类锥体相关蛋白TgCAHP1在弓形虫入侵宿主细胞过程中的功能及作用机制

弓形虫是专性细胞内寄生原虫，入侵宿主细胞是虫体寄生与致病的先决条件。弓形虫类锥体已被证明在虫体入侵中发挥重要角色作用，但机制一直不清。课题组前期发现，TgCAHP1定位于弓形虫类锥体，能与宿主细胞表面唾液酸结合并竞争抑制弓形虫入侵，其抗体也能部分抑制入侵，这提示TgCAHP1参与弓形虫入侵过程。项目拟通过CRISPR/Cas9技术构建CAHP1基因敲除和回补虫株，通过超高分辨率显微镜和透射电镜观察胞内和胞外弓形虫类锥体形态结构变化，解析TgCAHP1对类锥体形态结构稳定性作用；通过类锥体推出、滑移运动、微线体蛋白分泌及入侵等实验，确定TgCAHP1在弓形虫入侵细胞中的作用；通过生物素化标记技术、质谱鉴定、基因敲除等方法，构建TgCAHP1互作蛋白网络图并解析互作原理，旨在阐明TgCAHP1在弓形虫入侵宿主细胞过程中的功能及作用机制，为进一步揭示弓形虫对宿主细胞入侵及致病机理提供理论基础。

Toxoplasma gondii is an obligate intracellular parasitic protozoa. Host cell invasion is the precondition of parasitism and pathogenicity by T. gondii. The conoid has been shown to play an important role in the host cell invasion by T. gondii, but the mechanism has been unclear now. Our previous studies found that TgCAHP1 was located on the conoid of T. gondii, and could bind to sialic acid on the surface of host cells to compete to inhibit the invasion of T. gondii, its antibodies could partially inhibit the invasion by T. gondii, indicating that TgCAHP1 was involved in the host cell invasion process by T. gondii. In the present study, CAHP1 gene knockout and knockin strains of T. gondii will be constructed by CRISPR/Cas9 technology, then the effect of TgCAHP1 on the morphological and structural stability of conoid of T. gondii will be analyzed by morphological and structural changes of conoid in intracellular and extracellular T. gondii observed by ultra-high resolution microscopy and transmission electron microscopy; the role of TgCAHP1 in the invasion process by T. gondii will be determined by parasite conoid extrusion, gliding motility, microneme secretion and host cell invasion combined with phase contrast microscopy and video microscopy; the network of TgCAHP1 and interacted proteins will be constructed by analysis of biotinylated proteins (BioID) and mass spectrometry analysis (LC-MS/MS), and interactome analysis will be carried out by gene knockout and endogenous labeling techniques. The study will elucidate the function and mechanism of TgCAHP1 in the host cell invasion process by T. gondii, which will provide a theoretical basis for further revealing the host cell invasion and pathogenesis by T. gondii and lay a foundation for the identification of new drug targets and vaccine candidate antigens against T. gondii infection.