Bacterial invasion of of eukaryotic tissues

细菌侵入真核组织

• 批准号: 6433477
• 负责人: DENNIS J KOPECKO
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433477
• 关键词: bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; confocal scanning microscopy; enteric bacteria; eukaryote; fluorescence microscopy; gastrointestinal epithelium; gastrointestinal infection; messenger RNA; molecular cloning; molecular pathology; pathologic process; prokaryote; receptor binding; tissue /cell culture; transmission electron microscopy; video microscopy; virulence

Many bacterial pathogens of the intestinal tract have as an early and necessary step in initiation of disease the ability to penetrate gut epithelial cells. My lab's work in the early 1980's established that Shigella's genetic machinery to trigger invasion is encoded on a large virulence-associated plasmid. More recent studies have shown that the invasion ability of other enteric bacteria is chromosomally encoded. This project is aimed at understanding the prokaryotic and eukaryotic requirements for bacterial internalization, with the ultimate aim being a thorough molecular definition of the events involved in bacterial invasion of eukaryotic tissues. Current experimental approaches involve the use of assays measuring bacterial entry into cultured lines of human epithelial cells of various tissue origins. Biochemical inhibitors of prokaryotic structure/function or of eukaryotic cell processes are employed in these tissue culture invasion assays to examine the requirements for bacterial uptake. Direct visualization of bacterial entry is measured via transmission electron microscopy, video microscopy, fluorescent microscopy, and confocal microscopy. In addition to the approaches described above, genetic techniques are employed to clone the responsible bacterial genes. Eukaryotic receptors for bacterial ligands and specific eukaryotic cell responses to bacterial invasion are measured via inhibitor competition assays, ligand binding assays, and mRNA analyses of infected eukaryotic cells. The information gained from each of these approaches is integrated to provide a mechanistic understanding of bacterial entry for each pathway studied. Recent progress has revealed that Campylobacter jejuni invasion is dependent upon host microtubules but not microfilaments. Apparent signalling by the approaching bacterium triggers the host cell to extend a microtubule- based, fingerlike projection. C. jejuni interacts with a host receptor located in membrane caveolae at the tip of this membrane extension. This bacterial-host "ligand -receptor" interaction activates a signal transduction cascade that activates PI-3 kinase and releases Ca++ from intracellular stores, both events of which are required for C. jejuni internalization. The entering bacterium within an endosome moves via dynein and microtubules to the perinuclear region over 4 hrs.

许多肠道细菌病原体具有穿透肠上皮细胞的能力，这是疾病发生的早期和必要步骤。我的实验室在 1980 年代初期的工作证实，志贺氏菌触发入侵的遗传机制是在一个大型毒力相关质粒上编码的。最近的研究表明，其他肠道细菌的入侵能力是由染色体编码的。该项目旨在了解原核和真核细菌内化的要求，最终目标是对细菌侵入真核组织所涉及的事件进行彻底的分子定义。目前的实验方法涉及使用测定方法来测量细菌进入不同组织来源的人类上皮细胞培养系的情况。在这些组织培养物侵袭测定中采用原核结构/功能或真核细胞过程的生化抑制剂来检查细菌摄取的要求。通过透射电子显微镜、视频显微镜、荧光显微镜和共聚焦显微镜来测量细菌进入的直接可视化。除了上述方法之外，还采用遗传技术来克隆负责的细菌基因。通过抑制剂竞争测定、配体结合测定和受感染真核细胞的 mRNA 分析来测量细菌配体的真核受体和真核细胞对细菌入侵的特异性反应。从这些方法中获得的信息被整合起来，以提供对所研究的每个途径的细菌进入的机制的理解。 最近的进展表明，空肠弯曲杆菌的入侵依赖于宿主微管，而不是微丝。接近的细菌发出的明显信号会触发宿主细胞延伸基于微管的指状突起。 空肠弯曲杆菌与位于该膜延伸尖端的膜小窝中的宿主受体相互作用。 这种细菌-宿主“配体-受体”相互作用激活信号转导级联，激活PI-3激酶并从细胞内储存释放Ca++，这两个事件都是空肠弯曲菌内化所必需的。进入内体内的细菌在 4 小时内通过动力蛋白和微管移动到核周区域。