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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.

项目摘要结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病（TB）的病原体，是结核病的主要病原体。全世界死于细菌感染的原因。结核分枝杆菌的成功源于其规避能力通过巨噬细胞降解。最近的研究表明，巨噬细胞通过两种不同的溶酶体运输途径，涉及LC 3标记的细胞器（2，3）。食异是一个过程 LC 3标记的双膜细胞器捕获并降解入侵的微生物。LC 3相关吞噬作用（phagocytosis，简称PHD）与异种吞噬作用相似，但不涉及双膜，需要NADPH 氧化酶和活性氧（ROS），这是不必要的异食。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。激活许多PRR的Mtb未能引起实质性LC 3相关的吞噬溶酶体的运输尚不清楚。我们广泛的初步数据有力地表明，CpsA，一种由Mtb分泌的未表征的蛋白质，特异性地阻断β-内酰胺酶。我们假设CpsA干扰了激活NADPH氧化酶，从而阻断ROS的产生和LAP介导的Mtb向溶酶体与我们的假设一致，我们发现缺乏cpsA的结核分枝杆菌菌株表现出显著的增强与LC 3标记物的共定位，并且它们在巨噬细胞中高度减毒，小鼠此外，NADPH氧化酶和蛋白质特异性地需要的细胞是必要的，巨噬细胞来杀死cycpsA突变体。CpsA含有LytR-CpsA-Psr（LCP）结构域，其通常是发现于革兰氏阳性菌。在肺炎链球菌和枯草芽孢杆菌中，LCP结构域结合磷酸化聚类异戊二烯脂质。我们利用S. pneumoniae LCP蛋白，并发现所有的脂质磷酸结合残基在Mtb CpsA中是保守的。此外，我们发现CpsA可以与人T细胞白血病病毒I型结合蛋白1（TAX 1BP 1）结合，和核点蛋白52 kDa（NDP 52）。TAX 1BP 1和NDP 52是参与连接的旁系同源物自噬机器的细菌货物。因此，我们假设CpsA抑制肿瘤细胞增殖的能力， NADPH氧化酶和NADPH依赖于结合脂质磷酸和宿主蛋白TAX 1BP 1和NDP 52。到为了验证我们的假设，我们将（1）研究巨噬细胞杀死突变体的途径，（2）表征CpsA蛋白的作用机制，和（3）评估这种先天性的重要性，体内免疫逃避机制。结合这些研究，我们的研究将阐明一种新的免疫机制，躲避最可怕的病原体通过研究Mtb利用的分子机制，破坏宿主细胞功能，我们将进行基本的观察，这将有助于发展更好的治疗方法和疫苗。