Genetic and Molecular Basis of Bacterial Invasion

描述(由申请人提供)：我们建议的研究的主旨是使用携带抗nramp等位基因的动物来检查小鼠感染伤寒的模型。在这个感染的实验模型中，我们建议检查特征良好的细菌突变，以及重点放在细菌在感染的急性阶段和慢性携带期间的位置。由于持续感染是无症状的，我们依靠一种非侵入性的半定量光子检测系统来识别受感染的动物。我们将使用携带荧光报告分子的细菌菌株来跟踪细菌在组织中的位置，并使用感染组织的荧光激活分类(FACS)来定位与入侵细菌相互作用的宿主细胞群。利用免疫组织化学、原位杂交、抗体标记的报告分子共聚焦显微镜以及组织的生化分析方法，我们将确定持续感染沙门氏菌的宿主细胞的确切性质。我们已经构建了整个鼠伤寒沙门氏菌基因组的斑点DNA微阵列，以及一个由40000个元素组成的小鼠DNA微阵列。我们计划使用这些实验工具来监测细菌和宿主对感染的反应，特别强调微生物和宿主从急性感染过程到系统并最终持续感染的转变。最后，我们将使用基于签名标记转座子分析的遗传筛选方法，以及一种新的基于DNA微阵列的筛选方法，以确定对持续感染和细菌传播至关重要的细菌基因。

DESCRIPTION (provided by applicant): The thrust of our proposed research is to examine the typhoid model of mouse infection using animals carrying the nramp\resistant allele. In this experimental model of infection, we propose to examine well-characterized bacterial mutants, as well as focusing on the location of the bacteria during the acute phases of infection and during chronic carriage. Because persistent infection is asymptomatic, we rely upon a non-invasive, semi-quantitative photonic detection system to identify infected animals. We will employ bacterial strains carrying fluorescent reporter molecules to follow the location of the bacteria in the tissue, as well as using fluorescence activated sorting (FACS) of infected tissue to locate the host cell populations interacting with the invading bacteria. Using immunohistochemical methods, in-situ hybridization, confocal microscopy with antibody tagged reporter molecules, as well as biochemical assays of tissue, we will determine the precise nature of the host cells persistently infected by Salmonella. We have constructed a spotted DNA microarray of the entire S. typhimurium genome, as well as a 40000-element mouse DNA microarray. We plan to use these experimental tools to monitor both the bacterial response and the host response to infection with particular emphasis on the transition by both the microbe and the host from an acute infectious process to a systemic and eventually persistent infection. Finally, we will employ genetic screening methods based on signature tagged transposon analysis, as well as a new novel DNA microarray-based screen, to identify bacterial genes that are essential for persistent infection and bacterial transmission.