ECP1在弓形虫微孔内吞摄入营养中的作用机理

弓形虫是细胞内专性寄生虫，须从宿主获取营养。宿主细胞器在寄生虫囊泡膜内陷形成小泡而成为虫体的营养来源，然而这些小泡如何摄入有较多争议。近年弓形虫内溶酶体和大分子被摄入虫体的发现，使微孔的内吞作用受到关注，但对其机理完全未知。申请人在前期研究发现一个含EH结构域的新蛋白质ECP1，定位于微孔，且是必需蛋白质。EH结构域蛋白质参与内吞作用，能起动力蛋白的作用，并介导蛋白质的相互作用，推测ECP1是微孔内吞作用的关键蛋白质。因此，申请人拟开展研究：1）明确ECP1在微孔内吞宿主营养中的功能；2）利用免疫共沉淀、生物素标记技术和软件分析等方法，解析ECP1介导的蛋白质互作网络，探明该网络的作用方式，以期揭示微孔内吞作用的分子基础和机理；3）探讨缓殖子微孔内吞作用及其在慢性弓形虫病中的意义。本研究顺利实施将对慢性弓形虫病的药物靶点和疫苗抗原的筛选有重要意义，对其他顶复门原虫的研究也有直接影响。

Toxoplasma gondii is an obligate intracellular parasite, suggesting it must acquire nutrients from host cells. Host organelles invaginate on the parasitophorous vacuolar membrane, releasing vesicles in the vacuole which serve as a nutrient source for parasites. How these vesicles are up-taken into parasites is controversial. Recent advances that the Vacuolar Compartment (VAC) was identified as a parasite endo-lysosome and host proteins were ingested and digested in the VAC strongly argue for existence of endocytosis in T.gondii, potentially at the micropore. However, its mechanism is completely unknown. The applicant has discovered a novel EH-domain Containing Protein named ECP1, which is essential for parasite growth and localized at the micropore. EH domain proteins are associated with endocytosis by mediating protein-protein interactions and by acting as a dynamin-like protein. Therefore, the applicant strongly proposes, (1) to dissect the function of ECP1 on nutrient acquisition via endocytosis at the micropore; (2) to identify the protein-protein interactome mediated by ECP1, and the interactome’s role on endocytosis using various modern technologies such as co-immuno-precipitation, biotinylation, mass-spectrometry and bioinformatics, aiming to find out the molecular basis and mechanism of endocytosis via the micropore; (3) to further confirm the significance of endocytosis at the micropore on nutrient acquisition in chronic toxoplasmosis. This study will potentially provide drug targets and vaccine candidates against toxoplasmosis, and it will also lay solid foundation for studies on nutrient acquisitioon for other Apicomplexan parasites.