Bacterial invasion of of eukaryotic tissues

肠道的许多细菌病原体具有穿透肠道上皮细胞的能力，这是疾病发生的早期和必要步骤。我的实验室在20世纪80年代初的工作确立了S的工作，即志贺氏菌触发入侵的遗传机制是在一个与毒力相关的大质粒上编码的。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。本项目旨在了解原核生物和真核生物对细菌内化的要求，最终目的是对细菌入侵真核组织所涉及的事件进行彻底的分子定义。目前的实验方法包括使用检测细菌进入不同组织来源的人上皮细胞培养系的分析。在这些组织培养侵袭试验中使用原核结构/功能或真核细胞过程的生化抑制物来检查细菌摄取的要求。细菌进入的直接可视化是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的。除了上述方法外，还利用基因技术来克隆负责的细菌基因。通过抑制物竞争试验、配体结合试验和感染真核细胞的mRNA分析，检测细菌配体的真核细胞受体和对细菌入侵的特异性真核细胞反应。从这些方法中的每一种获得的信息被整合起来，以提供对所研究的每一条途径的细菌进入的机制的理解。最近的研究表明，空肠弯曲菌的侵袭依赖于宿主的微管，而不是微丝。接近的细菌发出的明显信号触发宿主细胞延伸以微管为基础的指状突起。空肠弯曲菌与位于膜延伸顶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”的相互作用激活了一系列信号转导，激活了PI-3激酶，并从细胞内的存储中释放出钙离子，这两个事件都是空肠弯曲菌内化所必需的。进入内小体的细菌通过动力蛋白和微管移动到核周区域，超过4小时。

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.