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.

项目摘要 结核病的病原体结核分枝杆菌(Mtb)在2017年造成140万人死亡， 比任何其他病原体都多。结核病的治疗仍然很困难，有效的疫苗还没有研究出来 过去一个世纪的努力。结束结核病大流行的一个主要障碍是对有效的 宿主免疫反应和结核分枝杆菌的免疫逃避策略。已经做了大量的体外工作来表征 结核分枝杆菌感染巨噬细胞，其主要细胞生态位。而活性氧(ROS)是一种 Mtb对多种病原体具有重要的抗微生物防御作用，对ROS具有抗药性。我们最近确认了CPSA 作为一种分泌的毒力因子，抑制NADPH氧化酶募集到含有Mtb的吞噬小体， 从而保护结核分枝杆菌免受氧化猝发。ROS也是与LC3相关的吞噬作用的重要信号 (LAP)，一种非规范的自噬形式。事实上，Δcpsa突变体在NADPH缺陷小鼠中得到了拯救。 氧化物酶和大腿。有趣的是，在小鼠中，ΔcPSA突变体在感染的头两周内严重减弱。 感染，并在6周内基本恢复，这表明CPSA在先天阶段最重要 在激活获得性免疫之前感染的可能性。这种表型与细胞类型的转变是一致的 感染和炎症反应。因此，我们假设CPSA特别保护结核分枝杆菌 ROS在细胞类型、感染和炎症环境中的先天免疫阶段。我们将测试我们的 利用流式细胞术研究体内不同髓系细胞内ΔcPSA突变体的假说 分选(FAC)和肺泡巨噬细胞、中性粒细胞或单核细胞来源缺陷的小鼠 巨噬细胞，以及未能对结核分枝杆菌或有细胞的小鼠产生适应性免疫反应的小鼠 键入LAP中的特定缺陷。KatG是一种过氧化氢酶-过氧化物酶，在结核分枝杆菌的ROS防御中也很重要。我们 将通过鉴定ΔcpsaΔkatG双重突变体来测试cpsa和katG是否在毒力上合作。我们 假设ΔcPSAΔkatG突变体将由于去抑制而比任何一个单一突变体更弱 宿主产生ROS，Mtb降低ROS解毒活性。KatG激活一线药物 异烟肼(INH)和katG突变使其对异烟肼产生耐药性。因此，CPSA可以允许传输异烟肼- 抗性katG突变体通过保护它们免受ROS的伤害。探讨ROS和多种髓系细胞的作用 参与结核分枝杆菌感染的细胞将影响宿主导向治疗的战略，针对耐药杆菌， 和新颖的疫苗设计。这一提议是Steven Grigsby在分子微生物学和 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在微生物发病机制和免疫学方面的优势使其非常适合进行研究 由史蒂文·格里斯比提出。他拥有所有必要的资源，来自一群杰出科学家的投入， 以及一个强有力的培训计划，这将支持他作为一名独立内科科学家的职业发展。