Genetic and Cellular Basics of H. pylori pathogenesis

描述(申请人提供)：幽门螺杆菌是一种独特的微生物，适合在人的胃中生存。它通常无症状地感染着世界上一半以上的人口。然而，15%的感染者会患上溃疡病或胃癌。我们建议的研究集中在单个细菌决定簇CagA上，这是一种由细菌分泌机构转位到胃上皮细胞的效应蛋白。CagA可以说是这种微生物最重要的毒力决定因素，因为它的存在与溃疡疾病和胃恶性肿瘤明显相关，它对宿主细胞生物学的影响使其处于微生物发病机制和癌症生物学之间的关键位置。我们建议解决三个基本问题，这些问题与幽门螺杆菌CagA蛋白之间的最初接触如何对微生物有利和对宿主不利。首先，CagA扰乱宿主上皮细胞生物学的分子机制是什么？第二，CagA在幽门螺杆菌与宿主细胞表面密切相关的生存中起什么作用？最后，在动物模型中，将CagA输送到胃粘膜的体内后果是什么？我们之前的研究将重点放在CagA蛋白和顶端连接复合体的宿主蛋白之间的相互作用上，顶端连接复合体是细胞的一个区域，控制细胞的极性，提供粘膜的屏障功能，并调节上皮细胞的分化、生长和伤口愈合能力。CAGA还触发一个或多个受体酪氨酸激酶信号通路。我们预测，这些功能并不是无关的，而是CagA篡夺了生长因子受体信号和心尖连接之间的内在联系。我们还假设幽门螺杆菌利用CagA来修饰宿主细胞表面以达到定植目的，将特定的宿主蛋白招募到细菌附着的位置，破坏细胞极性，并打开上皮连接。我们提出了一个CagA作用的假说，我们相信该假说可以在体外和体内进行测试。我们已经开发了新的显微工具和一个实验上容易处理的培养中极化细胞慢性感染的模型，以研究CagA-宿主细胞相互作用的细胞生物学。此外，我们还将广泛利用直接生化分离和转录分析在分子水平上剖析CagA的作用。最后，我们建议在更复杂但更相关的动物宿主的胃部环境中测试我们从精确的体外分析中收集的观察结果。

DESCRIPTION (provided by applicant): H. pylori is a remarkable microorganism that is uniquely adapted for survival in the human stomach. It infects, usually asymptomatically, over one half of the world's human population. However 15% of those infected will develop ulcer disease or gastric cancer. Our proposed research focuses on a single bacterial determinant, CagA, an effector protein translocated by a bacterial secretory apparatus into gastric epithelial cells. CagA is arguably the most important virulence determinant of this microbe, since its presence is clearly correlated with ulcer disease and gastric malignancy, and its effects on host cell biology place it at the crux between microbial pathogenesis and cancer biology. We propose to address three fundamental questions related to how the initial encounter between the H. pylori CagA protein works to the advantage of the microorganism and to the detriment of the host. First, what are the molecular mechanisms by which CagA perturbs host epithelial cell biology? Second, what is the role of CagA for H. pylori survival in close association with the host cell surface? And finally, what are the in vivo consequences of CagA delivery to the gastric mucosa in an animal model? Our previous research directs us to a clear focus on the interaction of the CagA protein and the host proteins of the apical junctional complex, a domain of the cell that controls cell polarity, provides the barrier function of the mucosa, and regulates the differentiation, growth and wound healing capacities of epithelia. CagA also triggers one, or possibly more, receptor tyrosine kinase signaling pathways. We predict that these are not unrelated functions, but rather that CagA usurps an intrinsic link between growth factor receptor signaling and the apical junctions. We have also hypothesized that H. pylori utilizes CagA to modify the host cell surface for colonization purposes, recruiting specific host proteins to the sites of bacterial attachment, disrupting cell polarity, and opening the epithelial junctions. We have proposed a hypothesis of CagA action that we believe can be tested both in-vitro and in-vivo. We have developed novel microscopic tools and an experimentally tractable model of chronic infection of polarized cells in culture to examine the cell biology of the CagA-host cell interaction. Moreover, we will also make extensive use of direct biochemical fractionation and transcriptional analysis to dissect the role of CagA at a molecular level. Finally we propose testing the observations we have gathered from precise in-vitro analysis in the more complex, but more relevant environment of the stomach in an animal host.