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慢性基础肺部疾病患者中很常见，如肺气肿和支气管扩张。尽管抗HIV-1治疗在减少禽分枝杆菌病的病例方面取得了成功，但最近发表的研究表明，在许多地区，禽分枝杆菌是一种环境细菌，对其来说，人类发病率的增加是可以预测的，因为具有易感条件的人口比例的增加需要严格的基因调控才能在宿主的不同环境中生存。由于禽型支原体对大多数抗生素标志物具有抗性，且不易被外界遗传物质转化，因此有必要建立专门的系统来研究其致病机理。在过去3.5年的资助期间，我们开发或调整了分子系统，如转座子突变、签名标签突变(STM)和GFP启动子融合库，使我们能够开始剖析禽类分枝杆菌与宿主相互作用的复杂方面。已确定了几个毒力基因，使细菌能够进入肠道上皮细胞，并在巨噬细胞中存活。此外，在体外鉴定的绝大多数基因也与体内的毒力有关。我们的假设是，鸟型支原体有特定的策略来颠覆宿主细胞。我们建议通过以下方式继续这项工作：A-研究禽流感分支杆菌毒力相关基因如何参与肠道粘膜细胞侵袭的机制。到目前为止，我们的研究已经确定细菌进入与小GTP酶Rho A和CDC42的激活以及N-WASP的磷酸化有关。我们现在详细地进行实验，以进一步剖析细菌吸收所需的宿主细胞途径，这是基于两条途径被用来进入上皮细胞的假设。B-分析毒力决定因素的功能，这些决定因素涉及巨噬细胞的生存和复制能力。我们建立了一种不抑制吞噬小体-溶酶体融合，也不能抑制空泡酸化的转座子诱变的禽类支原体细菌的分离方法。通过对3000个突变体的初步筛选，鉴定出了一些毒力决定因素，如PPE基因、聚酮合成酶、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.