BACTERIAL INVASION OF OF EURKARYOTIC 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。除了上述的机械方法，遗传技术被用来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。

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 mechanistic 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.