PhoP/PhoR双组份系统调控鸭疫里氏杆菌毒力的机制研究

双组份系统对细菌毒力的调控起着至关重要的作用。我们前期研究发现鸭疫里氏杆菌RAYM_RS09735/RAYM_RS09740是一对PhoP/PhoR双组份系统，该双组份系统为革兰阴性菌中首次报道。该双组份缺失株对雏鸭的毒力显著降低，但其调控毒力的机制不清楚。本研究拟在体外检测PhoR、PhoP的自磷酸化和磷酸基团转移，并确定PhoR、PhoP的磷酸化位点；通过构建该双组份单基因缺失株，挖掘其调控的毒力相关基因；通过PhoP超表达，运用ChIP-Seq、生物信息学和凝胶阻滞电泳实验等技术分析确定PhoP结合的靶基因启动子DNA motif，筛选其直接调控的毒力基因，并进一步构建相应的基因缺失突变株，对其毒力进行验证。阐明该双组份系统调控鸭疫里氏杆菌毒力的机制，为揭示鸭疫里氏杆菌的分子致病机制奠定基础，为新毒力因子、新药靶标、诊断标识和疫苗候选抗原等的发现提供理论依据。

Two component systems (TCSs) play important roles in the regulation of bacterial virulence. RAYM_RS09735/RAYM_RS09740 was confirmed as a PhoP/PhoR TCS in Riemerella anatipestifer by bioinformatics and RNA_Seq analysis in our previous studies, and PhoP/PhoR is first reported in gram-negative bacteria. The RAYM_RS09735/RAYM_RS09740 gene deletion strain lost the pathogenicity in ducklings, but its mechanism of regulation in virulence is not clear. In this study, we will perform In vitro phosphorylation assays to detect the autophosphorylation and phosphotransfer of PhoR and PhoP, and comfirm the sites of phosphorylation. To exploit the virulence genes which regulated by this TCS by constructing phoR or phoP deletion strain respectively. To investigate the target gene DNA motif and obtain the directly regulated virulence genes by PhoP with over expression of PhoP and using ChIP-Seq, bioinformatics and electrophoretic mobility shift assay (EMSA). Associated virulence gene deletion strains will be constructed to determine the virulence. Our study is aim to clarify the mechanism of regulation of virulence factors expression by this TCS, it will lay the foundation for revealing the molecular pathogenic mechanism of R. anatipestifer, and provide the theoretical basis for discovery of novel virulence factors，novel drug targets, diagnostic markers and vaccine candidate antigens.