禽致病性大肠杆菌Ⅲ型分泌系统2效应因子EspR1负向调控NF-κB通路的分子机制

Avian pathogenic E. coli (APEC) considered as zoonosis caused economically devastating to poultry industries, which also has important significance in public health. Injection of effectors by type III secretion system (T3SS) is a common infection strategy employed by pathogenic bacteria, to subvert nuclear factor-κB (NF-κB) signaling pathway and host responses. Our previous studies showed that APEC haboured 5 different isoforms of E. coli type III secretion system 2 (ETT2), which can secrete effectors and play roles in the pathogenicity of APEC. However, the precise pathogenic mechanisms of ETT2 and its effectors are unknown. Thus, this study attempts to examine the role and molecular mechanism of ETT2 effectors in the pathogenicity of APEC. The effectors of APEC ETT2 were identified by bioinformatics and comparative proteomics method. The important effector EspR1 was selected and its effects of on the growth characteristics and pathogenicity of APEC were determined. Then, whether effector EspR1 subvert the host cell NF-κB signaling pathway were detected through the Dual-Luciferase experiment, immunofluorescence, Western blot and point mutation technique. Using yeast two hybrid and co-immunoprecipitation (Co-IP) method, the host proteins interacting with effector EspR1 were identified, which aid in elucidating the molecular mechanism of ETT2 effector EspR1 in the APEC infection. This study will help us to understand the pathogenic mechanism of APEC. Furthermore, it will provide a new insight in the fuction of ETT2 in the APEC, which will contribute to the development of new treatment strategies and vaccines of colibacillosis.

禽致病性大肠杆菌（APEC）不仅给养禽业造成巨大损失，还威胁人类公共卫生安全。研究表明，病原菌利用III型分泌系统（T3SS）将效应因子注入宿主细胞内，扰乱宿主NF-κB信号通路等免疫反应，从而增强细菌致病力。申请人前期研究发现，APEC中含有5种Ⅲ型分泌系统2（ETT2）亚型，其可分泌效应因子发挥致病作用，然而致病机制尚不清楚。本研究拟通过生物信息学和比较蛋白组学技术筛选鉴定APEC ETT2效应因子；选择重要效应因子EspR1，探讨其对APEC的生长特性、致病力等的影响。进一步检测效应因子EspR1对宿主细胞NF-κB信号通路的影响；筛选并分析效应因子EspR1的宿主细胞互作蛋白及其蛋白修饰情况，深入研究效应因子EspR1调控NF-κB信号通路发挥致病作用的分子机制。本项目的开展不仅有助于加深对APEC分子致病机制的了解，还可为发展新的治疗策略和疫苗开发提供理论基础。

III型分泌系统（T3SS）与病原菌的致病性密切相关，而效应因子是T3SS的发挥作用的关键。病原菌通过T3SS将效应因子注入宿主细胞内，扰乱宿主信号通路，导致宿主免疫反应紊乱或细胞骨架重排，为细菌生存定殖及感染提供有利条件。研究表明，大肠杆菌III型分泌系统2（ETT2）与致病性大肠杆菌的毒力密切相关，且本项目前期研究发现ETT2存在于禽致病性大肠杆菌（APEC）中，但是其作用及分子机制尚不清楚。因此，本项目围绕APEC ETT2效应因子的筛选鉴定及分子致病机制开展了以下研究：（1）全面系统掌握了ETT2在APEC中的分子流行规律，发现ETT2广泛存在于APEC中，且有5种不同的ETT2亚型，为分析ETT2在APEC中的致病作用提供了临床数据；（2）解析了APEC ETT2核心组分ATPase EivC及调控蛋白EtrA的分子功能，为阐明ETT2的致病机制、毒力调控机制及筛选ETT2效应因子奠定了坚实基础；（3）筛选并鉴定了APEC ETT2的效应因子，并证实4个ETT2效应因子具有E3泛素连接酶活性；（4）确定了ETT2效应因子EspR1对APEC基本生物学特性及致病力的影响；（5）筛选鉴定了ETT2效应因子EspR1的宿主互作蛋白，解析了效应因子EspR1调控宿主免疫应答发挥致病作用的分子机制。由于T3SS对病原菌毒力的重要性，使其成为颇具前景的疫苗及药物靶点。在耐药病原菌不断出现、抗生素治疗面临挑战的情况下，通过本项目的实施解析了APEC ETT2效应因子EspR1的分子致病机制，对抗菌药物筛选研发及寻找新的抗菌药物靶点具有潜在的应用价值，可为动物及人的大肠杆菌病防治提供了理论依据，将产生很好的经济效益和社会效益。本项目发表研究论文16篇，其中SCI论文6篇；培养硕士研究生6名，培养优秀人才2人次。