BACTERIAL INVASION OF EUKARYOTIC TISSUES

真核组织的细菌入侵

• 批准号: 6543813
• 负责人: D 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/6543813
• 关键词: 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 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.

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~志贺氏菌~S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。除了上述的机械方法外，基因技术还被用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。