Granulysin, Granzymes and Perforin in Bacterial Immune Defense

细菌免疫防御中的颗粒溶素、颗粒酶和穿孔素

基本信息

批准号：
9222706
负责人：
Judy Lieberman
金额：
$ 44.25万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-15 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9222706
关键词：
AIDS-Related Opportunistic Infections Aerobic Bacteria Alpha Cell Anabolism Anaerobic Bacteria Animal Model Anti-Bacterial Agents Apoptosis Bacteria Bacterial Infections Caspase Cell Death Cell membrane Cells Cessation of life Characteristics Cholesterol Cleaved cell Complex Cytolysis Cytoplasmic Granules Cytotoxic T-Lymphocytes Data Electron Transport Enzymes Escherichia coli Fatty acid glycerol esters GZMA gene Genus Mycobacterium Goals Granzyme Hepatocyte Hip region structure Host Defense Mechanism Human Immune Immune System Diseases Immunity Immunologic Deficiency Syndromes Infection Interleukin-4 Killer Cells Listeria monocytogenes Liver diseases Lymphocyte Mammalian Cell Mammals Mediating Membrane Metabolic Metabolic Pathway Metabolic stress Metabolism Meteor Microbe Mitochondria Modeling Mus Mycobacterium tuberculosis Natural Killer Cells Outcome Oxidative Phosphorylation Oxidative Stress Parasites Pathway interactions Patients Peptide Hydrolases Physics Physiological Play Process Proteins Proteolysis Research Role Signal Transduction Skin Stimulus Stress Superoxides T-Cell Immunodeficiency T-Lymphocyte Time Transgenes VDAC1 gene Virulence Wild Type Mouse antimicrobial peptide bacterial metabolism bactericide base biological adaptation to stress catalase cytokine cytotoxic extracellular granulysin in vivo killings macrophage nanomolar novel novel strategies oxidative damage pathogen perforin programs public health relevance

项目摘要

DESCRIPTION (provided by applicant): Killer lymphocytes release their cytotoxic granule contents when they recognize cells infected with intracellular pathogens. Cytotoxic granule proteases (granzymes, Gzm), delivered into the target cell by the membrane perturbing protein perforin (PFN), trigger host cell apoptosis. However, what happens to intracellular bacteria during this process is unclear. Cytotoxic granules of humans and most other mammals, but not rodents, contain another pore-forming protein, granulysin (GNLY), which preferentially disrupts cholesterol-poor bacterial, fungal and parasite membranes, to lyse extracellular pathogens when applied at high concentrations. We recently found that GNLY delivers Gzms into intracellular and extracellular bacteria, where they rapidly kill bacteria. In aerobic bacteria, Gzs cleave bacterial electron transport chain (ETC) complex I components to disrupt electron transport and generate superoxide anion and at the same time cleave and disrupt oxidative stress defense enzymes that detoxify superoxide anion, rendering the bacteria defenseless. Anaerobic bacteria are also killed, but more slowly. The importance of this pathway in vivo was assessed using mice bearing a GNLY transgene (Tg), expressed, like the human protein, only in activated killer cells. GNLY-Tg mice cleared L. monocytogenes (Lm) much more effectively than wild-type (WT) mice. The goal of this application is to investigate the hypothesis that Gzms, GNLY and PFN in cytotoxic T cells and NK cells and other innate-like lymphocytes play an important role in bacterial immune defense and to define under what in vivo conditions it is important and the pathways that mediate bacterial cell death. Based on preliminary analysis of Gzm targets in bacteria, we hypothesize that the Gzms activate common programs of cell death in bacteria that disrupt key biosynthetic and metabolic pathways and stress responses. We will answer the following questions: Which types of lymphocytes express GNLY and do bacteria or bacterial products induce its expression? Are killer cells important against bacteria that do not use oxidative phosphorylation? Are extracellular bacteria targeted under some conditions? Is GNLY on its own used for NK and T cell-mediated elimination of extracellular bacteria, while all 3 effector molecules are employed to eliminate intracellular bacteria? Are there important bacterial substrates that contribute to bacterial elimination besides ETC complex I? We will answer these questions using a few model organisms. The in vivo consequences of GNLY and Gzm-dependent bacterial defense will be examined by comparing bacterial infections in WT and GNLY-Tg mice, focusing on the skin and gut, where bacteria enter the body. Our specific aims are to (1) define characteristics of bacterial death programs activated by Gzms and GNLY, (2) identify Gzm substrates and investigate whether and how the Gzms disrupt bacterial metabolism, biosynthesis and the stress response, and (3) determine the role of cytotoxic effector molecules (GNLY, Gzms, PFN) and classes of killer lymphocytes in protection from infection by diverse bacterial strains.

描述（由申请人提供）：杀伤淋巴细胞在识别被细胞内病原体感染的细胞时释放其细胞毒性颗粒内容物（颗粒酶，Gzm），通过膜扰动蛋白穿孔素（PFN）递送至靶细胞，触发宿主细胞。然而，人类和大多数其他哺乳动物（但啮齿类动物除外）的细胞内细菌在此过程中发生的情况尚不清楚。造孔蛋白颗粒溶素 (GNLY)，在高浓度下使用时，会优先破坏缺乏胆固醇的细菌、真菌和寄生虫膜，从而裂解细胞外病原体。我们最近发现，GNLY 将 Gzms 传递到细胞内和细胞外细菌中，并迅速杀死细菌。在需氧细菌中，Gzs 裂解细菌电子传递链 (ETC) 复合物 I 组件，以破坏电子传递并产生超氧阴离子，同时裂解并产生超氧阴离子。破坏氧化应激防御酶，使超氧阴离子解毒，使细菌失去防御能力。厌氧细菌也会被杀死，但体内这条途径的重要性是用携带 GNLY 转基因 (Tg) 的小鼠进行的，其表达与人类蛋白质一样。，仅在激活的杀伤细胞中，GNLY-Tg 小鼠比野生型 (WT) 小鼠更有效地清除单核细胞增生李斯特菌。细胞毒性 T 细胞、NK 细胞和其他先天样淋巴细胞中的 Gzms、GNLY 和 PFN 在细菌免疫防御中发挥着重要作用，并根据初步分析确定在体内条件下它是重要的以及介导细菌细胞死亡的途径。通过研究细菌中的 Gzm 靶点，我们帮助 Gzms 激活细菌中常见的细胞死亡程序，从而破坏关键的生物合成和代谢途径以及应激反应。我们将回答以下问题：哪些类型的淋巴细胞表达 GNLY，并且细菌表达 GNLY。或细菌产物诱导其表达？杀伤细胞对不使用氧化磷酸化的细菌是否重要？在某些条件下，GNLY 是否可以单独用于 NK 和 T 细胞介导的细胞外细菌消除，而所有 3 个效应子？分子用于消除细胞内细菌吗？除了 ETC 复合物 I 之外，是否还有有助于细菌消除的重要细菌底物？我们将使用一些模型生物来回答这些问题。 GNLY 和 Gzm 依赖性细菌防御的体内后果将是怎样的？通过比较 WT 和 GNLY-Tg 小鼠的细菌感染进行检查，重点关注细菌进入体内的皮肤和肠道，我们的具体目标是 (1) 定义 Gzms 和 GNLY 激活的细菌死亡程序的特征，(2)识别 Gzm 底物并研究 Gzms 是否以及如何破坏细菌代谢、生物合成和应激反应，以及 (3) 确定细胞毒性效应分子（GNLY、Gzms、PFN）的作用和杀伤淋巴细胞的类别防止多种细菌菌株的感染。