MOLECULAR MECHANISMS OF THE H. PYLORI VACUOLATING TOXIN

幽门螺杆菌是人类许多胃病的病原体，包括消化性溃疡和胃腺癌。早期对幽门螺杆菌致病机制的研究揭示了一种分泌型细菌毒素(VacA)的存在，越来越多的证据表明VacA是一个重要的毒力因子。VacA引起哺乳动物细胞的大量退行性空泡化，这与消化性溃疡疾病发生前的胃上皮侵蚀有关。这项研究计划的广泛目标是阐明VacA介导细胞毒性的基本机制。长期的里程碑包括鉴定VacA的生化活性和细胞内靶标。这一提议中的实验旨在验证VacA作为AB毒素介导细胞细胞毒性的假设。VacA作为AB毒素的模型很重要，因为它提出了与VacA进入宿主细胞并诱导空泡化的基本机制直接相关的特定假设。AB类毒素具有许多不同的特性，包括霍乱、白喉、破伤风和炭疽毒素等重要成员。这项建议的具体目的是为了阐明VacA的分子结构，并开始识别毒素的结构-功能关系。其具体目的是：[1]确定VacA细胞毒性的细胞机制。为了实现这些目标，将从基因上构建新的VacA融合蛋白，以确定VacA是否被运输到细胞质。此外，还将对放射性标记的VacA进行分析，以进行细胞内蛋白质分解处理。[2]鉴定对毒素细胞活性重要的VacA结构特征。为了实现这些目标，将从基因上构建VacA的突变形式和新型融合蛋白，以检验关于VacA结构-功能关系和生化活性的假说。据估计，发达国家幽门螺杆菌感染率为20%-50%，发展中国家为70%-90%，因此开发有效的幽门螺杆菌疫苗、化疗药物和诊断方法的重要性怎么强调都不为过。这些研究不仅有助于深入了解VacA介导的细胞毒性的基本机制，而且可能揭示在新的保护性疫苗中使用减毒VacA或VacA IS片段的新策略。

Helicobacter pylori is an etiologic agent of a number of gastric disorders in humans, including peptic ulceration and gastric adenocarcinomas. Early efforts to understand H. pylori pathogenesis revealed the presence of a secreted bacterial toxin (VacA) which mounting evidence indicates is an important virulence factor. VacA causes massive degenerative vacuolation of mammalian cells which has been implicated in the gastric epithelial erosion preceding the onset of peptic ulcer disease. The broad objective of this research program si to elucidate the fundamental mechanisms by which VacA mediates cellular cytotoxicity. Long-term milestones include identification of the biochemical activity and intracellular targets of VacA. Experiments in this proposal are designed to test the hypothesis that VacA mediates cellular cytotoxicity as an AB toxin. The model of VacA as an AB toxin is important because it suggests specific hypotheses directly relevant to the fundamental mechanism by which VacA enters host cells and induces vacuolation. The AB family of toxins share a number of distinct properties, and includes prominent members such as cholera, diphtheria, tetanus, and anthrax toxins. The specific aims in this proposal are designed to elucidate the VacA molecular structure and begin to identify structure-function relationships of the toxins. The specific aims are: [1] To identify cellular mechanisms of VacA cytotoxicity. To achieve these goals, novel VacA fusion proteins will be genetically constructed to determine if VacA is trafficked to the cytosol. In addition, radiolabeled VacA will be analyzed for intracellular proteolytic processing. [2] To identify VacA structural characteristics important to the toxin's cellular activities. To achieve these goals, mutant forms of VacA and novel fusion proteins will be genetically constructed to test hypotheses about VacA structure-function relationships and biochemical activities. Because it is estimated that the prevalence of H. pylori infection in developed countries is 20-50%, and 70-90% in developing countries, the importance of developing efficacious vaccines, chemotherapeutics, and diagnostics of H. pylori cannot be overstated. These studies will not only contribute to under understanding of the fundamental mechanisms of VacA-mediated cytotoxicity, but may reveal novel strategies for using attenuated VacA, or fragments of VacA is components in new protective vaccines.