微小隐孢子虫Cp2蛋白与宿主受体互作在虫体入侵中的作用机制研究

There are no effective specific pharmaceuticals and vaccines available for Cryptosporidiosis up to now. Limited knowledge on the pathogenesis of the parasite to host cells has hampered the development of targeted intervention strategies for this important disease. The previous studies suggested that the secreted proteins (Cp2) of C. parvum can interact with Integrin α2 of   host cell, then.activate actin accumulation signal pathway, and some key signal molecules among the pathway play important roles during C. parvum invasion. Based on current results and correlative research reports, we hypothesized that the activation of host cell actin accumulation signal pathway via interaction between C. parvum Cp2 and corresponding host cell receptors (Integrin α2) might be the key triggers.for C. parvum invasion. This project proposed to focus on the early stage of C.parvum invasion to the host cell. To understand the mechanism of interaction between C. parvum Cp2 and corresponding host cell receptors (Integrin α2) , Structure of C. parvum Cp2 will be analyzed through crystallization. Based on the above results, we will focus on several signal pathways that through C. parvum Cp2.-mediated activate the actin cytoskeleton and Integrin α2/β1 of the host cell. This project will reveal the mechanism of cellular nvasion by C. parvum via C.parvum Cp2 - mediated cellular actin polymerization signal pathway. The results of this project will strongly support us to further pursue the development of new classes of the drugs and vaccines, and to prevent and control cryptosporidiosis in human and animals.

隐孢子虫病目前尚无有效防治措施，其原因在于对其入侵等致病机制知之甚少。前期研究发现微小隐孢子虫入侵相关蛋白Cp2与宿主细胞对应受体Integrinα2互作，进而激活宿主细胞F-actin聚集重排通路，此通路与微小隐孢子虫入侵宿主细胞密切相关。基于此，以虫体入侵宿主细胞这一关键环节为基点，将微小隐孢子虫Cp2蛋白作为重点，利用结构生物学、分子对接分析，基因序列定点突变、免疫组化等技术研究微小隐孢子虫入侵相关蛋白Cp2蛋白与其宿主细胞受体Integrinα2的分子互作。在此基础上，利用Western Blot和磷酸化检测、药物阻断、细胞靶基因修饰、细胞培养等技术研究Cp2经Integrinα2介导的Actin聚集重排信号通路关键信号分子对隐孢子虫入侵的调控作用，进而阐明Cp2与细胞受体互作激活的Actin聚集重排介导的微小隐孢子虫入侵机制，研究结果对于揭示隐孢子虫的入侵和致病机制具有重要意义。

顶复门原虫入侵宿主细胞主要通过滑行运动的形式进行入侵，其中微线体蛋白TRAP家族是一种重要的入侵蛋白。本研究以微小隐孢子虫微线体蛋白TRAP-C1为研究对象，在前期建立的HCT-8细胞感染模型的基础上，筛选获得TRAP-C1受体蛋白CDH1，并证明TRAP-C1通过与受体CDH1互作经由CDH1-CTNNA1-F-actin信号通路介导隐孢子虫的入侵，取得了如下结果：通过构建重组质粒pGEX-4T-1-TRAP-C1并表达出带有GST标签蛋白的GST-TRAP-C1蛋白，利用GST pull-down技术共钓取出与TRAP-C1结合的HCT-8细胞蛋白939种，经过生信分析后最终筛选到TRAP-C1在HCT-8细胞上的受体CDH1。利用免疫共沉淀技术发现TRAP-C1能够与CDH1结合并被相应的抗体捕获，表明TRAP-C1与CDH1能够在体外发生互作。通过建立微小隐孢虫入侵HCT-8细胞模型，利用间接免疫荧光检测微小隐孢子虫TRAP-C1与HCT-8细胞CDH1的定位情况，发现TRAP-C1蛋白和CDH1蛋白在细胞膜表面子孢子入侵部位发生共定位，进一步表明TRAP-C1与CDH1蛋白存在相互作用。通过微小隐孢子虫感染率计算模型，分别对细胞CDH1基因沉默、抗体阻断和蛋白封闭等方法处理后检测隐孢子虫入侵率。结果显示，对HCT-8细胞CDH1基因沉默后，子孢子入侵率下降40%；对HCT-8细胞CDH1蛋白进行抗体阻断后子孢子入侵率下降约33%；利用重组蛋白TRAP-C1预先封闭HCT-8细胞CDH1受体后子孢子入侵率下降约87%，表明TRAP-C1与CDH1的互作能够促进子孢子的入侵。微小隐孢子虫和重组蛋白TRAP-C1分别作用HCT-8细胞后，检测细胞CDH1和CTNNA1的mRNA和蛋白水平变化，发现两者作用细胞后CDH1和CTNNA1基因的转录水平和蛋白水平均有不同程度的增加。同步激光共聚焦显微镜观察到在微小隐孢子虫子孢子入侵部位发生细胞骨架蛋白F-actin聚集，表明HCT-8细胞的CDH1-CTNNA1-F-actin信号通路可能是微小隐孢子虫诱导HCT-8细胞骨架重排并促进入侵的信号通路。综上，本研究结果为解析微小隐孢子虫入侵机制，研发新型抗隐孢子虫药物和疫苗奠定重要理论基础。