BINDING AND UPTAKE OF H PYLORI VACUOLATING CYTOTOXIN

描述（取自应用程序） 幽门螺杆菌感染是一个重要的危险因素， 消化性溃疡疾病和胃恶性肿瘤的发展。长期 本项目的目的是阐明H. 幽门螺杆菌引起的疾病，并制定有效的预防手段， 治疗感染。该提案关注的是一种重要的毒力 H的行列式pylori：空泡细胞毒素（VacA）。初步 在研究中，我们纯化了细胞毒素，克隆了vacA基因， vacA等位基因的嵌合现象，证明了s1型vacA 基因型和消化性溃疡，并证明VacA结合并进入 上皮细胞最近，我们已经证明VacA是一个十二聚体， 90 kDa亚基，其解离成功能活性单体， 暴露于酸性pH值。该建议的假设是（i）VacA 与上皮细胞上的特异性受体结合，（ii）M1和M2型 VacA蛋白结合不同的细胞表面受体，（iii）VacA 寡聚体通过网格蛋白介导的内吞作用进入细胞，以及（iv）当 酸化后，VacA插入膜中形成离子传导通道。的 具体目标是（i）表征VacA与血浆的结合 上皮细胞膜，（ii）鉴定和分离VacA 受体，和（iii）研究上皮细胞对VacA的摄取。在 第一个目标，我们将开发直接标记VacA的方法， 表征其与几种不同类型的上皮细胞的结合， 比较VacA寡聚体和单体的结合特性，并比较 m1和m2型VacA蛋白的结合特性。第二个目标，我们 将通过以下技术鉴定推定的VacA受体： 交联或免疫沉淀方法，并寻求部分纯化 受体。在第三个目标中，我们将使用深蚀刻电子显微镜 成像技术，以确定是否VacA插入膜，并使用 药理剂和动力蛋白突变细胞系来确定途径 从而VacA进入细胞。了解VacA结合的机制 进入细胞最终可能会导致 预防和治疗H.与幽门相关的胃十二指肠疾病。

DESCRIPTION (taken from the application) Helicobacter pylori infection is a significant risk factor for the development of peptic ulcer disease and gastric malignancies. The long-term objectives of this project are to elucidate pathogenic mechanisms whereby H. pylori causes disease, and to develop effective means for prevention and treatment of infection. This proposal focuses on an important virulence determinant of H. pylori: the vacuolating cytotoxin (VacA). In preliminary studies, we have purified the cytotoxin, cloned the vacA gene, demonstrated mosaicism in vacA alleles, demonstrated an association between type s1 vacA genotypes and peptic ulceration, and demonstrated that VacA binds and enters epithelial cells. Most recently, we have shown that VacA is a dodecamer of 90 kDa subunits that dissociates into functionally active monomers following exposure to acidic pH. The hypotheses of this proposal are (i) that VacA binds to a specific receptor on epithelial cells, (ii) that type m1 and m2 VacA proteins bind to different cell-surface receptors, (iii) that VacA oligomers enter cells via clathrin-mediated endocytosis, and (iv) that when acidified, VacA inserts into membranes to form ion-conductive channels. The specific aims will be (i) to characterize the binding of VacA to the plasma membrane of epithelial cells, (ii) to identify and isolate the VacA receptor, and (iii) to investigate uptake of VacA by epithelial cells. In the first aim, we will develop methods for directly labelling VacA, characterize its binding to several different types of epithelial cells, compare the binding properties of VacA oligomers and monomers, and compare binding properties of type m1 and m2 VacA proteins. In the second aim, we will identify the putative VacA receptor by techniques involving cross-linking or immunoprecipitation methods, and seek to partially purify the receptor. In the third aim, we will use deep-etch electron microscopic imaging techniques to determine whether VacA inserts into membranes, and use pharmacologic agents and dynamin-mutant cell lines to determine the pathway whereby VacA enters cells. Understanding the mechanisms whereby VacA binds to and enters cells may ultimately result in the development of strategies to prevent and treat H. pylori-associated gastroduodenal illnesses.