肠炎沙门氏菌感染输卵管过程中与鸡TLRs间的互作、调控基因的鉴定和功能研究

Salmonella Enteritidis (SE) is a major environmental stressor in the poultry production. SE could enter the chicken gut via chicken mouth and colonize in the intestinal tract. And it could enter oviduct to contaminate chicken eggs and cause human salmonellosis through consuming contaminated eggs. Toll-like receptors (TLRs) are important components of innate immune response and play a vital role in early recognition of pathogen. Previous studies show that the mRNA level of TLRs has been significantly changed following SE infection. However, the molecular interaction between SE and chicken TLRs during SE infection in oviduct is still unclear. In this proposal, SPF white leghorn laying chicken will be orally infected with SE. The animal model of chicken oviduct infected with SE will be constructed. The transcriptome of SE will be analyzed through next generation sequencing. The mRNA expression of chTLRs will be analyzed through quantitative real-time PCR. The molecular interaction network and signaling pathway of SE during infection will be analyzed. The key virulence genes will be identified through bioinformatics tools. The interaction of key regulating genes of SE with chicken TLRs and other proteins will be uncovered through yeast two-hybrid system. The key regulating genes will be knocked out through lambda red recombination method. Chicken primary oviduct epithelial cells will be cultured and infected with mutant and wild SE. The cell invasion of SE will be evaluated. The findings in the current study will pave the foundation and scientific guide to control salmonella in environment and prevent chicken salmonellosis.

肠炎沙门氏菌（SE）是家禽养殖环境中主要生物性应激源。该菌经口腔进入鸡体内，在肠道增殖，进入输卵管污染鸡蛋，并可引发人肠炎沙门氏菌病。鸡TLRs在病原菌的识别中发挥着关键作用。前期研究结果表明SE感染可以显著改变鸡盲肠和脾脏组织TLRs基因mRNA表达。然而关于SE感染鸡输卵管过程中与鸡TLRs间的互作关系尚不明确，本项目拟用SE口腔接种SPF白来航开产蛋鸡，构建SE经口腔感染鸡输卵管模型；检测SE转录组，鸡TLRs基因mRNA表达；构建感染过程中SE调控网络，鉴定信号通路和关键调控基因；利用酵母双杂交系统分析SE关键调控基因与鸡TLRs及其它蛋白的互作；利用λRed重组系统敲除SE关键调控基因，分析关键调控基因对SE侵染鸡原代输卵管上皮细胞能力的影响；阐明肠炎沙门氏菌与鸡TLRs间的分子互作关系。研究结果将为养殖环境中肠炎沙门氏菌的控制以及鸡肠炎沙门氏菌病的防控提供理论基础和科学依据。

肠炎沙门氏菌（SE）是家禽养殖环境中主要生物性应激源。鸡TLRs在病原菌识别中发挥着关键作用。前期研究结果表明SE感染可以显著改变鸡盲肠和脾脏组织TLRs基因mRNA表达。然而SE感染鸡输卵管过程中与鸡TLRs间的互作关系尚不明确，本项目用SE口腔接种SPF白来航和海兰褐初开产蛋鸡，构建SE感染鸡输卵管模型，发现SE感染可以造成组织器官的病理变化，毒力基因invH、lpfC、sefA的表达量在感染宿主后迅速下降，与盲肠和输卵管中TLRs、卵巢中TLR4、TLR5的表达量呈负向调控，随着感染时间的推移，TLRs的表达量下降；SE感染过程中TLR1A受gga-mir-1662的调控；同时TLRs与NLRs间表现为协同调控作用；SE的感染受TGF-β、FoxO、神经受体-配体作用过程、细胞因子-因子受体作用等信号通路的甲基化调控，鉴定了HOX家族的甲基化调控；SE沙门氏菌影响了鸡肠道微生物菌落变化，丰度最高的如粪杆菌属，梭菌科和拟杆菌属；构建了毒力基因ssrAB缺失株和酵母双杂交系统，为进一步深入研究SE感染中的调控奠定基础。本研究成功构建了SE感染鸡输卵管模型、毒力基因ssrAB缺失菌株、酵母双杂交系统，分析了SE感染过程中毒力基因与TLRs的互作关系，鉴定了沙门氏菌感染中的甲基化调控通路。为养殖环境中肠炎沙门氏菌与宿主的互作以及鸡肠炎沙门氏菌病的防控提供理论基础和科学依据。