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

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