PERP-p53-MDM2反馈环路在肠炎沙门氏菌致鸭颗粒细胞凋亡的作用机制

Salmonella enteritidis (SE) can be colonized persistently in the follicular granulosa cells and accompanied follicular degradation, which not only affect egg production, but also cause eggs and water pollution. Recently, we found SE effector protein SipC was combined with the surface transmembrane protein PERP of granulosa cells, differentially expressed genes between normal and SE-infected duck follicular granulosa cells were highly enriched in the PERP-p53-MDM2 signaling pathway and the apoptosis of granulosa cells. However, the specific regulatory mechanism was not clear. This project will focus on the effect of PERP on the apoptosis of dGCs and the downstream gene expression of PERP-p53-MDM2 signaling pathway will be confirmed by using the SipC deletion strain of SE. Firstly, the combination mode of key nodes of PERP-p53-MDM2 signal pathway will be detected by chromatin immunoprecipitation. And then gene overexpression and knockdown of key nodes of PERP-p53-MDM2 signal pathway will be used to regulate the genes transcription to investigate the change of upstream and downstream genes expression and the apoptosis of granulosa cells. Finally, co-immunoprecipitation and western blot will be used to explore the effect of PERP negative feedback regulation on the formation of p53-MDM2 module in order to elucidate the mechanism of PERP-p53-MDM2 feedback loop in the apoptosis of dGCs challenged by SE-infected. The results will provide a reference for reducing the infection of SE and improving the hygiene of eggs.

鸭肠炎沙门菌伴随产蛋周期持续定植于卵泡颗粒细胞，引起卵泡退化，不仅影响鸭产蛋量，还会造成鸭蛋和水体污染。本课题前期发现肠炎沙门菌的效应蛋白SipC与鸭卵泡颗粒细胞（dGC）表面跨膜蛋白PERP相结合，感染肠炎沙门菌的dGCs差异表达基因高度富集在PERP-p53-MDM2信号通路，并发生凋亡，但具体调控机制不清楚。本项目将利用肠炎沙门菌的SipC缺失株感染dGCs，验证PERP对dGCs凋亡及PERP-p53-MDM2信号通路基因表达的影响；采用COIP-WB和ChIP技术检测PERP-p53-MDM2信号通路关键节点的互作方式，解析其基因表达的变化对dGCs凋亡的影响；深入探究PERP负反馈对p53-MDM2模块形成的机制。研究将有助于全面阐明PERP-p53-MDM2反馈环路在肠炎沙门菌感染dGCs致凋亡的调控机制，同时也可为降低鸭肠炎沙门菌感染，提高禽蛋的安全性提供一定的参考依据。

肠炎沙门氏菌（Sallmonella enteritidis，SE）常定植于成年家禽生殖道的卵巢等组织，造成禽蛋的直接污染和垂直传播难于清除和预防，不仅影响了产蛋量，还造成禽蛋和环境污染，危害人类健康。卵巢和输卵管的抗菌作用对于禽蛋免受感染以及产生卫生蛋是必需的，鉴于SE感染鸭生殖道过程中，宿主的应答反应及关键分子的调控机制尚不清楚。本研究以高产蛋鸭绍鸭为研究对象，构建人工感染模型，研究表明SE感染易定植于鸭卵巢基质、小卵泡、输卵管峡部和阴道组织中，并导致TLR2、TLR4-5、TLR15、TLR21、NOD1、NLRX1、NLRP12、AvβD4-5、AvβD7、AvβD12、IL-6、IL-1β、IFN-γ和MyD88显著上调；而TLR3、TLR7、NLRC3和TNF-α在这些组织中显著下调。通过RNA-Seq技术，筛选出了鸭卵巢感染SE的应答基因，差异基因主要富集在细胞凋亡p53信号通路。SE感染中，p53信号传导途径的关键基因PERP通过影响通路中p53、MDM2、PERP、caspace-3和Bcl-2的表达，促进dGC凋亡；抑制dGC PERP表达，导致p53、MDM2、PERP、caspace-3和Bcl-2的表达量下调，促进dGC的增殖，减少SE对dGC的黏附和侵袭量。通过酵母双杂交系统从鸭卵泡颗粒细胞cDNA文库中筛选到12个可能与SE SipC相互作用的蛋白，其中PERP的阳性克隆出现频率高、速度快。采用一对一酵母回复性杂交和GST-pull down试验，验证了SipC与PERP结合关系，且利用λRed同源重组系统构建sipC基因缺失株，体外证实过表达PERP可以促进SE MY1 SipC黏附dGC。研究结果为揭示SE的致病机理及宿主抗病机制奠定基础，同时为提高禽蛋食品安全性提供一定的参考依据。