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

结核分枝杆菌的氧化还原防御和活性氧逃避介导的宿主免疫

• 批准号: 10481829
• 负责人: Steven Joseph Grigsby
• 金额: $ 3.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10481829
• 关键词: Alveolar Macrophages; Attenuated; Autophagocytosis; Bacillus; Bacteria; Bone Marrow; C57BL/6 Mouse; Cause of Death; Cell Separation; Cells; Cessation of life; Chemicals; Chronic; Complement; Defect; Doctor of Philosophy; Drug Targeting; Drug resistance; Genetic; Immune; Immune Evasion; Immune response; Immunity; Immunology; In Vitro; Infection; Infection Control; Inflammatory; Inflammatory Response; Innate Immune Response; Interferons; Isoniazid resistance; Knockout Mice; Lung; Mediating; Mediator of activation protein; Medical; Microbe; Microbiology; Molecular; Mus; Mutation; Mycobacterium tuberculosis; Myeloid Cells; NADPH Oxidase; Natural Immunity; Neutrophil Infiltration; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Peroxidases; Persons; Phagocytosis; Phagosomes; Pharmaceutical Preparations; Phase; Phenotype; Physicians; Plasmids; Play; Production; Proteins; Pulmonary Tuberculosis; Reactive Oxygen Species; Reporter; Research; Resistance; Resources; Respiratory Burst; Role; SCID Mice; Scientist; Signal Transduction; Signaling Molecule; Stress; T-Lymphocyte; Testing; Tetanus Helper Peptide; Therapeutic; Time; Training; Training Programs; Tuberculosis; Universities; Vaccine Design; Vaccines; Virulence; Virulence Factors; Washington; Wild Type Mouse; Work; acute infection; adaptive immune response; adaptive immunity; antigen-specific T cells; antimicrobial; career development; catalase; cell type; chronic infection; conditional knockout; design; experimental study; immunoregulation; in vivo; inflammatory milieu; isoniazid; macrophage; medical schools; microbial; microbicide; monocyte; mutant; neutrophil; novel therapeutics; novel vaccines; pandemic disease; pathogen; promoter; recruit; transmission process; tuberculosis drugs; tuberculosis treatment

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.

项目概要 结核病 (TB) 的病原体结核分枝杆菌 (Mtb) 在 2017 年导致 140 万人死亡， 比任何其他病原体都多。结核病治疗仍然困难，有效的疫苗尚未被研究出来 上个世纪的努力。结束结核病大流行的一个主要障碍是对有效措施的了解有限。 宿主免疫反应和结核分枝杆菌的免疫逃避策略。已经做了很多体外工作来表征 Mtb 感染巨噬细胞，其主要细胞生态位。而活性氧 (ROS) 是一种 Mtb 是针对多种病原体的重要抗菌防御手段，对 ROS 具有抵抗力。我们最近发现了 CpsA 作为一种分泌的毒力因子，抑制 NADPH 氧化酶招募到含有 Mtb 的吞噬体， 从而保护 Mtb 免受氧化爆发。 ROS 也是 LC3 相关吞噬作用的重要信号 （LAP），一种非规范形式的自噬。事实上，ΔcpsA 突变体在 NADPH 缺陷的小鼠中得到了拯救 氧化酶和LAP。有趣的是，在小鼠中，ΔcpsA 突变体在治疗的前两周内严重减弱。 感染并在 6 周时基本恢复，表明 CpsA 在先天阶段最为重要 适应性免疫激活之前的感染。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 专门保护 Mtb ROS 在感染细胞类型和先天免疫阶段的炎症环境中。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骨髓细胞内的 ΔcpsA 突变体来提出假设 分选 (FACS) 以及肺泡巨噬细胞、中性粒细胞或单核细胞来源缺陷的小鼠 巨噬细胞，以及未能对 Mtb 产生适应性免疫反应或具有细胞的小鼠 LAP 中的类型特定缺陷。 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 将通过表征 ΔcpsA ΔkatG 双突变体来测试 CpsA 和 KatG 是否在毒力上合作。我们 假设 ΔcpsA ΔkatG 突变体由于去抑制而比任一单一突变体的减毒程度更高 宿主产生 ROS，并降低 Mtb 的 ROS 解毒活性。 KatG激活一线药物 异烟肼 (INH) 和 katG 突变会导致 INH 耐药。因此，CpsA 可能允许 INH- 的传播 通过保护它们免受 ROS 的影响，产生抗性 katG 突变体。研究 ROS 和多种骨髓细胞的作用 参与结核分枝杆菌感染的细胞将影响针对宿主的治疗策略，针对耐药杆菌， 和新颖的疫苗设计。该提案是 Steven Grigsby 的分子微生物学博士论文的主题 华盛顿大学医学院医学科学家培训项目中的微生物发病机制 （WUSM）。 WUSM 在微生物发病机制和免疫学方面的实力使其非常适合这些研究 由史蒂文·格里格斯比提出。他拥有所有必要的资源，来自一群杰出科学家的投入， 以及强有力的培训计划，这将支持他作为独立医师科学家的职业发展。