弓形虫新毒力因子MAPK的表达特性及功能分析

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. Mitogen-activated protein kinase (MAPK) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including proliferation, differentiation, apoptosis and survival. Even though these proteins are highly conserved, MAPKs from closely related species often possess distinct signature sequences, making them well suited as drug discovery targets. Toxoplasma gondii has been found two MAPK proteins, including MAPK1 and MAPK2, which are the novel virulence factors. MAPK1 is a stress response protein associated with T. gondii growth and development. However, the expression and functions of MAPK in T. gondii are still unclear. In the present study, we will determine the expression characteristics of MAPK1 and MAPK2 in T. gondii, and construct MAPK2-deficient strain, and MAPK1/2 double genes-deficient strain or conditional knockout of MAPK2 on the basis of MAPK1-deficient strain, whose biological characteristics will be further determined. By comparison of cell adhesion, invasion, proliferation, virulence in mice, cyst formation, and related protein expression changes of these gene-deficient strains, the functions and possible mechanisms of T. gondii MAPK1/2 will be clarified, which 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。丝裂原活化蛋白激酶（MAPK）是一类保守的丝氨酸／苏氨酸蛋白激酶，调节细胞增殖、分化、凋亡等过程。弓形虫编码MAPK1和MAPK2，其功能还未见报道。我们通过敲除弓形虫MAPK1，发现其与虫体生长发育及致病密切相关，是一种新的毒力因子。本研究拟在前期研究结果基础上，确定弓形虫在不同应激条件下MAPK1和MAPK2的表达特性；构建弓形虫MAPK2缺失株以及MAPK1/2双基因缺失株或MAPK1缺失/MAPK2条件表达虫株，比较弓形虫MAPK基因缺失株的细胞粘附、侵入、增殖、小鼠致病力、包囊形成等的变化，分析弓形虫MAPK1/2的功能及作用特点，为弓形虫基因缺失疫苗及MAPK靶向药物研制奠定基础。

弓形虫是一种全球性分布、可感染人及多种动物的机会性致病原虫，在世界人群中的感染率约为5％～25％，我国平均感染率为7.9%，在某些特殊人群中感染率高达40%以上。近年来，随着艾滋病等免疫抑制人群的逐年上升，弓形虫病发病率也随之升高，已成为一个严重的、世界性的公共卫生问题。目前尚无理想的治疗药物。安全、高效的弓形虫疫苗研制对于控制弓形虫病具有重要意义，弓形虫基因缺失疫苗是未来疫苗研制的发展趋势。.丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPKs）是真核细胞内的一类丝氨酸／苏氨酸蛋白激酶，将细胞外刺激信号转导至细胞内，在调节细胞增殖、分化、转化及凋亡等过程中起重要作用。寄生性原虫作为一类低等真核生物，现已发现存在MAPK信号通路，在虫体增殖、分化、致病性等方面起重要作用。由于寄生虫MAPK结构与其宿主MAPK存在明显差异，MAPK作为寄生虫病药物治疗靶标而备受关注。.弓形虫体外诱导缓殖子形成过程中MAPK1表达水平明显增高，MAPK2蛋白位于虫体顶端膜表面。通过基因打靶方法构建了MAPK1及MAPK2缺失株，弓形虫MAPK1缺失株体外缓殖子形成能力减弱，体外细胞粘附能力及增殖明显减弱。弓形虫MAPK1缺失株对小鼠的毒力减弱，存活小鼠体内弓形虫数量明显减少。TgMAPK2缺失株的增殖能力显著性减弱，感染小鼠后对小鼠不致死，并且在小鼠体内的增殖数明显减少。上述研究结果表明，MAPK1及MAPK2是弓形虫的毒力因子。本项目分析了弓形虫MAPK的功能及作用特点，为弓形虫基因缺失疫苗及MAPK靶向药物研制奠定基础。