Metacaspase调控弓形虫凋亡的分子机制

Apoptosis, a basic phenomenon of physiology, is not only essential to normal development of mammals and other living organisms but also important in the process of diseases. Caspases are central executioners of apoptosis in mammals and many other species. Previous studies showed Toxoplasma gondii exhibited apoptosis upon exposure to apoptosis inducers, and apoptotic parasites were also identified in the peritoneal lavage fluid of mice infected with T. gondii by intraperitoneal injection, but the mechanism of apoptosis in T. gondii has not been elucidated. Our group has conducted preliminary researches on apoptosis in T. gondii. We found that caspases were not existent in T. gondii but metacaspase, which shares structural similarity to caspases, are present in the parasite. Metacaspases are involved in apoptosis in Saccharomyces and Arabidopsis. We have demonstrated that metacaspase is a key factor of apoptosis in T. gondii. Further to our above findings and to elucidate the molecular mechanisms and pathways of apoptosis of T. gondii, we propose to generate metacaspase functional domain knock-out lines and amino acid mutants and compare these genetically modified lines with the wild type. In addition, we will also study the mitochondria，endoplasmic reticulum and death receptor of the parasite lines to understand the pathway and mechanism of metacaspase activation. Downstream molecules of metacaspase will be explored as well to understand the roles of activated metacaspase and downstream molecules in apoptosis. The proposed study findings would enable us to have a comprehensive understanding of the molecular mechanism of and the role of metacaspase in the apoptosis of T. gondii.

凋亡是细胞的基本生物学现象，在生命活动和多种疾病过程中发挥重要作用。Caspases是哺乳动物及多种生物中调控细胞凋亡的关键因子。研究发现凋亡诱导剂可以成功诱导弓形虫的凋亡，从感染小鼠腹腔分离的弓形虫也存在凋亡现象，但未见关于弓形虫凋亡关键因子及凋亡机制的报道。本课题组前期已经开展弓形虫凋亡研究，通过生物信息学分析发现，虽然其不存在Caspases，但存在与其类似的Metacaspase，后者在酵母、拟南芥等生物中具有调控凋亡的功能，我们随后证实其是调控弓形虫凋亡的关键因子。本项目拟在此基础上，构建弓形虫Metacaspase（TgMCA）功能域调控虫株及氨基酸突变虫株，比较不同虫株的凋亡，明确TgMCA发挥凋亡作用的分子基础；检测凋亡虫体的线粒体、内质网及死亡受体变化，确认TgMCA被激活的上游路径；挖掘TgMCA作用的靶蛋白，明确其下游路径。全面解析弓形虫凋亡的分子机制。

利用数据库和软件分析发现刚地弓形虫中存在三个Metacaspases（MCAs），分别命名为TgMCA1、TgMCA2和TgMCA3。.首先，确认胞内虫体（RH株）能够表达MCA1且定位于的虫体胞质；胞外虫体的MCA1转移并最终聚集于虫体核内；分裂过程中弓形虫的MCA1定位于子代虫体的内膜复合体。分别构建了MCA1的单基因缺失株Δmca1、Δmca2和双基因缺失株（Δmca1Δmca2）以及过表达株（mca1-OE）。比较研究发现Δmca1、Δmca2的增殖能力无明显变化，Δmca1Δmca2的增殖能力显著下降，mca1-OE的增殖能力下降，后者的分裂延迟，提示MCA1参与调控虫体分裂。在胞外缓冲液4 h时，Δmca1虫株的凋亡率显著下降，mca1-OE的凋亡率则显著上升，说明MCA1是调节弓形虫的凋亡的重要因子。将各虫株分别接种小鼠，与细胞培养上表现一致，说明两种蛋白的功能有部分重叠。双基因缺失株的大部分纳虫空泡内的虫体不能正常分裂出子代虫体，出现多核体且子代虫体的内膜复合体表达和定位异常，释放后的大部分虫体在胞外变圆。双基因缺失株IMC1的成熟受到抑制，证实弓形虫MCA1和MCA2共同参与虫体IMC1的加工成熟。.同时，还发现缓殖子的MCA2转录水平明显上调，推测其在缓殖子中发挥重要作用。分别构建了RH虫株的MCA2过表达虫株（RHΔku80 mca2-oe）和Pru虫株的MCA2缺失株（PruΔmca2）。发现RHΔku80 mca2-oe的增殖能力下降，而PruΔmca2增殖能力上升，缺失MCA2的Pru虫株不能在细胞培养上被诱导成囊。检测细胞培养下诱导成囊的PruΔmca2缓殖子的特异性基因BAG1、LDH2和SAG4A等的转录水平，发现PruΔmca2比Pru的LDH2、SAG4A和BAG1转录水平明显下降。提示MCA2可能影响了LDH2、SAG4A和BAG1的转录，导致虫体成囊能力下降。.综上所述，MCA1是调节弓形虫自身凋亡的重要因子；两种蛋白在虫体分裂过程中共同发挥作用，参与弓形虫分裂过程中内膜复合体蛋白IMC1的形成；单一缺失MCA1或MCA2对子代虫体分裂的影响不显著。MCA2在Pru虫株的成囊过程中发挥重要作用。