Bacterial invasion of of eukaryotic tissues

肠道的许多细菌病原体具有穿透肠上皮细胞的能力，作为疾病起始的早期和必要步骤。我的实验室在20世纪80年代早期的工作确定了志贺氏菌引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染色体编码的。该项目旨在了解细菌内化的原核和真核要求，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。目前的实验方法涉及使用测定细菌进入各种组织来源的人上皮细胞的培养系的测定。在这些组织培养侵入测定中使用原核结构/功能或真核细胞过程的生物化学抑制剂，以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、配体结合测定和感染真核细胞的mRNA分析来测量细菌配体的真核受体和对细菌入侵的特异性真核细胞应答。从这些方法中的每一种获得的信息被整合，以提供对所研究的每个途径的细菌进入的机械理解。 最近的进展表明，空肠弯曲菌的侵袭依赖于宿主微管，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以微管为基础的指状突起. C.空肠与位于该膜延伸末端处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，其激活PI-3激酶并从细胞内储存释放Ca++，这两个事件都是C.空肠内化进入核内体的细菌在4小时内通过动力蛋白和微管移动到核周区域。

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.