Rip1 controlled stress resistance and virulence in Mycobacterium tuberculosis

Rip1 控制结核分枝杆菌的应激抵抗力和毒力

• 批准号: 10084263
• 负责人: Michael Stephen Glickman
• 金额: $ 46.33万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-07 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10084263
• 关键词: Acids; Active Sites; Air; Anabolism; Antibiotics; Bacteria; Chelating Agents; Cleaved cell; Complement; Copper; Country; Defect; Development; Diffuse; Disease; Elements; Environment; Gene Expression Profiling; Genetic Epistasis; Genetic Transcription; Growth; Homeostasis; Hypoxia; Infection; Link; Mediating; Mediator of activation protein; Membrane; Metalloproteases; Metals; Molecular; Morbidity - disease rate; Mus; Mycobacterium tuberculosis; NOS2A gene; Nitric Oxide; Operon; Oxidative Stress; Pathogenesis; Pathway interactions; Peptide Hydrolases; Peptides; Phenotype; Phosphorylation; Phosphotransferases; Production; Protein-Serine-Threonine Kinases; Proteolysis; RIPK1 gene; RNA; RNA Binding; Resistance; Role; Sigma Factor; Signal Transduction; Signaling Molecule; Stress; Structure; System; Testing; Tuberculosis; Vaccines; Virulence; attenuation; base; biological adaptation to stress; cofactor; gene repression; genome sequencing; insight; mortality; mouse model; mutant; novel; pathogen; peptide synthase; programs; response; sensor; whole genome

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.

项目摘要 结核分枝杆菌毒力的分子基础仍未完全理解。在主机内，M。 结核病必须应对并抵抗过多的压力。最近使用遗传定义的研究 细菌突变体已确定几种信号转导系统对感官至关重要 主机环境，包括两个组件系统，跨膜丝氨酸苏氨酸激酶和蛋白水解 系统。 RIP1是膜内金属蛋白酶（S2P类），用于结核分枝杆菌毒力 老鼠。膜内蛋白酶是膜结合的蛋白酶，在膜中有活性位点，该蛋白酶 切割跨膜底物。 RIP1具有四个已知的底物，膜跨越抗sigma因子。 对于SIGK，L，M和D。但是，结核分枝杆菌滴管的严重毒力缺陷并未被M. 结核病DSIGKLM三重突变体或DSIGD，提高了额外的下游途径的可能性 由RIP1控制的是毒力的关键介体。我们已经确定RIP1是必需的 对与发病机理有关的几种应力的抗性，包括铜，一氧化氮和缺氧。然而， 这些表型与四个RIP1控制的Sigma因子途径无关。遗传上毒分析 表明由RIP1蛋白酶控制的铜和无电阻途径与已知 铜外排和Cu/no诱导的转录系统，表明金属/NO的新机制 反抗。令人惊讶的是，抗sigd的丢失（但不是SIGD，请参见上文），RIP1底物会导致严重的铜 灵敏度，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背景中具有孤立的自发突变体，可抑制DRIP1铜敏感性 表型，并通过对这些突变体进行针对其他应力的筛选，整个基因组测序和 互补已确定PDTA/PDTAR传感器激酶/响应调节剂对作为中介体 RIP1依赖铜，无电阻。转录分析表明RIP1/PDTA/PDTAR系统 通过转录抑制，控制应力诱导编码非透射体的操纵子的表达 最近显示的肽合酶直接直接合成铜螯合肽（白垩菌），这表明 与铜稳态的合理直接链接。我们还发现滴水1菌株的毒力缺陷为 在NOS2缺乏的小鼠中实质上逆转，证实对一氧化氮的敏感性是有助于 DRIP1菌株的衰减。基于这些新颖的发现，我们提出了一个实验程序 解剖这种新发现的结核分枝杆菌RIP1/PDTA/PDTAR信号传导的分子机制 级联控制应力响应途径，该途径整合了对铜，一氧化氮和一氧化氮和 缺氧，白垩纪生物合成对这一途径的贡献以及该途径在M中的作用。 结核病毒力。通过以下特定目标表征该途径将产生重要的 对结核分枝杆菌宿主 - 病原体相互作用的洞察力。