Rip1 controlled stress resistance and virulence in Mycobacterium tuberculosis

项目摘要 结核分枝杆菌毒力的分子基础仍不完全清楚。在寄主内，M。 结核病必须对过多的压力做出反应，并加以抵抗。最近的研究使用基因定义的 细菌突变体已经确定了几个信号转导系统，它们对感知和响应 寄主环境，包括两个组成系统，跨膜丝氨酸苏氨酸激酶和蛋白水解酶 系统。RIP1是一种膜内金属蛋白酶(S2P类)，是结核分枝杆菌毒力所必需的。 老鼠。膜内蛋白水解酶是一种膜结合的蛋白水解酶，其活性部位在膜上 切开跨膜底物。RIP1有四种已知底物，膜上有抗Sigma因子 对于SigK、L、M和D来说，结核分枝杆菌Drip1的严重毒力缺陷并不是M。 结核DsigKLM三重突变或DsigD，增加了额外下游途径的可能性 由RIP1控制的是毒力的关键介体。我们已确定需要RIP1 对几种与发病相关的胁迫的抵抗，包括铜、一氧化氮和低氧。然而， 这些表型与RIP1控制的四条Sigma因子通路无关。遗传上位性分析 表明RIP1酶控制的铜和NO抗性途径是独立于已知的 铜外流和铜/NO诱导的转录系统，提示了一种新的金属/NO机制 抵抗。令人惊讶的是，RIP1底物抗SigD(但不是SigD，见上)的丢失会导致严重的铜 敏感性，提示这个抗Sigma的信号作用独立于它的同源Sigma。我们 在Drip1背景中分离出抑制Drip1铜敏感性的自发突变体 表型，并通过对这些突变体进行反筛选以对抗其他胁迫，全基因组测序，以及 互补作用，已经确定PdtaS/PdtaR传感器激酶/反应调节因子对是 依赖于RIP1的铜，没有电阻。转录图谱显示RIP1/PdtaS/PdtaR系统 对照，通过转录抑制，应激诱导编码非核糖体的操纵子的表达 最近发现的多肽合成酶可以直接合成一种铜络合肽(一种胆红素)，这表明 这似乎与铜的动态平衡有直接联系。我们还发现，Drip1株的毒力缺陷是 NOS2缺陷小鼠的情况基本相反，证实了对一氧化氮的敏感性是导致 Drip1菌株的减毒作用。基于这些新发现，我们提出了一项实验计划，以 剖析新发现的结核分枝杆菌RIP1/PdtaS/PdtaR信号转导的分子机制 CASCADE控制一条应激反应通路，该通路整合了细胞对铜、一氧化氮和 低氧，白炭黑生物合成在这一途径中的贡献，以及这一途径在微囊藻中的作用。 肺结核的致死性。通过以下具体目标对这一途径进行表征将产生重要的 结核分枝杆菌宿主-病原体相互作用的洞察。

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.