Structure-function analysis of H. pylori VacA

描述(申请人提供)：人胃的定植 幽门螺杆菌感染的粘膜与罹患幽门螺杆菌相关 消化性溃疡和远端胃腺癌的发生。研究 在幽门螺杆菌感染小鼠模型中表明毒素的表达 (VacA)增强幽门螺杆菌在胃内定植的能力，以及 用VacA免疫小鼠可产生保护性免疫。Vaca贡献 幽门螺杆菌VacA等位基因对胃粘膜损伤的影响 提示VacA在消化性溃疡的发病机制中起作用 疾病。VacA对真核细胞的影响包括空泡化，改变 内吞途径内的运输、膜通道的形成以及 细胞凋亡。VacA的作用机制仍不完全清楚。基座 在我们的初步研究中，我们假设成熟的Vaca毒素可能是 分为三个功能区：(I)N-末端疏水区 (氨基酸1-32)参与膜插入，跨膜蛋白 二聚和膜通道的形成；(Ii)N-末端区域(氨基 酸33-422)是细胞内毒素活动(细胞)所必需的 (Iii)C-末端结构域(氨基酸423-821)。 参与VacA与真核细胞的结合。这份提案概述了计划 对这三个领域进行深入的结构-功能分析。我们将使用 多种实验方法，包括几种诱变策略， 体外多肽分析系统--VacA通道活性测定 一种将重组VacA插入膜中表达的系统 VacA胞内表达及重组定位VacA结构 抗VacA抗体，并使用小鼠模型检测VacA的功能 在活体内。这些研究应该会对Vaca有更好的理解 结构和功能，以及对Vaca作用机制的洞察。 最终，这些研究可能会在治疗或预防方面取得进展。 幽门螺杆菌相关的人类疾病。

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.