宿主细胞互作蛋白介导的弓形虫ROP18毒力机制研究

Toxoplasma gondii is a widely distributed intracellular parasitic protozoa, and may cause severe illness and even death in immunosuppressive patients and fetal infection. The processes of Toxoplasma gondii invading host cells and causing pathological injury, depend on the interaction of the parasite molecules with the host cell proteins. The current researches on Toxoplasma virulence mechanism are focused on the discovery and identification of virulence mediating factors , only few of them are involved deeply into the host cell regulation etc. ROP18 is an important virulence mediating factor of Toxoplasma gondii, which is closely related to the strain virulence, and exhibits polymorphism in different strains. This project aims to screen the human proteins interacting with the ROP18s from different strains of Toxoplasma gondii from the human protein library in high-throughput, with the interaction protein screening platform based on the technology of Biomolecular Fluorescence Complementation Assay (BiFC). CO-IP and FRET will be used to identify its interaction; the profiles of the host proteins interacting with the ROP18s from different strains will be compared and the interaction network diagram will be drawn. ROP18 interaction protein genes will be knocked out from the host cell to detect the virulence efficiency of Toxoplasma gondii in infection. Co-localization of ROP18 with its interaction host proteins and the effects on the host cell signaling will be observed. From above researches, the mechanism of ROP18 virulence medicated by the host interaction proteins will be more clear, and it is important for understanding of Toxoplasma gondii pathogenesis mechanism, and finding of the effective drug targets.

弓形虫是广泛分布的细胞内寄生原虫，在免疫抑制病人及胎儿感染中引起严重疾病甚至死亡。弓形虫感染宿主并产生病理损伤，依赖于寄生虫分子与宿主细胞蛋白的互作。目前对弓形虫毒力调节因子的发现及鉴定是弓形虫毒力机制研究的热点，却少有研究深入到毒力调节因子对宿主细胞的调控等方面。ROP18是弓形虫重要的毒力调节因子，与虫株毒力密切相关，在不同毒株中具有多态性。本项目拟应用基于双分子荧光互补技术(BiFC)的平台从人蛋白库中高通量地筛选弓形虫不同毒株ROP18的互作蛋白，并用CO-IP及FRET验证其互作；比较不同毒株ROP18互作蛋白谱的差异并绘制其互作网络图；敲除细胞ROP18互作蛋白基因测试其对弓形虫毒力效率的影响；观察互作蛋白对在细胞内的共定位及对宿主细胞信号通路活化的作用。从而阐明宿主细胞互作蛋白介导的弓形虫ROP18毒力的机制，对理解弓形虫的致病机制及寻找有效的药物靶点具有重大的意义。

应用BiFC技术，我们在18,000余个人蛋白中，筛选到492及141个蛋白分别与ROP18I及ROP18II互作。用 FRET、Co-IP、IFA等实验证实NMI可与ROP18I强互作，但不与ROP18II互作；ROP18I与宿主细胞P2X1、IL20RB, IL21, UBC,和Vimentin均互作。GO分析发现ROP18的互作蛋白的功能与天然免疫与细胞凋亡有关。在感染CEP+ROP18I的HFF中细胞核内的NMI的量明显减少, ChIP实验结果显示，在感染CEP+ROP18I的HFF中，结合在IFN-γ/STAT1通路下游基因irf1的启动子区域的GAS element的NMI显著低于对照组，且ROP18I抑制IFN-γ/STAT1通路下游基因irf1及nmi的转录及表达，说明ROP18I在宿主细胞质中与STAT1竞争结合NMI，阻遏NMI向胞核转移并结合于GAS element上，最终抑制人细胞中的IFN-γ/STAT1信号通路实现免疫逃避。用RH-WT及RH-∆ROP18株感染SF268细胞，我们观察到TgROP18能够抑制ATP所诱导的线粒体膜去极化和线粒体内细胞色素C的释放，促进Bcl2在线粒体膜上表达，同时抑制Caspase 3和7活化。在COS-7细胞内分别过表达TgROP18或P2X1，ATP作为P2X1的配体刺激细胞后，过表达P2X1的胞质内钙离子浓度增加；然而在共表达TgROP18和P2X1的细胞中，TgROP18通过降低P2X1蛋白水平抑制P2X1所诱导的钙离子内流。以上结果提示TgROP18可能通过P2X1-Ca2+-Cyt C-caspase路径抑制细胞凋亡。我们发现TRIM21的PRY-SPRY结构域为其与ROP18互作所必需，互作后TRIM21发生磷酸化并使ROP18发生蛋白酶体途径的降解，而ROP18促进TRIM21的降解依赖于溶酶体途径。体外实验证明TgROP18磷酸化并降解CLUL1，且这种作用依赖于ROP18的激酶活性；TgROP18促进CLUL1的泛素化，从而通过蛋白酶体途径促进CLUL1的降解,促进SF268细胞的凋亡。截短的ROP18（仅保留激酶区）突变体仍可与CLU1蛋白互作，但使CLUL1的降解减弱，提示ROP18激酶结构域是与CLUL1相互作用的区域。