MOLECULAR MECHANISMS OF THE H. PYLORI VACUOLATING TOXIN

幽门螺杆菌是人类许多胃疾病的病因，包括消化性溃疡和胃腺癌。早期了解幽门螺杆菌发病机制的努力揭示了一种分泌细菌毒素（VacA）的存在，越来越多的证据表明这是一个重要的毒力因素。VacA引起哺乳动物细胞的大量退行性空泡化，这与消化性溃疡发病前的胃上皮侵蚀有关。本研究计划的主要目的是阐明VacA介导细胞毒性的基本机制。长期里程碑包括确定VacA的生化活性和细胞内靶标。本实验旨在验证VacA作为AB毒素介导细胞毒性的假设。VacA作为AB毒素的模型是重要的，因为它提出了与VacA进入宿主细胞并诱导空泡形成的基本机制直接相关的特定假设。AB毒素家族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。本提案的具体目标是阐明VacA分子结构，并开始确定毒素的结构-功能关系。具体目的是：b[1]确定VacA细胞毒性的细胞机制。为了实现这些目标，新的VacA融合蛋白将被遗传构建，以确定VacA是否被运输到细胞质中。此外，放射性标记的VacA将用于分析细胞内的蛋白水解过程。[2]鉴定VacA的结构特征对毒素的细胞活动很重要。为了实现这些目标，将构建VacA的突变形式和新的融合蛋白，以测试有关VacA结构-功能关系和生化活性的假设。据估计，幽门螺杆菌感染在发达国家的患病率为20-50%，在发展中国家为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再怎么强调也不为过。这些研究不仅有助于了解VacA介导的细胞毒性的基本机制，而且可能揭示使用减毒VacA的新策略，或VacA片段作为新的保护性疫苗的成分。

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.