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

• 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
• 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

 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(申请人提供)：当杀手淋巴细胞识别感染了细胞内病原体的细胞时，他们会释放出细胞毒颗粒内容物。细胞毒性颗粒蛋白(GZM)通过膜扰动蛋白穿孔素(PFN)递送到靶细胞，触发宿主细胞的凋亡。然而，在这个过程中，细胞内的细菌发生了什么还不清楚。人类和大多数其他哺乳动物(但不包括啮齿动物)的细胞毒性颗粒含有另一种造孔蛋白-颗粒溶素(GNLY)，当高浓度应用时，颗粒溶素优先破坏低胆固醇的细菌、真菌和寄生虫膜，以溶解细胞外病原体。我们最近发现，GNLY将GZM输送到细胞内和细胞外的细菌中，在那里它们可以迅速杀死细菌。在需氧细菌中，GZS裂解细菌电子传输链(ETC)复合体I组分，扰乱电子传递，产生超氧阴离子，同时裂解和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，但速度更慢。使用携带GNLY转基因(TG)的小鼠评估了这一途径在体内的重要性，该转基因与人类蛋白一样，仅在激活的杀伤细胞中表达。GNLY-TG小鼠比野生型(WT)小鼠更有效地清除单核细胞增多性乳杆菌(LM)。本应用的目的是探讨细胞毒性T细胞、NK细胞和其他类天然淋巴细胞中的GZMS、GNLY和PFN在细菌免疫防御中起重要作用的假说，并确定在什么体内条件下它是重要的，以及介导细菌细胞死亡的途径。基于对细菌中GZM靶标的初步分析，我们假设GZM激活了细菌中常见的细胞死亡程序，从而扰乱了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，细菌或细菌制品是否诱导其表达？杀伤细胞对不使用氧化磷酸化的细菌重要吗？胞外细菌在某些条件下是靶标吗？GNLY是否单独用于NK和T细胞介导的细胞外细菌清除，而所有三种效应分子都用于消除细胞内细菌？除了ETC复合体I外，是否还有重要的细菌底物有助于消除细菌？我们将使用几个模型生物来回答这些问题。依赖GNLY和GZM的细菌防御在体内的后果将通过比较WT和GNLY-TG小鼠的细菌感染来检验，重点是细菌进入人体的皮肤和肠道。我们的具体目标是(1)确定GZMS和GNLY激活的细菌死亡程序的特征，(2)确定GZM底物，并研究GZMS是否以及如何扰乱细菌的新陈代谢、生物合成和应激反应，以及(3)确定细胞毒效应分子(GNLY、GZMS、PFN)和不同类型的杀伤淋巴细胞在保护不同细菌菌株感染中的作用。