弓形虫棒状体激酶调控宿主NF-κB信号通路及其致病分子机制研究

Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. The period of tachyzoite is the main pathogenic stage. The ROP18 kinase has been identified as a key virulence determinant conferring a high mortality phenotype characteristic of type I Toxoplasma gondii strains. This major effector molecule is secreted by the rhoptries into the host cells during invasion; however, the molecular mechanisms by which this kinase exerts its pathogenic action remain poorly understood. We identified that the host I κ B is a binding partner with the ROP18 using yeast two-hybrid and pull-down assays. In this project, we speculate that ROP18 phosphorylate I κ B to control the host NF-κB pathway. We will use the transgenic parasites expressing wild type ROP18 and parasites expressing kinase death ROP18 to infect HFF cells, using the luciferase report assay,ChIP and EMSA experiment to analyze the host NF-κB pathway of the infected cells; phosphorylation assay in vitro, IP and mutation technique to identify the phosphorylated sites; PCR-Array, WB and Real-time PCR to analyze the modulation of host cell genes in NF-κB pathway by transgenic parasites infection; TUNEL and Annexin-V/PI to analyze apoptosis. Our date will delineate the molecular mechanism utilized by the intracellular pathogen capable of manipulating the NF-κB pathway, and provide the theory and experiment basis for the treatment of Toxoplasmosis.

弓形虫是一种重要的机会致病性病原体，速殖子期是其主要的致病阶段。ROP18 是由速殖子棒状体分泌的丝/苏氨酸蛋白激酶，是决定虫体毒力和增殖能力的关键蛋白，但确切机制不清。我们通过酵母双杂交及亲和实验鉴定出宿主的IκB是 ROP18的作用底物。故我们推测：弓形虫可能通过ROP18磷酸化宿主IκB，进而激活NF-κB信号通路，参与宿主转录调控。为此，拟建立野生型和激酶失活型ROP18转基因虫株并感染细胞，通过萤光素酶报告、ChIP和EMSA实验分析感染不同转基因虫株的宿主NF-κB转录活性变化；体外磷酸化实验、免疫共沉淀及点突变技术明确ROP18 和IκB之间的相互作用和磷酸化位点；检测ROP18激活p65转录调控的下游炎症因子和凋亡相关蛋白的变化；TUNEL和Annexin-V/PI检测感染细胞的凋亡变化。本研究将完善弓形虫参与宿主转录调控的分子机制，为弓形虫病的防治提供新的理论和实验依据。

课题组前期实验通过酵母双杂交及体外亲和实验鉴定出一系列与ROP18互作的宿主蛋白， 其中一个宿主作用蛋白是NF-κB信号通路的的核心转录因子p65。本课题建立了野生型和激酶失活型 ROP18 转基因虫株并感染细胞，通过荧光素酶报告分析发现野生型ROP18虫株可抑制NF-κB信号通路、激酶失活型 ROP18无此抑制作用； 反转录qPCR 和ELISA 实验分析发现ROP18可抑制κB靶向的炎症因子如IL-6，IL-12和TNF-α的表达； 酵母双杂交和pull down实验发现ROP18通过其N端的27-250aa与NF-κB的核心转录因子p65的二聚化域相互作用。体外生化研究结果表明：ROP18磷酸化p65的468位丝氨酸位点将其泛素化，通过蛋白酶体依赖的途径将p65蛋白降解。尤为重要的是，ROP18的激酶活性是导致p65降解的关键因素。与ROP18激酶失活型转基因虫株相比，野生型ROP18虫株可抑制NF-κB信号通路，抑制κB靶向的炎症因子表达，从而驱动巨噬细胞向M2应答偏移/极化。结果首次证明：弓形虫I型虫株的毒力因子ROP18通过磷酸化宿主NF-κB转录因子p65使其发生泛素化降解，进而抑制NF-κB通路的活化和靶向的炎症因子表达；驱动巨噬细胞向M2偏移/极化，这是I型RH株在宿主细胞内赖以生存和快速增殖的机制之一。该研究为筛选弓形虫增殖的新药靶标奠定基础。在本项目的支持下，我们还发现ROP18可以促进宿主神经细胞凋亡并发现了其促凋亡的分子机制。已发表研究论文13篇（SCI收录11篇，核心2篇）。