Bacterial invasion of of eukaryotic tissues

许多肠道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年代早期的工作确定了志贺氏菌触发入侵的遗传机制是在一个大的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。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的事件进行彻底的分子定义。目前的实验方法包括使用测定细菌进入培养的各种组织来源的人上皮细胞系的方法。原核结构/功能或真核细胞过程的生化抑制剂被用于这些组织培养入侵试验，以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测量的。除上述方法外，还采用遗传技术克隆负责的细菌基因。通过抑制剂竞争试验、配体结合试验和感染真核细胞的mRNA分析来测量细菌配体的真核受体和特定真核细胞对细菌入侵的反应。从这些方法中获得的信息被整合起来，为所研究的每个途径提供细菌进入的机制理解。最近的研究表明，空肠弯曲杆菌的入侵依赖于宿主微管而不是微丝。接近的细菌发出明显的信号，触发宿主细胞伸出一个基于微管的手指状突起。空肠梭菌与位于膜小囊顶端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中释放Ca++，这两个事件都是空肠梭菌内化所必需的。进入核内体的细菌通过动力蛋白和微管在4小时内移动到核周区域。

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.