弓形虫依赖唾液酸途径入侵宿主细胞机制研究

弓形虫是专性细胞内寄生原虫，寄生于人和温血动物，引发人兽共患病，世界约1/3人群感染，我国约8%人群感染。作为胞内寄生原虫，弓形虫在感染过程中，其在细胞外仅有非常有限的存活能力，因此虫体对宿主细胞的入侵是虫体得以存活、增殖和不断扩大传播感染的前提条件。虫体与宿主细胞结合（粘附）是入侵过程的首要环节。目前有关弓形虫在宿主细胞表面的受体还不很清楚。本项目旨在通过亲和层析结合SWATH质谱鉴定技术全面解析弓形虫与唾液酸结合的蛋白质；通过体外重组蛋白与唾液酸结合及抑制试验验证弓形虫与唾液酸特异性结合的蛋白质；通过免疫荧光、免疫电镜与抗体抑制实验、CRISPR/Cas9基因敲除技术阐明弓形虫与唾液酸结合的关键蛋白质在虫体的表达定位与功能，最终阐明弓形虫依赖唾液酸途径入侵宿主细胞的分子机理。研究结果不但有助于揭示虫体对宿主细胞入侵及致病机理，还能够为确定新的抗弓形虫感染药物靶标和疫苗候选抗原奠定基础。

Toxoplasma gondii is an obligate intracellular protozoon, which can infect warm-blooded animals, including human, and cause zoonosis. Around one third of the human population worldwide are infected, and about 8% in China. Being an obligate intracellular parasite, T. gondii has a limited capacity to survive outside of a cell during infection, invading a host cell is therefore crucial for its survival, multiplication and the expansion of infection. The first step of the cell invasion by T. gondii is the attachment (adhesion) of the parasite to the host cell. At present, the receptors on host cell's surface recognised by T. gondii remain largely unknown. In this study, T. gondii proteins binding with sialic acid will be analyzed by affinity purification coupled with SWATH mass spectrometry technology; T. gondii specific sialic acid-binding proteins will be verified by recombinant proteins and sialic acid combination and inhibition test; cellular localization and function of key proteins will be verified by using immunofluorescence, immune electron microscopy, antibody inhibit assay and CRISPR/Cas9 gene knockout technology. This will illuminate the molecular mechanism of cell invasion by T. gondii through sialic acid-dependent pathway. Research results will not only help to reveal the mechanism of host cell invasion and pathogenesis by T. gondii, but also lay the foundation for the determination of the new anti-parasite drug targets and candidate vaccine antigens.