胞内劳森菌蛋白靶向宿主细胞MAPK信号转导通路的机制研究

Porcine proliferative enteropathy is a contagious disease, caused by L. intracellularis and characterized by the thickening of the intestinal mucosa and associated with acute necrotic diarrhea or chronic proliferative enteritis which results in huge economic losses. L. intracellularis can’t be cultivated in axenic media and has no methods available for genetic manipulation hinders the knowledge of its pathogenesis. The genome sequence of L. intracellularis indicates that it possesses a type III secretion system, but its effectors, the main virulence, was never reported. Effector proteins always inhibit yeast growth. In a S. cerevisiae model system, we also found three L.intracellularis proteins, LI0677, LI1035, LIB005 individually expression resulted in a severe growth defect of yeast that was suppressed by some yeast haploid deletion strains respectively. 9 genes are located in four different pathways. In this study, reporter assay and qRT-PCR were performed to identify the specific pathways which are attenuated by these three proteins, MAPK phosphorylation assays were used to further localize the targets, cell morphogenesis and cell cycle progression were used to analyze the functions of these three proteins. The study will help elucidate their molecular mechanisms, understand the disease mechanisms and provide theoretical foundations for diagnosis, prognosis and treatment.

由胞内劳森菌感染引起的猪增生性肠炎，广泛流行，以肠粘膜增厚为特征，引起急性坏死性腹泻或慢性增生型肠炎，造成巨大经济损失。胞内劳森菌专性胞内寄生，无法进行遗传操作，严重阻碍了其致病机理的认识。序列分析表明胞内劳森菌存在Ⅲ型分泌系统，但其底物-效应蛋白还未见报道。效应蛋白多会抑制酵母生长。本课题组也筛选到胞内劳森菌蛋白LI0677，LI1035和LIB005均会抑制酵母生长，多个酵母缺失菌株能够挽救其生长抑制表型，9个位于四条的MAPK信号转导通路中。本研究以LI0677，LI1035和LIB005为研究对象，拟用转录报告基因和qRT-PCR来确定其靶向的通路；通过MAPK蛋白磷酸化水平来定位其靶标；通过观察细胞形态和检测细胞周期进程来分析其功能。本研究不仅有助于阐明胞内劳森菌蛋白调控宿主细胞的分子机制，还可促进对猪增生性肠炎致病机制的了解，为疾病的进一步诊断、预后和治疗提供重要的理论依据。

胞内劳森菌是猪增生性肠炎的病原，其分子致病机理尚不明确。尽管序列分析表明，胞内劳森菌含有编码3型分泌系统（T3SS）所需的整个装置的基因，并且在实验感染期间检测到其中一些成分的表达，但胞内劳森菌T3SS底物（效应蛋白）的鉴定受到阻碍。本项目旨在阐明前期筛选到的三个T3SS候选效应蛋白---抑制酿酒酵母生长的胞内劳森菌基因LI0677、LI1035和LIB005靶向宿主细胞MAPK信号转导通路的机制研究。因实验过程中碰到瓶颈，LI0677和LIB005两个基因研究停滞，只对LI1035进行了较深入的研究。我们的结果表明，LI1035靶向酵母和动物细胞的MAPK通路，调节其微丝组装，该结果发表于Vet Microbiol (2019), 235: 127-135。进一步的研究结果表明，LI1035可被耶尔森氏菌T3SS分泌，与ERK体/内外互作，导致细胞周期停滞，其机制正进一步深入研究。该项目还资助了一套酵母荧光定位报告菌株的开发，这些菌株表达定位于特定亚细胞结构的红色荧光融合蛋白，为观察细胞器的动态变化和识别未知蛋白质在酵母中的定位提供了基本工具，该结果发表于《微生物学报》(2020), 60(4): 667–678。同时，本项目资助了胞内劳森菌外膜蛋白LI0902在宿主中的互作蛋白筛选、鉴定和作用机制的研究，我们的研究结果表明，LI0902与磷脂酰肌醇激酶PIP4Kγ的相互作用，抑制PIP4Kγ的酶活。本项目还资助了胞内劳森菌LI0666酪氨酸磷酸化机制的研究。我们的研究结果表明，LI0666抑制酵母生长，其作用与EPIYA中的酪氨酸有关；LI0666可被耶尔森氏菌T3SS分泌；LI0666 EPIYA基序中的酪氨酸在动物细胞中会被磷酸化；LI0666位于酵母和动物细胞的细胞膜上；纯化的LI0666与多个角蛋白互作。后两项工作均已投稿，审稿中。上述结果可大大促进人们对胞内劳森菌感染和致病机理的认识。本项目资助发表1篇SCI和1篇A类核心期刊，2篇SCI审稿中，1篇SCI待整理。本项目培养硕士生7名，5名已取得硕士学位，2名在读。本项目投入经费25万元，支出17.3314万元，各项支出基本与预算相符，剩余经费7.6686万元计划用于本项目研究后续支出。