Rip1 controlled stress resistance and virulence in Mycobacterium tuberculosis

项目概要 结核分枝杆菌毒力的分子基础仍不完全清楚。在主机内，M. 结核病必须对过多的压力做出反应并抵抗。最近的研究使用基因定义 细菌突变体已经确定了几种信号转导系统对于感知和响应细菌至关重要 宿主环境，包括两个组成系统，跨膜丝氨酸苏氨酸激酶和蛋白水解 系统。 Rip1 是结核分枝杆菌毒力所需的膜内金属蛋白酶（S2P 类） 鼠标。膜内蛋白酶是在膜上具有活性位点的膜结合蛋白酶， 切割跨膜底物。 Rip1 有四种已知底物，即跨膜抗西格玛因子 然而，结核分枝杆菌 Drip1 的严重毒力缺陷并未被分枝杆菌表型复制。 结核病 DsigKLM 三重突变体或 DsigD，增加了其他下游途径的可能性 由 Rip1 控制的病毒是毒力的关键调节因子。我们确定需要 Rip1 对与发病机制相关的几种应激的抵抗力，包括铜、一氧化氮和缺氧。然而， 这些表型独立于四个 Rip1 控制的 sigma 因子途径。遗传上位分析 表明 Rip1 蛋白酶控制的铜和 NO 抗性途径与已知的无关 铜流出和 Cu/NO 诱导转录系统，表明金属/NO 的新机制 阻力。令人惊讶的是，Rip1 底物抗 SigD（但不是 SigD，见上文）的损失会导致严重的铜 敏感性，表明该抗 Sigma 具有独立的信号传导作用，与其同源 Sigma 无关。我们 在 Drip1 背景中分离出自发突变体，抑制 Drip1 铜敏感性 表型，并通过针对其他应激反筛选这些突变体、全基因组测序，以及 互补，已确定 PdtaS/PdtaR 传感器激酶/反应调节器对作为 Rip1 依赖于铜和 NO 电阻。转录分析表明 Rip1/PdtaS/PdtaR 系统 通过转录抑制，控制应激诱导的编码非核糖体操纵子的表达 最近显示肽合酶可以直接合成铜螯合肽（一种chakophore），这表明 与铜稳态的可能直接联系。我们还发现 Drip1 菌株的毒力缺陷是 在 NOS2 缺陷小鼠中，这种情况基本上逆转，证实对一氧化氮的敏感性是导致 Drip1 菌株的衰减。基于这些新颖的发现，我们提出了一个实验计划 剖析新发现的结核分枝杆菌 Rip1/PdtaS/PdtaR 信号传导的分子机制 级联控制应激反应途径，整合细胞对铜、一氧化氮和 缺氧、chakophore 生物合成对该途径的贡献以及该途径在 M. 结核病毒力。通过以下具体目标来表征该途径将产生显着的效果 深入了解结核分枝杆菌宿主与病原体的相互作用。

Project Summary The molecular basis of M. tuberculosis virulence is still incompletely understood. Within the host, M. tuberculosis must respond to, and resist, a plethora of stresses. Recent studies using genetically defined bacterial mutants have identified several signal transduction systems as critical to sense and respond to the host environment, including two component systems, transmembrane serine threonine kinases, and proteolytic systems. Rip1 is an intramembrane metalloprotease (S2P class) required for M. tuberculosis virulence in the mouse. Intramembrane proteases are membrane bound proteases with active sites in the membrane which cleave transmembrane substrates. Rip1 has four known substrates, the membrane spanning anti-sigma factors for SigK,L,M and D. However, the severe virulence defect of M. tuberculosis Drip1 is not phenocopied by an M. tuberculosis DsigKLM triple mutant or DsigD, raising the likelihood that additional downstream pathways controlled by Rip1 are the critical mediators of virulence. We have determined that Rip1 is required for resistance to several stresses relevant to pathogenesis, including copper, nitric oxide, and hypoxia. However, these phenotypes are independent of the four Rip1 controlled sigma factor pathways. Genetic epistasis analysis indicates that the copper and NO resistance pathway controlled by the Rip1 protease is independent of known copper efflux and Cu/NO induced transcriptional systems, indicating a novel mechanism of metal/NO resistance. Surprisingly, loss of anti-SigD (but not SigD, see above), a Rip1 substrate, causes severe copper sensitivity, suggesting an independent signaling role for this anti-Sigma, independent of its cognate sigma. We have isolated spontaneous mutants in the Drip1 background that suppress the Drip1 copper sensitivity phenotype and, by counterscreening these mutants against other stresses, whole genome sequencing, and complementation, have identified the PdtaS/PdtaR sensor kinase/response regulator pair as mediator of the Rip1 dependent copper and NO resistance. Transcriptional profiling reveals that the Rip1/PdtaS/PdtaR system controls, through transcriptional repression, stress induced expression of an operon encoding a nonribosomal peptide synthase recently shown to direct synthesis of a copper chelating peptide (a chalkophore), suggesting a plausible direct link to copper homeostasis. We also find that the virulence defect of the Drip1 strain is substantially reversed in NOS2 deficient mice, confirming that sensitivity to nitric oxide is the a contributor to the attenuation of the Drip1 strain. Based on these novel findings, we propose an experimental program to dissect the molecular mechanisms by which this newly discovered M. tuberculosis Rip1/PdtaS/PdtaR signaling cascade controls a stress response pathway that integrates cellular response to copper, nitric oxide, and hypoxia, the contribution of chalkophore biosynthesis to this pathway, and the role of this pathway in M. tuberculosis virulence. Characterization of this pathway through the following specific aims will yield significant insight into M. tuberculosis host-pathogen interactions.