Mycobacteria Invasion and Persistence

分枝杆菌入侵和持续存在

• 批准号: 6669669
• 负责人: Eric J. Brown
• 金额: $ 18.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6669669
• 关键词: Mycobacterium; bacteria infection mechanism; bacterial genetics; bactericidal immunity; clinical research; gene targeting; genetic mapping; genetic models; genetic screening; human subject; laboratory mouse; leukocyte activation /transformation; macrophage; mutant; plasmids; transposon /insertion element

DESCRIPTION (provided by applicant): Mycobacteria infections remain a major problem in human health. Mycobacterium tuberculosis infects approximately 1/3 of the world's population; Mycobacterium leprae (ML) is an endemic infection in many parts of the world. M. marinum (Mm) is a natural pathogen of fish and can cause chronic disease in frogs with many features of tuberculosis. Because of this, its rapid growth in vitro, its suitability for forward genetics, its minimal pathogenicity for laboratory" workers, and its ability to grow in mammalian macrophages in vitro, Mm can be a facile model to discover genes involved in pathogenesis of Mycobacteria infections. We have developed a transposon mutagenesis plasmid for use in Mm that has proven to provide an excellent library of random mutations. From that random insertion library, we have screened >1000 mutants for ability to grow in macrophages and have characterized >25 mutants that fail to grow normally. For each mutant, we have sequenced the transposon insertion site, and, using this information, we have discovered several genes required for intracellular growth of Mm. We propose to focus on a more detailed characterization of two most interesting mutants to understand the roles of the targeted genes in host cell invasion by, and intracellular growth of, Mycobacteria. In each case, we have shown that the Mtb homologue of the targeted Mm genes will complement the phenotypic defects discovered in the mutant. This allows a rapid and thorough investigation of the roles of these Mtb genes in a model that is capable of intracellular growth and ultimately of extension to in vivo models of infection. The purpose of the current proposal is to use these mutants to develop a better understanding of Mycobacteria entry into and survival in macrophages, and of macrophage response to Mycobacteria infection, critical events in the pathogenesis of disease. Our specific aims are: 1. Characterize the mip locus, required by Mm for host cell invasion and intracellular survival in macrophages. 2. Determine how the GDP-mannose synthesis operon regulates macrophage activation and intracellular survival. 3. Carry out a genetic screen for Mm genes specifically required for intracellular growth in macrophages.

描述（由申请人提供）：分枝杆菌感染仍然是人类健康的一个主要问题。结核分枝杆菌感染了世界上大约 1/3 的人口；麻风分枝杆菌 (ML) 是世界许多地区的地方性感染。海分枝杆菌 (Mm) 是鱼类的天然病原体，可引起青蛙的慢性疾病，具有多种结核病特征。因此，Mm 在体外快速生长，适合正向遗传学，对实验室工作人员的致病性极低，并且能够在体外哺乳动物巨噬细胞中生长，因此 Mm 可以成为发现分枝杆菌感染发病机制相关基因的简便模型。我们开发了一种用于 Mm 的转座子诱变质粒，已证明可以提供出色的随机突变文库，我们从该随机插入文库中筛选出超过 1000 个突变体的生长能力。巨噬细胞并鉴定了超过 25 个无法正常生长的突变体，对于每个突变体，我们对转座子插入位点进行了测序，并且利用这些信息，我们发现了 Mm 细胞内生长所需的几个基因。两个最有趣的突变体的详细表征，以了解目标基因在分枝杆菌宿主细胞入侵和细胞内生长中的作用，在每种情况下，我们都表明目标 Mm 基因的 Mtb 同源物将互补。在突变体中发现的表型缺陷。这使得能够在能够细胞内生长并最终扩展到体内感染模型的模型中快速、彻底地研究这些 Mtb 基因的作用。当前提案的目的是利用这些突变体更好地了解分枝杆菌进入巨噬细胞并在巨噬细胞中存活，以及巨噬细胞对分枝杆菌感染的反应，这是疾病发病机制中的关键事件。我们的具体目标是： 1. 表征 Mip 基因座，这是 Mm 宿主细胞侵袭和巨噬细胞胞内存活所需的。 2. 确定GDP-甘露糖合成操纵子如何调节巨噬细胞活化和细胞内存活。 3. 对巨噬细胞细胞内生长特别需要的 Mm 基因进行遗传筛选。