利用小肠结肠炎耶尔森氏菌作为替代宿主鉴定胞内劳森菌三型分泌系统效应蛋白

Procine proliferative enteropathy（PPE） is an infectious disease characterized by thickening of the intestinal epithelium due to enterocyte proliferation. Lawsonia intracellularis，an obligate intracellular Gram-negative bacterium， is the etiologic agent of proliferative enteropathy (PE). Although the published genome sequence suggests that L. intracellular encodes a Type III secretion system （T3SS），translocating several effector proteins across the host cell plasma membrane and the inclusion membrane，playing a significant role in the pathogenicity， and three genes highly homologous to the component of the prototypic T3SS of Yersinia spp. difficulties in vitro cultivation and the current inability to genetically manipulate L. intracellular have hampered the examination of this potentially important class of virulence genes. Our earlier studies by using yeast growth inhibition due to the expression of uncharacterized proteins have led to the identification of 30 L. intracellularis genes as canidate effector proteins. In this study, a study will be initiated to identify and characterize L. intracellularis gene products that are effector proteins by Yersinia. enterocolitica T3S assay and translocation assays. Secondly, yeast two-hybrid assay will be assayed for specific interactions between the effector proteins with target eukaryotic protein(s). co-immunoprecipitation will be used to test for the association of native proteins in Hela cells. Thirdly, to find the effects on target protein(s) as a consequence of interaction with effector proteins, the target proteins will be examined directly to evaluate differences in steady-state levels or covalent modification. Finally, the cytoseletal and cell proliferation changes will be examined. The study will help elucidate the full complement of L. intracellularis T3SS substrates and their respective host targets, which will likely contribute significantly to understanding the pathogenesis of this complex and important parasite.

猪增生性肠炎是由肠上皮细胞增殖引起的肠上皮增生为主要特征的传染性疾病，其病原为胞内劳森菌，一种严格胞内寄生的革兰氏阴性细菌。基因组序列预示胞内劳森菌编码3型分泌系统（分泌效应蛋白穿过细胞膜和内吞膜，与细菌的致病性密切相关），其组分与耶氏菌高度同源，但无法在培养基上培养和无合适的遗传操作系统阻碍了胞内劳森菌效应蛋白的鉴别。在前期研究工作中，通过检测胞内劳森菌功能未知蛋白在酿酒酵母中表达，我们已筛选到30个引起酵母生长抑制的候选效应蛋白。本研究拟先用小肠结肠炎耶尔森氏菌T3S试验和移位试验来筛选和鉴定胞内劳森菌效应蛋白；用酵母双杂交筛选效应蛋白的真核靶标蛋白，用免疫共沉淀验证其相互联系；用靶蛋白的蛋白质水平或共价修饰差异、细胞形态和细胞增殖的变化来评价效应蛋白对靶蛋白和宿主细胞的影响。本研究不仅有助于阐明胞内劳森菌的T3S底物和它们各自的靶标，还将显著加深对这一复杂重要的胞内菌致病机制的了解。

严格胞内寄生的劳森菌是引起猪增生性肠炎的病原，其分子致病机理和毒力因子尚不明确。本研究的初衷旨在利用耶尔森氏菌作为替代宿主，筛选和鉴定胞内劳森菌效应蛋白，并进一步阐明效应蛋白在感染和致病中的作用机制。结果表明，胞内劳森菌移位蛋白SctE（LI1158）可被耶尔森氏菌分泌。但这一系统用于其它候选基因时无效，且体内感染易位细胞实验未获成功，因此，在致力于研究胞内劳森菌致病机理的大目标下，我们适当调整了研究内容。本项目资助研究了LI035的作用机制，研究结果表明，LI1035靶向酿酒酵母和动物细胞的MAPK通路，调节肌动蛋白组织，并发表于Vet Microbiol, 235: 127-135。本项目资助研究了外膜蛋白LI0902互作蛋白和作用机制，研究结果表明，LI0902与磷脂酰肌醇激酶PIP4Kγ的相互作用，并抑制PIP4Kγ的酶活。该工作已投稿Vet Microbiol，审稿中。本项目还资助研究了LI0666 EPIYA基序中酪氨酸磷酸化作用机制，研究结果表明，LI0666抑制酿酒酵母生长，其作用与EPIYA中的酪氨酸残基有关；LI0666 EPIYA基序中的酪氨酸残基在动物细胞中会被磷酸化，纯化的LI0666与多个角蛋白互作,该工作预备投稿中。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。本项目还资助研究了4个临床研究工作，均已发表在国内核心期刊上。