Rip1 controlled stress resistance and virulence in Mycobacterium tuberculosis

项目摘要 对M.结核病的毒力仍然不完全清楚。在宿主中，M。 结核病必须对过多的压力作出反应并加以抵抗。最近的研究使用基因定义的 细菌突变体已经鉴定了几种信号转导系统，它们对于感知和响应细菌的细胞周期是至关重要的。 宿主环境，包括双组分系统，跨膜丝氨酸苏氨酸激酶和蛋白水解酶， 系统. Rip 1是M.结核病毒力 老鼠.膜内蛋白酶是在膜中具有活性位点的膜结合蛋白酶， 切割跨膜底物。Rip 1有四种已知的底物，即跨膜抗σ因子 对于SigK、L、M和D。然而，M.结核分枝杆菌Drip 1不被M. 结核病DsigKLM三重突变体或DsigD，增加了额外的下游途径 由Rip 1控制的是毒力的关键介质。我们已经确定Rip 1是必需的， 对与发病机制相关的几种应激的抗性，包括铜、一氧化氮和缺氧。然而，在这方面， 这些表型独立于四种Rip 1控制的σ因子途径。遗传上位性分析 表明由Rip 1蛋白酶控制的铜和NO抗性途径不依赖于已知的 Cu外排和Cu/NO诱导的转录系统，表明金属/NO的新机制 阻力令人惊讶的是，抗SigD（但不是SigD，见上文）（Rip 1底物）的损失导致严重的铜 敏感性，这表明一个独立的信号作用，这种反西格玛，独立于其同源西格玛。我们 在Drip 1背景中分离出抑制Drip 1铜敏感性的自发突变体 表型，并通过对其他压力，全基因组测序， 互补，已经确定PdtaS/PdtaR传感器激酶/反应调节剂对作为介导的 Rip 1依赖于铜，无电阻。转录谱显示Rip 1/PdtaS/PdtaR系统 通过转录抑制控制应激诱导的编码非核糖体蛋白的操纵子的表达， 最近显示肽合酶指导铜螯合肽（chalkophore）的合成，表明 与铜稳态的直接联系我们还发现Drip 1菌株的毒力缺陷是 在NOS 2缺陷小鼠中基本上逆转，证实对一氧化氮的敏感性是导致这种情况的一个因素 Drip 1菌株的减毒。基于这些新的发现，我们提出了一个实验方案， 剖析这种新发现的M.结核Rip 1/PdtaS/PdtaR信号通路 级联控制应激反应途径，整合细胞对铜，一氧化氮， 缺氧、白垩体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对这一途径进行表征， 对M.结核病宿主-病原体相互作用

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.