Mechanisms of Innate Immune Evasion by Mycobacterium Tuberculosis

结核分枝杆菌先天免疫逃避的机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要结核分枝杆菌 (Mtb) 是结核病 (TB) 的病原体，也是结核病的主要致病菌。结核分枝杆菌的成功源于其逃避细菌感染的能力。最近的研究表明，巨噬细胞通过巨噬细胞清除微生物。涉及 LC3 标记细胞器的两种不同的溶酶体运输途径 (2, 3)。带有 LC3 标记的双膜细胞器可以捕获并降解与 LC3 相关的入侵微生物。吞噬作用（LAP）与异体吞噬相似，但不涉及双层膜并且需要 NADPH 氧化酶和活性氧（ROS），它们对于异体吞噬不是必需的。降解途径由刺激病原体识别受体（PRR）的微生物配体激活。 Mtb 能激活大量 PRR，但未能引起实质性 LC3 相关的原因我们广泛的初步数据强烈表明 CpsA 是一种吞噬溶酶体贩运。 Mtb 分泌的未表征的蛋白质，专门阻断 LAP 我们努力解决 CpsA 干扰的问题。激活 NADPH 氧化酶，从而阻断 ROS 的产生以及 LAP 介导的 Mtb 递送与我们的假设一致，我们发现缺乏 cpsA 的 Mtb 菌株表现出显着的变化。与 LAP 的 LC3 标记物的共定位增强，并且它们在巨噬细胞中高度减弱，此外，NADPH 氧化酶和 LAP 特异所需的蛋白质对于小鼠来说是必需的。巨噬细胞杀死 cpsA 突变体，CpsA 包含一个 LytR-CpsA-Psr (LCP) 结构域，这是常见的。在革兰氏阳性菌中发现，LCP 结构域与肺炎链球菌和枯草芽孢杆菌结合。我们使用晶体结构和 S 来模拟 Mtb CpsA 的结构。肺炎链球菌 LCP 蛋白，发现所有脂质磷酸结合残基在 Mtb CpsA 中都是保守的。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（TAX1BP1），核点蛋白 52 kDa (NDP52) 和 NDP52 是参与连接的旁系同源物。因此，我们认为 CpsA 具有抑制自噬机制的能力。 NADPH 氧化酶和 LAP 依赖于脂质磷酸盐和宿主蛋白 TAX1BP1 和 NDP52 的结合。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 蛋白的作用机制，并且 (3) 评估这种先天性的重要性结合体内的免疫逃避机制，我们的研究将阐明一种新的免疫机制。通过研究结核分枝杆菌利用的分子机制来逃避最强大的病原体之一。破坏宿主细胞功能，我们将进行基本观察，这将有助于开发更好的结核分枝杆菌治疗方法和疫苗。