与EHEC O157:H7 Ⅳ菌毛互作宿主蛋白的筛选及鉴定

EHEnterohemorrhagic Escherichia coli (EHEC) O157:H7 produces long bundles of type Ⅳ pili (TFP) called hemorrhagic coli pili (HCP). HCP is an important and key virulence of EHEC O157:H7,but its pathogenic mechanism is unknown. We construct cDNA library by extracting RNA from intestinal epithelial cells of gnotobiotic newborn piglets, and screen host proteins interacted with HCP by yeast two-hybrid and bacterial two-hybrid to identify HCP receptors and receptor-like host proteins. The interactions between HCP and host proteins are confirmed by the GST pull-down and the co-immunoprecipitation technology. Receptor functions of host proteins are further identified. Parent EHEC O157: H7,EHEC O157: H7 (△ hcpA) and HB101(pJX22)are chosen to design adherence and adherence inhibition experiments by adhering to HT-29 cells, IPEC-2 cells and CHO cells transfected with the candidate host gene. In sum, we aim at deeply explain HCP pathogenic mechanisms of EHEC O157: H7 and lay the substantial foundation for the prevention and control of human O157:H7.

Ⅳ型菌毛HCP是肠出血性大肠杆菌O157:H7的重要毒力因子，是其定植和致病的关键因子，但其致病机制尚不明确。本项目用新生仔猪肠上皮细胞提取RNA，构建cDNA文库，酵母双杂交和细菌双杂交两种方法筛选与HCP互作的宿主蛋白因子，以期筛选到HCP的受体或直接作用的膜蛋白等。然后用GST pull down和免疫共沉淀技术验证筛选的阳性克隆。再针对阳性克隆的受体功能进行鉴定。选用亲本株EHEC O157:H7、缺失株EHEC O157:H7（△hcpA）和HB101（pJX22）对允许细胞HT-29和IPEC-2以及转染有阳性克隆基因的CHO细胞进行粘附和粘附抑制试验。综合分析，深入阐释EHEC O157:H7 HCP致病的机制，为人类O157:H7的防控奠定基础。

本项目研究Ⅳ型菌毛HCP与宿主互作致病机制，运用酵母双杂交和pull down技术从结肠cDNA文库中筛选与HCP互作的宿主因子，并对宿主因子功能进行初步鉴定。从人结肠cDNA文库中筛选到6个阳性克隆，pull-down技术验证其中3个因子，表明2个宿主因子与IV菌毛互作，分别为TLR2和FCN3。HCP与TLR2结合诱导宿主细胞促炎因子IL-8和TNF-a的分泌，而与FCN3结合后，抑制凝集素补激活系统，具有双重功能，表明HCP是EHEC O157:H7重要的毒力因子，且与免疫逃逸密切相关，有待深入阐释。HCP与宿主互作机制的研究，将为人EHEC O157:H7的防控提供重要的基础数据。在本项目的资助下，发表文章3篇，其中SCI 2篇，授权1个。