鸭疫里默氏杆菌九型分泌系统(T9SS)分泌蛋白B739_0093的功能鉴定

Roles of B739_0093 as a protein secreted by type IX secretion system(T9SS)in Riemerella anatipestifer

【目的】鸭疫里默氏杆菌是引起鸭浆膜炎的一种革兰氏阴性病原菌,该菌编码的九型分泌系统(type IX secretion system,T9SS)参与滑动、致病等过程。前期研究结果表明,鸭疫里默氏杆菌CH-1株B739_0093基因在限铁培养条件明显上调。序列分析表明,B739_0093编码蛋白含有一个T9SS分泌蛋白保守的C端结构域,然而其具体功能未知。本研究旨在鉴定该基因编码蛋白是否被T9SS分泌,以及在该菌致病中的作用。【方法】用荧光定量PCR检测B739_0093是否被铁离子和铁转运调节子(ferric uptake regulator,Fur)调控;用大肠杆菌表达重组B739_0093截短蛋白制备多克隆抗体,通过Western blotting检测是否由T9SS分泌;构建B739_0093基因缺失株,通过毒力和定殖试验鉴定B739_0093在鸭疫里默氏杆菌致病中的功能。【结果】荧光定量PCR结果表明,B739_0093基因在铁离子限制性培养基明显上调,此调控是由调控蛋白Fur介导的;Western blotting结果显示,B739_0093基因编码蛋白在亲本株RA CH-1主要定位在分泌物中,而在T9SS缺失株定位在菌体且不能在分泌物被检测到;与亲本株RA CH-1相比,RA CH-1ΔB739_0093对雏鸭的致病力减弱,在雏鸭各组织器官的定殖能力明显降低。【结论】B739_0093基因编码蛋白是由鸭疫里默氏杆菌T9SS分泌的,其表达受铁离子及Fur调控,并且参与了该菌的致病。

[Objective]Riemerella anatipestifer(RA)is a Gram-negative pathogen that can cause duck serositis.The type IX secretion system(T9SS)of this bacterium is involved in sliding and pathogenic processes.The previous study showed that the expression of B739_0093 in R.anatipestifer CH-1 was significantly up-regulated under the iron-limited condition.The sequence analysis showed that the protein encoded by B739_0093 contained a conserved C-terminal domain of the proteins secreted by T9SS,while its function remained unknown.This study aims to identify whether the protein encoded by B739_0093 is secreted by T9SS and the role of this protein in the pathogenesis of this bacterium.[Methods]qPCR was conducted to determine whether the transcription of B739_0093 was regulated by iron and the ferric uptake regulator(Fur).The recombinant truncated B739_0093 protein was expressed in Escherichia coli,and the polyclonal antibody was prepared.Western blotting was employed to detect whether the protein was secreted by T9SS.Furthermore,we deleted B739_0093 from RA CH-1 and identified the role of B739_0093 in the pathogenicity of R.anatipestifer by virulence and colonization tests in ducklings.[Results]The expression of B739_0093 was significantly up-regulated in the iron-restricted medium,which was mediated by Fur.Western blotting results showed that the protein encoded by B739_0093 was localized in the secretion of the parent strain RA CH-1,while it was localized in the bacterial lysate and not detected in the secretion of the T9SS-deleted strain.Compared with the parent strain RA CH-1,RA CH-1ΔB739_0093 demonstrated attenuated pathogenicity and reduced colonization ability in various tissues and organs of ducklings.[Conclusion]The protein encoded by B739_0093 is secreted by T9SS and involved in the pathogenicity of R.anatipestifer,and its expression is regulated by iron and Fur.