弓形虫丝裂原活化蛋白激酶调控虫体毒力的分子基础

Toxoplasmosis, caused by Toxoplasma gondii, is an important zoonotic parasitic disease, having great impact on human health, food safety, and animal husbandry production. There is still no ideal method to control the disease in humans and animals. T. gondii encodes mitogen-activated protein kinase 1 (MAPK1) and MAPK2, which regulate the growth and differentiation of the parasite and the activation of inflammasomes, and play an important role in the pathogenesis of T. gondii. Therefore, MAPKs are considered as important regulation factors of virulence in T. gondii. In the present study, we will quantitatively analyze phosphoproteomics of wild and MAPK-deleted strains of T. gondii by liquid chromatography-mass spectrometry (LC-MS), and to identify phosphorylated proteins by MAPKs in vivo. The recombinant MAPKs were expressed in E. coli, and purified for further in vitro kinase assay. After the parasite lysates from wild strain are used to dephosphorylate endogenous phosphoproteins with alkaline phosphatase, the target kinase is added to phosphorylate its substrates. A common phosphoproteomics workflow including peptide desalting, phosphopeptide enrichment and LC-MS analysis is sequentially applied. The phosphoproteomic experiments generate a set of proteins whose phosphorylation is dependent on MAPKs. The overlap between the direct substrates identified from an in vitro kinase reaction and the in vivo kinase-dependent phosphorylation identified from global phosphoproteomics represents the candidate substrates of MAPKs. The antibodies against MAPKs will be prepared and the interaction proteins with MAPKs will identified using immunoprecipitation and mass spectrometry technology. The candidate substrates of MAPKs will functionally be verified by gene deletion using the gene editing system CRISPR/cas9. The study will solve to the molecular basis of virulence regulation by MAPKs in T. gondii, which will lays the foundation for development of Toxoplasma gene-deleted vaccines and MAPK targeting drugs.

弓形虫病是一种重要的人兽共患寄生虫病，严重危害人类健康、食品安全和畜牧业生产。目前仍无理想的防治方法。弓形虫编码丝裂原活化蛋白激酶MAPK1和MAPK2，调节虫体生长分化及宿主炎症小体活化，在虫体致病过程中起重要作用，是弓形虫的毒力调控因子。本研究拟在前期研究基础上，通过弓形虫MAPKs缺失株与野生株磷酸化蛋白质组定量分析，确定MAPKs体内磷酸化蛋白；体外表达弓形虫MAPKs,经体外蛋白激酶反应，确定其体外磷酸化蛋白。制备抗弓形虫MAPKs抗体，通过免疫共沉淀及质谱技术鉴定MAPKs互作蛋白。综合分析MAPKs体内磷酸化蛋白、体外磷酸化蛋白及互作蛋白，确定弓形虫MAPKs候选作用底物，并构建底物缺失虫株，鉴定其生物学特性，并与MAPKs缺失株比较，从功能上验证弓形虫MAPKs底物，解析弓形虫MAPKs调控虫体毒力的分子基础,为弓形虫基因缺失疫苗及MAPKs靶向药物研制奠定基础。

弓形虫病是一种重要的人兽共患寄生虫病，严重危害人类健康、食品安全和畜牧业生产。目前仍无理想的防治方法。本研究运用CRISPR-cas9基因编辑技术构建了弓形虫MAPK1、MAPK2基因敲除株；建立了弓形虫卵囊/包囊感染途径鉴定ELISA方法；进行了弓形虫感染犬中性粒细胞胞外诱捕网纤维网状结构分析，分析了弓形虫诱导中性粒细胞NETs形成与时间的关联性，证实弓形虫诱导NETs形成依赖于ROS的产生并可逃逸NETs的捕获，弓形虫体具有逃避NETs的DNAse I类似酶，为弓形虫体DNase I类似酶的结构分析与鉴定奠定了基础。上述研究结果对于人兽共患弓形虫病的传播途径研究提供了诊断技术，并开辟了弓形虫免疫逃避机制研究的新方向。. 此外，在我国东北地区发现一种与人发热病相关的新型分节段病毒—阿龙山病毒。并阐明了疾病的临床特征，基因组结构及流行病学，明确了其传播媒介及宿主动物，目前该病毒在其它国家也有发现，揭示其重要的公共卫生意义。阿龙山病毒的发现，拓展了我国虫媒疫病病原谱，对于新发传染病的防控具有重要意义。