; voltage /patch clamp

DESCRIPTION (provided by the applicant): Colonization of the human gastric mucosa by Helicobacter pylori is associated with an increased risk for development of peptic ulcer disease and distal gastric adenocarcinoma. Studies in a mouse model for H. pylori infection indicate that expression of a toxin (VacA) enhances the capacity of H. pylori to colonize the stomach, and immunization of mice with VacA results in protective immunity. VacA contributes to gastric mucosal damage, and analysis of vacA alleles in H. pylori isolates from humans suggests that VacA plays a role in the pathogenesis of peptic ulcer disease. The effects of VacA on eukaryotic cells include vacuolation, altered trafficking within the endocytic pathway, membrane channel formation, and apoptosis. The VacA mechanism of action remains incompletely understood. Based on our preliminary studies, we hypothesize that the mature VacA toxin can be divided into three functional domains: (i) an N-terminal hydrophobic region (amino acids 1-32) involved in membrane insertion, transmembrane protein dimerization, and membrane channel formation; (ii) an N-terminal region (amino acids 33-422) that is required for intracellular toxin activities (cell vacuolation and apoptosis); and (iii) a C-terminal domain (amino acids 423-821) involved in binding of VacA to eukaryotic cells. This proposal outlines plans for in-depth structure-function analysis of these three domains. We will use multiple experimental approaches, including several mutagenesis strategies, assays of VacA channel activity, an in vitro system for analyzing peptide insertion into membranes, expression of recombinant VacA, a system for intracellular VacA expression, mapping of VacA structure with recombinant anti-VacA antibodies, and use of a mouse model to examine the functions of VacA in vivo. These studies should result in a better understanding of VacA structure and function, and insights into the VacA mechanism of action. Ultimately, these studies may lead to advances in the treatment or prevention of H. pylori-associated human diseases.

描述（由申请人提供）：人类胃的定殖 幽门螺杆菌的粘膜与增加的风险有关 消化性溃疡疾病和胃远端腺癌的发展。研究 在幽门螺杆菌感染的小鼠模型中，表明毒素的表达 （vaca）增强了幽门螺杆菌在胃定居的能力，并增强 带有VACA的小鼠免疫会导致保护性免疫。 Vaca贡献 对胃粘膜损伤以及幽门螺杆菌分离株中VACA等位基因的分析 来自人类的表明，VACA在消化性溃疡的发病机理中起作用 疾病。 VACA对真核细胞的影响包括空泡，改变 内吞途径，膜通道形成和 凋亡。 VACA作用机理仍然尚未完全理解。基于 在我们的初步研究中，我们假设成熟的Vaca毒素可以是 分为三个功能域：（i）N末端疏水区域 （氨基酸1-32）参与膜插入，跨膜蛋白 二聚化和膜通道的形成； （ii）N末端区域（氨基 酸33-422）是细胞内毒素活性所需的（细胞 液泡和凋亡）； （iii）C末端结构域（氨基酸423-821） 参与VACA与真核细胞的结合。该提案概述了计划 对于这三个域的深入结构 - 功能分析。我们将使用 多种实验方法，包括几种诱变策略， VACA通道活动的测定，一种用于分析肽的体外系统 插入膜，表达重组VACA，一个系统的系统 细胞内VACA表达，重组VACA结构的映射 抗VACA抗体，并使用小鼠模型检查VACA的功能 体内。这些研究应该使对Vaca的了解更好 结构和功能，以及对VACA作用机理的见解。 最终，这些研究可能会导致治疗或预防的进步 幽门螺杆菌相关的人类疾病。