禽致病性大肠杆菌ETT2关键毒力效应蛋白对上皮细胞致病作用的研究

The interaction between virulence effector proteins of pathogen and epithelial cells is an important mechanism of host infection. Avian pathogenic Escherichia coli is a potential pathogen of zoonosis and the its prevention and control is still a worldwide problem. The new discovered type three secretion system 2(ETT2) of E.coli makes the pathogenicity of APEC more complex. In order to study the pathogenic role of APEC-ETT2 key virulence effector proteins to epithelial cells, firstly, the different virulence genes of APEC-ETT2 will be screened by suppression subtractive hybridization(SSH) and homologous sequence analysis. Secondly, we will use Red homologous recombination system to construct APEC-ETT2 mutant strains and evaluate the role of APEC-ETT2 key virulence genes by testing some biological characteristics. Thirdly, to study the differential proteomics of APEC-ETT2 mutants on the tracheal epithelial cells, we need to establish the chicken tracheal epithelial cell infection model and conduct relative and absolute quantitation(iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS) identification on the APEC-infected and uninfected chicken tracheal epithelial cells. The obtained differentially expressed proteome data will be further analyzed by bioinformatics, which can help us to find new targets. Fourthly, the cDNA library of the tracheal epithelial cell will be constructed by using the key effector proteins bait and the systematic yeast two-hybrid library screens for the key effectors proteins of APEC-ETT2 will be performed. Then the partial results will be further confirmed by coimmunoprecipitation (Co-IP). Through our project, we will find some new clues about APEC effectors functions and mechanisms which will not only lay a foundation for the elucidation of the pathogenesis of APEC, but also provide a new perspective for the prevention and treatment of APEC.

病原毒力效应蛋白与上皮细胞的相互作用是引起宿主感染的重要机制。禽致病性大肠杆菌（APEC）是人兽共患病的潜在病原体，其防治仍是当今世界性的难题。新近发现的Ⅲ型分泌系统2（ETT2）使其致病性更为复杂。为探讨APEC-ETT2毒力效应蛋白对上皮细胞的致病作用，本项目通过抑制差减杂交（SSH）和生物信息学方法，筛选关键毒力效应蛋白基因，Red同源重组构建其突变株及生物学特性评价；采用iTRAQ 标记-结合2D LC-MS/MS技术开展差异蛋白质组学研究;以关键毒力效应蛋白基因为诱饵,构建cDNA文库，酵母双杂交筛选上皮细胞蛋白，免疫共沉淀（CO-IP）验证蛋白相互作用，揭示APEC-ETT2关键毒力效应蛋白致病作用，为探寻以ETT2为主要靶点的宿主靶向抗感染提供切实的应用基础，进一步为防治APEC感染提供新思路。

病原毒力效应蛋白与上皮细胞的相互作用是引起宿主感染的重要机制。禽致病性大肠杆菌（APEC）是人兽共患病的潜在病原体，其防治仍是当今世界性的难题。新近发现的Ⅲ型分泌系统2（ETT2）使其致病性更为复杂。.本项目以APEC-ETT2关键毒力因子对宿主上皮细胞的致病作用及机制为主要内容，进行病原细菌学及病原菌与宿主互作方向研究。首先检测APEC-ETT2基因簇在禽致病大肠杆菌的流行情况；此后通过生物信息学软件预测编码关键毒力蛋白的基因，利用Red同源重组系统和 CRISPR/Cas 9系统构建毒力蛋白基因缺失株、回复株，开展生物学特性及致病性评价、转录组学分析、鸡气管上皮细胞感染后天然免疫因子转录水平检测，并进行ETT2基因缺失株和野生株分泌蛋白质组学检测，全面分析受ETT2影响的分泌蛋白。继而在禽源大肠杆菌分离株中评价ETT2基因簇及毒力效应蛋白EspE3 (Escherichia coli. secretion protein E3 ubiquitin ligase )基因的流行关联性情况，通过毒力效应蛋白AE81-EspE3的自制鼠源多克隆抗体表征并验证效应蛋白的分泌及感染侵入鸡气管上皮细胞情况，最终使用GST-pull down技术筛选毒力效应蛋白EspE3在鸡气管上皮细胞的相互作用蛋白。.通过以上研究，共筛选到7个APEC-ETT2关键毒力基因：ETT2伴侣蛋白基因ETT2-yegG, 5个转录调控子基因ETT2-yqeH、ETT2-yqeI、ETT2-ygeK、ETT2-eivF、ETT2-etrA和一个分泌效应蛋白基因espE3，并筛选出107个AE81-EspE3在鸡气管上皮细胞的互作蛋白，为禽致病性大肠杆菌ETT2致病作用的研究提供有效分子靶标，揭示了APEC-ETT2 多个关键毒力因子的调控机制和致病机理，为解决以 ETT2 为靶标的禽致病性大肠杆菌防治提供新思路。