Mycobacterium tuberculosis Survival Regulatory Genes

描述(申请人提供)：结核分枝杆菌是全球领先的艾滋病相关传染病杀手。结核病是一种多病理阶段的疾病，因此结核分枝杆菌。必须拥有多种适应性遗传策略才能在这些不同的环境中生存。了解机体在这些疾病阶段的发病机制是开发更好的诊断、疫苗和药物的最可靠途径，这些都是结核病和结核病/艾滋病毒患者迫切需要的。我们已经从基因上中断了500万结核杆菌。Sigma因子基因，利用微阵列技术鉴定它们调控的基因，并评估它们在小鼠身上的毒力。在这些可供选择的西格玛因子中，存在功能冗余，因为在感染小鼠时，5个突变体中有4个呈现免疫病理表型的衰减，其中分枝杆菌计数保持在较高水平，但在肺部的死亡率和疾病进展方面有显着延迟。在第一个目标中，我们将探索几个Sigma因子基因敲除突变体所表现出的免疫病理缺陷。已知的结核病控制介质，如一氧化氮、肿瘤坏死因子-α、干扰素-伽马和吞噬细胞氧化酶的作用将使用结核分枝杆菌进行检测。DeltasigH和其他表现出免疫病理表型的敲除突变体。其次，我们将通过研究胁迫条件下的表达谱，通过转录的生化分析，通过构建双敲除突变体，以及通过条件表达西格玛因子，来完善我们对这些西格玛因子调节因子的理解。我们将讨论是否有足够的ECF启动子共识简并来允许依赖基因的冗余Sigma因子控制，或者是否有有待发现的远端介体。最后，在第三个目标中，我们将通过研究反西格玛因子来探索西格玛因子活性的调节。我们将研究ASIA的作用，这是一种噬菌体编码的抗Sigma因子，它与RNA聚合酶结合，改造它，并改变其启动子的特异性。我们将寻求确定亚洲或其部分是否对分枝杆菌具有转录特异性修饰作用。我们还将研究一种新的西格玛因子调节因子在结核分枝杆菌Rv1364c中的作用，我们的数据表明，这是抵抗十二烷基硫酸钠胁迫所必需的。增进我们对FM的理解。结核西格玛因子及其相关调节因子将有助于在结核病发病机制中建立关键的适应机制。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is a leading AIDS-related infectious disease killer worldwide. Tuberculosis is a disease of multiple pathologic stages, and hence M. tb. must possess multiple adaptive genetic strategies to survive in these differing environments. Understanding the organism's pathogenesis mechanisms during these disease stages is the surest way to develop better diagnostics, vaccines, and drugs which are critically needed for patients with TB and TB/HIV. We have genetically interrupted 5 M. tb. sigma factor genes, identified the genes which they regulate by using microarray technology and assessed their virulence in mice. Among these alternative sigma factors there is functional redundancy in that upon infection of mice 4 of the 5 mutants show the immunopathology phenotype of attenuation in which mycobacterial counts are maintained at high level but there is a significant delay in mortality and in disease progression in the lungs. In the first aim, we will explore the immunopathology defect demonstrated by several of the sigma factor knockout mutants. The roles of known mediators of TB control such as nitric oxide, TNF-alpha, interferon-gamma and phagocyte oxidase will be examined using the M. tb. deltasigH and other knockout mutants which display the immunopathology phenotype. Second, we will refine our understanding of these sigma factor regulons by studying expression profiles under stress conditions, by biochemical analysis of transcription, by constructing double knockout mutants, and through conditional expression of sigma factors. We will address whether there is sufficient ECF promoter consensus degeneracy to permit redundant sigma factor control of dependent genes or whether there are distal mediators which remain to be discovered. Finally, in the 3rd aim we will explore the modulation of sigma factor activity by studying anti-sigma factors. We will study the effect of AsiA, a phage-encoded anti-sigma factor which binds to RNA polymerase, remodels it, and alters its promoter specificity. We will seek to determine whether AsiA or portions of it have a transcription-specificity modifying effect in mycobacteria. We will also study the role of a novel sigma factor-regulator in M. tb, Rv1364c, which our data show is required for resistance to SDS stress. Advancing our understanding ofM. tb sigma factors and their related regulators will help establish key adaptive mechanisms in the pathogenesis of TB.