Mechanisms of Innate Immune Evasion by Mycobacterium Tuberculosis

结核分枝杆菌先天免疫逃避的机制

• 批准号: 10078851
• 负责人: JENNIFER A PHILIPS
• 金额: $ 54.45万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078851
• 关键词: Attenuated; Autophagocytosis; Bacillus subtilis; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biological Assay; C Type Lectin Receptors; Cause of Death; Cell physiology; Cells; Cessation of life; Characteristics; Crystallization; Data; Degradation Pathway; Development; Disease; Drug Targeting; Exhibits; Generations; Goals; Gram-Positive Bacteria; Human; Human T-Cell Leukemia Viruses; Immune; Immune Evasion; Immunity; Impairment; Infection; Inflammatory; Innate Immune Response; Invaded; Ligands; Link; Lipid Binding; Lipids; Lysosomes; Mediating; Membrane; Microbe; Molecular; Mus; Mycobacterium tuberculosis; NADPH Oxidase; Nuclear; Organelles; Organism; Pathway interactions; Phagocytosis; Phagocytosis Inhibition; Phosphoric Monoester Hydrolases; Play; Population; Process; Production; Proteins; Reactive Oxygen Species; Relapse; Role; Streptococcus pneumoniae; Structural Models; Structure; Testing; Therapeutic; Toll-like receptors; Tuberculosis; Tuberculosis Vaccines; Vaccines; Virulence; Virulence Factors; Work; acute infection; base; catalase; chronic infection; experience; immune clearance; in vivo; innate immune mechanisms; innate immune pathways; inorganic phosphate; insight; macrophage; microbial; microorganism; mutant; new therapeutic target; novel; novel therapeutics; novel vaccines; paralogous gene; pathogen; prevent; receptor; stem; success; therapy development; trafficking; translational impact

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB) and the leading cause of death worldwide from a bacterial infection. The success of Mtb stems from its ability to evade degradation by macrophages. Recent studies have revealed that macrophages clear microorganisms through two distinct lysosomal trafficking pathways that involve LC3-marked organelles (2, 3). Xenophagy is a process by which LC3-marked, double-membrane organelles capture and degrade invading microbes. LC3-associated phagocytosis (LAP) is similar to xenophagy, but does not involve a double membrane and requires NADPH oxidase and reactive oxygen species (ROS), which are not necessary for xenophagy. These lysosomal degradative pathways are activated by microbial ligands that stimulate pathogen recognition receptors (PRRs). The reason why Mtb, which activates numerous PRRs, fails to provoke substantive LC3-associated phagolysosomal trafficking is not understood. Our extensive preliminary data strongly suggest that CpsA, an uncharacterized protein secreted by Mtb, specifically blocks LAP. We hypothesize that CpsA interferes with the activation of NADPH oxidase, thereby blocking the generation of ROS and the LAP-mediated delivery of Mtb to the lysosome. Consistent with our hypothesis, we found that Mtb strains lacking cpsA exhibit dramatically enhanced colocalization with the LC3 marker of LAP and that they are highly attenuated in macrophages and mice. Moreover, NADPH oxidase and the proteins specifically required for LAP are necessary for macrophages to kill the cpsA mutant. CpsA contains a LytR-CpsA-Psr (LCP) domain, which is commonly found in Gram-positive organisms. In Streptococcus pneumoniae and Bacillus subtilis, the LCP domain binds phosphorylated polyisoprenoid lipids. We modeled the structure of Mtb CpsA using the crystal structures an S. pneumoniae LCP protein and found that all of the lipid phosphate-binding residues are conserved in Mtb CpsA. In addition, we found that CpsA can bind the human T-cell leukemia virus type I binding protein 1 (TAX1BP1), and nuclear dot protein 52 kDa (NDP52). TAX1BP1 and NDP52 are paralogs that are involved in linking bacterial cargo to the autophagy machinery. Thus, we hypothesize that the ability of CpsA to inhibit the NADPH oxidase and LAP depends upon binding lipid phosphate and host proteins TAX1BP1 and NDP52. To test our hypotheses, we will (1) study the pathway by which macrophages kill the cpsA mutant, (2) characterize the mechanism of action of the CpsA protein, and (3) evaluate the importance of this innate immune evasion mechanism in vivo. Combined, our studies will elucidate a novel mechanism of immune evasion by one of the most formidable pathogens. By studying the molecular mechanisms Mtb utilizes to sabotage host cellular functions, we will make fundamental observations that will aid in the development of better therapeutics and vaccines for Mtb.

项目摘要 结核分枝杆菌（MTB）是疾病结核病（TB）的病因和领先 全世界因细菌感染而导致的死亡原因。 MTB植物从逃避的能力中取得了成功 巨噬细胞降解。最近的研究表明，巨噬细胞通过 两个涉及LC3标记细胞器的两种不同的溶酶体运输途径（2，3）。 Xenophagy是一个过程 LC3标记的双膜细胞器捕获并降解了入侵微生物。 LC3相关 吞噬作用（LAP）类似于Xenophapy，但不涉及双膜，需要NADPH 氧化物和活性氧（ROS），这对于异种含量不是必需的。这些溶酶体 降解途径被刺激病原体识别受体（PRR）的微生物配体激活。 激活众多PRR的MTB无法引起实质性LC3相关的原因 吞噬物质贩运尚不清楚。我们广泛的初步数据强烈表明CPSA是 MTB分泌的未表征的蛋白质，特别是阻塞圈。我们假设CPSA干扰了 NADPH氧化酶的激活，从而阻断ROS的产生和MTB的lap介导的递送 溶酶体。与我们的假设一致，我们发现缺乏CPSA的MTB菌株急剧暴露 与LAP的LC3标记增强了共定位，并且它们在巨噬细胞中高度衰减 老鼠。此外，需要专门为膝盖所需的NADPH氧化物和蛋白质 巨噬细胞杀死cpsa突变体。 CPSA包含一个LYTR-CPSA-PSR（LCP）域，通常是 在革兰氏阳性生物体中发现。在肺炎链球菌和枯草芽孢杆菌中，LCP结构域结合 磷酸化的聚异型脂质。我们使用晶体结构和S.对MTB CPSA的结构进行了建模。 肺炎LCP蛋白，发现所有脂质磷酸盐结合的保留均在MTB CPSA中保守。 此外，我们发现CPSA可以结合人类T细胞白血病病毒I型结合蛋白1（税务1BP1）， 和核点蛋白52 kDa（NDP52）。税务1BP1和NDP52是链接涉及的旁系同源物 细菌货物到自噬机械。这是我们假设CPSA抑制的能力 NADPH氧化酶和LAP取决于结合脂质磷酸盐和宿主蛋白税1BP1和NDP52。到 检验我们的假设，我们将（1）研究巨噬细胞杀死cpsa突变体的途径，（2） 表征CPSA蛋白的作用机理，（3）评估这种先天的重要性 体内免疫逃避机制。结合在一起，我们的研究将阐明一种新颖的免疫机制 逃避最强大的病原体之一。通过研究分子机制MTB利用 破坏宿主的细胞功能，我们将进行基本观察，以帮助发展 MTB的更好的治疗和疫苗。