Redox defenses and evasion of reactive oxygen species mediated host immunity in Mycobacterium tuberculosis

项目摘要 结核分枝杆菌（Mtb）是结核病（TB）的病原体，2017年造成140万人死亡， 比任何其他病原体都多。结核病治疗仍然困难，有效的疫苗也没有研究出来 在过去的世纪里，结束结核病大流行的一个主要障碍是对有效治疗结核病的认识有限。 宿主免疫反应和结核杆菌的免疫逃避策略。已经做了大量体外工作来表征 结核分枝杆菌感染的巨噬细胞，其主要的细胞生态位。而活性氧（ROS）是一种 Mtb是针对多种病原体的重要抗微生物防御，其对ROS具有抗性。我们最近发现了CpsA 作为抑制NADPH氧化酶募集到含Mtb的吞噬体的分泌毒力因子， 从而保护Mtb免于氧化爆发。ROS也是LC 3相关吞噬作用的重要信号 (LAP)自噬的一种非典型形式。事实上，ΔcpsA突变体在NADPH缺陷的小鼠中被拯救， 氧化酶和过氧化物酶。有趣的是，在小鼠中，ΔcpsA突变体在前两周内严重减弱， 感染并在6周内基本恢复，这表明CpsA在先天期最重要 在获得性免疫激活之前感染。这种表型与细胞类型的转变相一致， 感染和炎症反应。因此，我们假设CpsA特异性地保护Mtb免受 ROS在细胞类型感染和炎症环境的先天免疫阶段。我们将测试我们的 通过使用流动辅助细胞在体内表征不同髓样细胞内的ΔcpsA突变体， 分选（FACS）和在肺泡巨噬细胞、嗜中性粒细胞或单核细胞来源的 巨噬细胞，以及未能建立针对Mtb的适应性免疫应答或具有细胞免疫应答的小鼠， 具体缺陷类型KatG是一种过氧化氢酶-过氧化物酶，在Mtb的ROS防御中也很重要。我们 将通过鉴定ΔcpsA ΔkatG双突变体来检验CpsA和KatG是否协同致病。我们 假设ΔcpsA ΔkatG突变体由于抑制解除， ROS生产的主机和减少ROS解毒活性Mtb。KatG激活一线药物 异烟肼（INH），katG的突变赋予INH抗性。因此，CpsA可能允许INH的传播- 抗katG突变体通过保护他们免受ROS。研究ROS和多种骨髓细胞的作用 参与Mtb感染的细胞将影响宿主定向治疗的策略，靶向耐药杆菌， 新型疫苗设计。这个建议是史蒂芬·格雷斯比博士论文的主题， 华盛顿大学医学院医学科学家培训计划中的微生物致病机制 （WUSM）。WUSM在微生物发病机理和免疫学方面的优势使其非常适合研究 史蒂文·格雷斯比提出的他拥有所有必要的资源，来自一群杰出科学家的投入， 和一个强大的培训计划，这将支持他的职业发展，作为一个独立的物理学家，科学家。

Project Summary Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), caused 1.4 million deaths in 2017, more than any other pathogen. TB treatment remains difficult, and an effective vaccine has eluded research efforts for the past century. A major barrier to ending the TB pandemic is a limited understanding of effective host immune responses and Mtb’s immune evasion strategies. Much in vitro work has been done to characterize Mtb infection of macrophages, its predominant cellular niche. Whereas reactive oxygen species (ROS) are an important antimicrobial defense against diverse pathogens, Mtb is resistant to ROS. We recently identified CpsA as a secreted virulence factor that inhibits NADPH oxidase recruitment to the Mtb-containing phagosome, thereby protecting Mtb from an oxidative burst. ROS is also an essential signal for LC3-associated phagocytosis (LAP), a noncanonical form of autophagy. Indeed, the ΔcpsA mutant is rescued in mice defective in the NADPH oxidase and LAP. Interestingly, in mice the ΔcpsA mutant is severely attenuated during the first two weeks of infection and recovers substantially by 6 weeks, suggesting that CpsA is most important during the innate phase of infection before the activation of adaptive immunity. This phenotype coincides with a shift in cell types that are infected and the inflammatory response. Therefore, we hypothesize that CpsA specifically protects Mtb against ROS in the cell types infected and inflammatory environment of the innate immune phase. We will test our hypothesis by characterizing the ΔcpsA mutant within different myeloid cells in vivo using flow-assisted cell sorting (FACS) and in mice that are deficient in alveolar macrophages, neutrophils, or monocyte-derived macrophages, as well as in mice that fail to mount an adaptive immune response against Mtb or that have cell type specific defects in LAP. KatG is a catalase-peroxidase that is also important in ROS defense in Mtb. We will test whether CpsA and KatG cooperate in virulence by charactering a ΔcpsA ΔkatG double mutant. We hypothesize that the ΔcpsA ΔkatG mutant will be more attenuated than either single mutant due to disinhibited ROS production by the host and reduced ROS detoxifying activity by Mtb. KatG activates the first-line drug isoniazid (INH), and mutations in katG confer INH resistance. CpsA, therefore, may permit transmission of INH- resistant katG mutants by protecting them against ROS. Investigating the roles of ROS and the diverse myeloid cells involved in Mtb infection will impact strategies for host-directed therapies, targeting drug-resistant bacilli, and novel vaccine design. This proposal is the topic of Steven Grigsby’s PhD thesis in Molecular Microbiology & Microbial Pathogenesis in the Medical Scientist Training Program at Washington University School of Medicine (WUSM). The strength at WUSM in microbial pathogenesis and immunology makes it a perfect fit for the studies proposed by Steven Grigsby. He has all of the necessary resources, input from a group of outstanding scientists, and a robust training plan, which will support his career development as an independent physician-scientist.