Granulysin, Granzymes and Perforin in Bacterial Immune Defense

 描述（由申请方提供）：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。细胞毒性颗粒蛋白酶（granzymes，Gzm）通过膜扰动蛋白穿孔素（PFN）递送到靶细胞中，触发宿主细胞凋亡。然而，在这个过程中细胞内细菌发生了什么尚不清楚。人类和大多数其他哺乳动物的细胞毒性颗粒，但不是啮齿动物，含有另一种成孔蛋白，颗粒溶素（GNLY），它优先破坏胆固醇贫乏的细菌，真菌和寄生虫膜，溶解细胞外病原体时，在高浓度应用。我们最近发现GNLY将Gzms递送到细胞内和细胞外细菌中，在那里它们迅速杀死细菌。在需氧细菌中，Gz切割细菌电子传递链（ETC）复合物I组分以破坏电子传递并产生超氧阴离子，同时切割并破坏使超氧阴离子解毒的氧化应激防御酶，使细菌失去防御能力。厌氧细菌也被杀死，但速度较慢。使用携带GNLY转基因（Tg）的小鼠评估了该途径在体内的重要性，该转基因与人类蛋白质一样仅在活化的杀伤细胞中表达。GNLY-Tg小鼠清除L.单核细胞增多症（Lm）小鼠比野生型（WT）小鼠有效得多。本申请的目的是研究细胞毒性T细胞和NK细胞和其他先天性样淋巴细胞中的Gzms、GNLY和PFN在细菌免疫防御中起重要作用的假设，并确定在何种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靶点的初步分析，我们假设Gzms激活了细菌中细胞死亡的常见程序，这些程序破坏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，细菌或细菌产物诱导其表达？杀伤细胞对不使用氧化磷酸化的细菌重要吗？在某些条件下，细胞外细菌是否是目标？GNLY是否单独用于NK和T细胞介导的细胞外细菌消除，而所有3种效应分子均用于消除细胞内细菌？除了ETC复合物I之外，是否存在有助于细菌消除的重要细菌底物？我们将用几种模式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通过比较WT和GNLY-Tg小鼠中的细菌感染来检查，重点是皮肤和肠道，细菌进入体内。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）鉴定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）和杀伤淋巴细胞类别在保护免受不同细菌菌株感染中的作用。

 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.