Genes Associated with M .avium Pathogenesis

描述（由申请人提供）： 由鸟分枝杆菌复合体引起的感染在艾滋病患者和患有慢性潜在肺部疾病（如肺气肿和支气管扩张）的患者中很常见。尽管抗HIV-1治疗在减少鸟分枝杆菌病的病例方面取得了成功，但最近发表的工作表明，在许多领域，鸟分枝杆菌是一种环境细菌，其人类疾病发病率的增加可通过具有易感条件的人口百分比的增加来预测。由于鸟分枝杆菌对大多数抗生素标记具有抗性，并且难以通过外部遗传物质转化，因此有必要建立特定的系统来研究致病机制。在过去的3.5年中，我们已经开发或改造了分子系统，如转座子诱变，标记诱变（STM）和GFP启动子融合文库，使我们能够开始剖析鸟分枝杆菌与宿主相互作用的复杂方面。几个毒力基因被确定，允许细菌进入肠上皮细胞，并在巨噬细胞中存活。此外，体外鉴定的绝大多数基因也与体内毒力相关。我们的假设是鸟分枝杆菌有特定的策略来破坏宿主细胞。我们建议通过以下方式继续这项工作：A-研究鸟分枝杆菌毒力相关基因如何参与肠粘膜细胞的侵袭机制。迄今为止，我们的研究已经确定细菌进入与小GTP酶Rho A和Cdc 42的活化以及N-WASP的磷酸化有关。我们现在详细的实验，以进一步剖析宿主细胞的途径所需的细菌摄取，基于假设，两个途径是用来进入上皮细胞。B-分析参与巨噬细胞存活和复制能力的毒力决定因子的功能。我们已经开发了一种筛选转座子诱变的鸟分枝杆菌细菌的分离，其不抑制吞噬体-溶酶体融合，并且不能抑制液泡酸化。3000个突变体的初步筛选导致鉴定出许多毒力决定因素，如PPE基因、聚酮酶、MmpL蛋白、ABC转运蛋白和几个功能未知的基因。

DESCRIPTION (provided by applicant): Infections caused by Mycobacterium avium complex are common in AIDS patients, and in patients with chronic underlying lung disease, such as emphysema and bronchiectasy. Despite of the success of anti-HIV-1 therapy in reducing the cases of M.avium disease, recent published work has demonstrated that in many areas M.avium is M.avium is an environmental bacteria for which increasing incidence of disease in humans is predictable by the increased percent of the population with predisposing conditions The ability to survive in different environments in the host requires tight gene regulation. Because M.avium is resistant to most of the antibiotic markers and is difficult to transform by external genetic material, it was necessary to create specific systems to study pathogenesis. During the past 3.5 years of this grant we have developed or adapted molecular systems, such as a transposon mutagenesis, signature tagged mutagenesis (STM), and GFP promoter fusion library, that allowed us to begin dissecting the complex aspects of M.avium interaction with the host. Several virulence genes were identified that allow the bacterium to enter intestinal epithelial cells, and to survive in macrophages. In addition, the great majority of the genes identified in vitro are also associated with virulence in vivo. Our hypothesis is that M.avium has specific strategies to subvert the host cells. We propose to continue this work by: A- Investigating how M.avium virulence-related genes are involved in the mechanism of invasion of intestinal mucosal cells. Our studies thus far have determined that bacterial entry is associated with the activation of small GTPases Rho A, and Cdc 42, and phosphorylation of N-WASP. We now detail experiments to further dissect the host cells pathways needed for bacterial uptake, based on the hypothesis that two pathways are used to enter epithelial cells. B- Analyzing the function of virulence determinants that are involved in the ability to survive and replicate in macrophages. We have developed a screening for the isolation of transposon-mutagenized M.avium bacteria that does not inhibit phagosome-lysosome fusion, and fails to suppress vacuole acidification. An initial screen of 3000 mutants resulted in the identification of a number of virulent determinants, such as PPE genes, polyketide synthases, MmpL proteins, ABC transporters, and several genes of unknown function.