弓形虫终末宿主发育阶段裂殖子的蛋白质组学研究

Toxoplasma gondii has a complex life cycle, and is global distribution. Felids are its only definitive hosts. Currently, abundant literatures have reported the tachyzoites studies on invasion, proliferation and evasion immunity within intermediate hosts, and very little research on the developmental mechanism and the initiation of sexual reproduction in definitive hosts has been done that seriously hindered to reveal the pathogenic mechanism of T. gondii. Merozoite is the first developmental stage of T.gondii in cat enterocytes, it carries out several rounds of asexual division and amplification followed by differentiation into macro- and microgamonts. And so it is the key stage for sexual reproduction of T. gondii. Here, we choose merozoite which developes in definitive hosts as the object to carry out its proteomics research. We have successfully established a effective method of isolating and culturing the cat intestinal epithelial cells in vitro, and we will construct the infection system of cat intestinal epithelial cells in vitro and in vivo to culture the merozoite. Then we use two-dimensional electrophoresis, gel-liquid chromatography linked tandem mass spectrometry and MudPIT to analyze the proteome of merozoite. Subcellular localization and function of proteins are predicted to identify virulence factors that are concerned with invasion, proliferation, and regulation of sexual reproduction of merozoite in the definitive host to reveal the growth mechanism and the regulation mechanism of sexual reproduction. The study have a great significance for prevention and treatment of toxoplasmosis.

刚地弓形虫生活史复杂，全球分布，猫科动物是其唯一的终末宿主。目前，文献多见速殖子对中间宿主入侵、增殖及逃避其免疫等方面的研究，对于弓形虫在终末宿主体内的发育及启动有性生殖转化机制了解较少，阻碍了其致病机制的揭示。裂殖子是弓形虫进入终末宿主后发育的第一个虫体形态，经数代无性增殖后可转化为雌雄配子体，是弓形虫进入有性生殖发育的关键阶段。本项目选择弓形虫终末宿主内裂殖子为研究对象，开展其蛋白质组学研究，在对猫肠上皮细胞成功体外分离、培养的基础上，构建体内体外感染猫肠上皮细胞培养纯化裂殖子体系，应用2-D电泳技术、凝胶液相色谱串联质谱技术、多维蛋白鉴定技术（MudPIT）对裂殖子蛋白质组分离鉴定，并进行功能预测及亚细胞定位，以确定裂殖子入侵、增殖及调控有性生殖转化相关的蛋白因子。该研究有助于揭示弓形虫在终末宿主内的生长发育及有性生殖调控机制，对弓形虫病控制意义重大。

弓形虫病是目前全球共同面临的一个潜在危害人类健康和畜牧业生产的公共卫生问题。我国具备弓形虫病传播的大量潜在的易感人群及动物，而猫作为弓形虫重要终末宿主，其数量众多，位居世界第二位，且感染弓形虫较为普遍。其粪便内的卵囊是造成我国畜牧动物弓形虫病流行及人类弓形虫病死亡的主要原因。但目前，关于弓形虫在猫体内的有性生殖开启机制仍是未知。裂殖子是弓形虫在终末宿主内进行有性生殖的一个关键虫体形态，它是决定弓形虫雌雄配子形成及有性生殖过程的唯一虫体形态。所以为揭示弓形虫在终末宿主内的生长发育及无性/有性生殖转化机制，推动弓形虫致病机制研究。本研究通过蛋白质组学技术筛选鉴定裂殖子期特异表达蛋白。首先Pru株弓形虫卵囊及缓殖子的培养、鉴定及纯化。然后体外弓形虫感染猫IECs体系的建立，观察弓形虫对猫IECs的入侵及其发育情况。接下来弓形虫感染家猫体系的建立及裂殖子的纯化；感染猫后，每天收集其粪便镜检；一旦镜检到卵囊后，对猫小肠IECs内弓形虫裂殖子进行检测及纯化。最后采用蛋白质组学技术及生物信息学分析技术对裂殖子差异表达蛋白筛选鉴定。研究结果显示，本研究成功建立了体外分离培养原代猫IECs系统, 为弓形虫在终末宿主内的研究提供了细胞模型；同时在培养纯化Pru株缓殖子的基础上，建立了弓形虫体外感染猫IECs体系。在体外，弓形虫成功入侵猫肠IECs，并完成胞内分裂增殖发育，未观察到无性/有性转换过程和形成的裂殖体/雌雄配子体。通过体内感染，可在猫粪便中检测到卵囊，在其IECs内检测到类裂殖体结构，说明弓形虫在猫体完成了有性生殖过程，且效率远远高于猫自然感染的情况，为弓形虫在终末宿主内的相关研究提供了一个成熟的感染体系。本研究共鉴定出15种裂殖子期差异表达蛋白，其中A0A125YP41属于RNA结合蛋白8A家族的1种成员可能参与调控裂殖子向雌雄配子体的转化。鉴定出的差异蛋白推动弓形虫无性/有性生殖转化机制的研究。