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种重要的危险因素 消化性溃疡疾病和胃恶性肿瘤的发展。长期的 本项目的目标是阐明H. 幽门螺杆菌致病，并开发有效的预防和治疗手段 感染的治疗。这项提议关注的是一种重要的毒性 幽门螺杆菌的决定因素：空泡毒素(VacA)。在预赛中 研究表明，我们已经提纯了细胞毒素，克隆了VacA基因，证明了 VacA等位基因中的嵌合体，表明S1型VacA之间存在关联 基因分型和消化性溃疡，并证明VacA结合并进入 上皮细胞。最近，我们已经证明了Vaca是一种 90 kDa亚基解离为以下功能活性单体 暴露在酸性酸碱度中。这项提议的假设是：(I)Vaca 与上皮细胞上的一种特定受体结合，(Ii)m1和m2型 VacA蛋白结合不同的细胞表面受体，(Iii)VacA 寡聚体通过网状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。这个 具体目标将是(I)表征VacA与血浆的结合 上皮细胞膜，(II)鉴定和分离VacA (Iii)研究上皮细胞对VacA的摄取。在……里面 第一个目标，我们将开发直接标记Vaca的方法， 鉴定其与几种不同类型的上皮细胞的结合， 比较VacA齐聚物和单体的结合性能，并比较 M1型和m2型VacA蛋白的结合特性。在第二个目标中，我们 将通过以下技术识别可能的VacA受体 交联法或免疫沉淀法，并寻求部分纯化 感受器。在第三个目标中，我们将使用深蚀刻电子显微镜 确定VacA是否插入到膜中的成像技术，并使用 药理药物和动力蛋白突变细胞系确定该途径 Vaca由此进入细胞。了解VacA结合的机制 进入和进入细胞可能最终导致策略的发展 预防和治疗幽门螺杆菌相关性胃十二指肠疾病。

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.