弓形虫丝/苏氨酸蛋白激酶调控宿主细胞凋亡的线粒体途径机制研究

Toxoplasma gondii is an obligate intracellular parasite and could have a great proliferation in host cells. The parasites could suppress the host cell apoptosis and make the host immune system can not eliminate the infected cells. However, the mechanism of host cell apoptosis suppression is not very clearly. Base on phenomenon of host mitochondria association with the parasitophorous vacuole and the important role of mitochondria in cell apoptosis, we speculate that T. gondii could regulate the host mitochondria function and suppress cell apoptosis by rhoptry protein with serine/threonine protein kinases activity. In order to confirm our hypothesis, we want to study the function of T. gondii ROP16, ROP18 and ROP38 (ROP16/18/38) in the host cell apoptosis suppression. The distribution of ROP16/18/38 in the host cells would be studied by indirect immunofluorescence assay and immuno-electron microscope. The impacts of ROP16/18/38 on host mitochondria function are studied by the methods of overexpression of ROP16/18/38 in HeLa cell and knockout ROP16/18/38 genes in T. gondii. We will find out the mechanism of host cell apoptosis via mitochondria pathway regulated by serine/threonine protein kinases in T. gondii.

弓形虫是专性细胞内寄生虫，能在细胞内大量增殖的原因是，虫体抑制宿主细胞凋亡，阻止宿主免疫系统清除被感染细胞。然而，弓形虫抑制细胞凋亡的机制尚不明确。根据弓形虫诱导宿主细胞线粒体向纳虫泡聚集的现象以及线粒体在调控细胞凋亡中的关键作用，我们推测弓形虫通过自身分泌具有丝/苏氨酸蛋白激酶活性的棒状体蛋白，调控细胞线粒体功能，抑制宿主细胞凋亡。为证实该假说，我们选取具有丝/苏氨酸蛋白激酶活性的ROP16、ROP18和ROP38（ROP16/18/38）作为研究对象，采用基因标记、间接免疫荧光和免疫电镜等技术研究ROP16/18/38在被感染细胞中分布；结合HeLa细胞过表达技术和弓形虫基因敲除技术探讨ROP16/18/38对宿主细胞线粒体功能的影响，证明弓形虫丝/苏氨酸蛋白激酶可以调控宿主细胞线粒体功能，阐明弓形虫丝/苏氨酸蛋白激酶调控宿主细胞凋亡的线粒体途径机制。

弓形虫是一种专性细胞内寄生原虫，能侵入宿主所有的有核细胞并大量增殖破坏细胞，最终造成宿主组织器官的损伤。抑制细胞凋亡是弓形虫在宿主体内大量增殖一个重要策略，虫体通过抑制被感染细胞的凋亡，阻止宿主免疫系统清除被感染细胞，为虫体创造一个能够长时间增殖的良好生存环境。本项目中，我们研究了弓形虫分泌的丝/苏氨酸蛋白激酶ROP16和ROP18调控宿主细胞凋亡的能力以及调控机制。采用真核细胞转染技术在293T细胞中过表达弓形虫ROP16和ROP18，检测转染前后293T细胞的凋亡率和细胞线粒体外膜电位差变化，以及线粒体细胞色素C向胞浆外泄程度。研究结果表明，弓形虫ROP16和ROP18可以显著抑制宿主293T细胞凋亡。其抑制细胞凋亡是通过维持细胞线粒体膜电位差和完整性，防止内部细胞色素C等物质泄露启动线粒体途径凋亡。