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

摘要:

【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。

Abstract:

[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