Granzyme and Granulysin Mediated Death of Trypanosoma cruzi

 描述(申请人提供)：当杀手淋巴细胞识别出被细胞内病原体感染的细胞时，它们会释放其细胞毒颗粒内容物，以诱导受感染目标细胞的凋亡。然而，在这一过程中，细胞内寄生虫发生了什么尚不清楚。宿主细胞的凋亡是由细胞毒性颗粒蛋白(GZM)触发的，GZM由胆固醇依赖的膜扰动蛋白穿孔素(PFN)传递到靶细胞。人类和其他一些哺乳动物的细胞毒性颗粒，而不是啮齿类动物，含有另一种造孔蛋白，颗粒溶素(GNLY)，它优先破坏低胆固醇的细菌、真菌和寄生虫膜。我们最近发现，当人类杀伤淋巴细胞识别受细菌感染的细胞时，它们会迅速杀死细胞内的细菌。GNLY将GZM传递到细菌中，在那里它们蛋白水解性地攻击细菌电子传递链复合体I，产生有毒的超氧阴离子，并蛋白水解性地破坏细菌的氧化防御酶。初步数据显示，GNLY还将GZM传递到原生动物寄生虫(克氏锥虫、弓形虫、大利什曼原虫)中，这些寄生虫也以超氧化物依赖的方式迅速被杀死。细胞内寄生虫的破坏依赖于所有三个细胞毒效应分子-GZMS、PFN和GNLY的协同作用。在杀手淋巴细胞中表达GNLY转基因的小鼠能够在对野生型(WT)小鼠致命的克氏锥虫挑战中存活下来。该方案以克氏锥虫为模型，研究GNLY、GZM和PFN介导的杀灭胞内原生动物寄生虫对胞内寄生虫具有强大的免疫保护作用。我们假设克氏锥虫的无鞭毛体在宿主细胞之前被杀死，这限制了感染的传播。我们还假设，在克鲁兹锥虫中，杀伤细胞迅速诱导细胞死亡程序，这与哺乳动物细胞或细菌中GZMS激活的死亡途径有一些共同的特征。我们还假设，直接杀死寄生虫有助于控制急性感染，增强疫苗保护，并减少慢性感染的突变病理。这项建议加入了两位主要研究人员的专业知识，他们是研究(1)杀手细胞如何摧毁受感染的宿主细胞及其病原体和(2)对克氏锥虫的免疫反应的领导者。该提案的目的是通过比较不表达GNLY的WT小鼠和GNLY转基因小鼠的感染情况，定义杀伤细胞介导的寄生虫死亡的特征，识别在导致死亡中重要的克氏锥虫GZM底物，并探索直接杀死寄生虫如何有助于免疫防御。

 DESCRIPTION (provided by applicant): When killer lymphocytes recognize cells infected with intracellular pathogens, they release their cytotoxic granule contents to induce apoptosis of the infected target cell. However, what happens to intracellular parasites during this process is unclear. Host cell apoptosis is triggered by cytotoxic granule proteases (granzymes, Gzm), delivered into the target cell by the cholesterol-dependent membrane perturbing protein, perforin (PFN). Cytotoxic granules of humans and some other mammals, but not rodents, contain another pore-forming protein, granulysin (GNLY), which preferentially disrupts cholesterol-poor bacterial, fungal and parasite membranes. We recently found that when human killer lymphocytes recognize bacteria-infected cells, they rapidly kill intracellular bacteria. GNLY delivers Gzms into bacteria where they proteolytically attack bacterial electron transport chain complex I to generate toxic superoxide anion and also proteolytically destroy bacterial oxidative defense enzymes. Preliminary data show that GNLY also delivers Gzms into protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii, Leishmania major), which are rapidly killed, also in a superoxide-dependent manner. Destruction of intracellular parasites relies on the concerted action of all three cytotoxic effector molecules - Gzms, PFN and GNLY. Mice that express a GNLY transgene in killer lymphocytes are able to survive a T. cruzi challenge that is lethal to wild-type (WT) mice. This proposal uses T. cruzi as a model to investigate the hypothesis that GNLY, Gzm and PFN-mediated killing of intracellular protozoan parasites provides strong immune protection against intracellular parasites. We hypothesize that T. cruzi amastigotes are killed before the host cell, which limits the spread of infection. We also hypothesize that killer cells rapidly induce programs of cell death in T. cruzi that share some features with death pathways activated by Gzms in mammalian cells or bacteria. We also hypothesize that direct parasite killing helps control acute infection, enhance vaccine protection and reduce the chagasic pathology of chronic infection. This proposal joins the expertise of two principal investigators, who are leaders in studying (1) how killer cells destroy infected host cels and their pathogens and (2) the immune response to T. cruzi. The aims of the proposal are to define the features of killer cell-mediated parasite death, identify T. cruzi Gzm substrates that are important in causing death and explore how direct parasite killing contributes to immune defense by comparing infection in WT mice that do not express GNLY with GNLY-transgenic mice.