Characterization of RaeE-RaeF-RopN, a putative RND efflux pump system in Riemerella anatipestifer

Resistance-nodulation-division (RND) efflux systems are ubiquitous in Gram-negative bacteria and play a predominant role in antimicrobial resistance and other diverse phenotypes, but the knowledges of RND efflux systems are poorly understood so far in Riemerella anatipestifer. According to the sequence annotation, RIA_1117-RIA_1118-RIA_1119 operon in RA-GD strain encodes a putative tripartite RND efflux system. RIA_1117, RIA_1118 and RIA_1119 genes encode an outer member protein (OMP), an inner membrane pump protein (pump transporter), and a periplasmic membrane fusion protein (MFP), respectively. Furthermore, RIA_1119 protein is annotated as a MexE component. In this work, the biological functions of RIA_1117-RIA_1118-RIA_1119 proteins were studied. The antibiotic susceptibility testing showed that the inactivation of RIA_1117, RIA_1118 and RIA_1119 genes all raised susceptibility to amikacin, streptomycin and SDS. By induction with the above antimicrobial agents, the transcription levels of RIA_1117 and RIA_1118 genes were up-regulated significantly using qRT-PCR detection, but no significance difference was observed for the transcription level of RIA_1119 gene. CCCP inhibitor assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins mediated amikacin, streptomycin and SDS resistance depending on proton motive force (PMF). Spot assay and streptomycin accumulation assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins contributed to export streptomycin, and CCCP increased the accumulation of streptomycin. Furthermore, RIA_1117, RIA_1118 and RIA_1119 proteins also were involved in the fitness and virulence of RA-GD strain. These results showed that RIA_1117-RIA_1118-RIA_1119 operon encoded a RND efflux system, which has the substrate specificity for streptomycin, amikacin and SDS and contributed to the growth and virulence of RA-GD. RIA_1117-RIA_1118-RIA_1119 was designated RaeE-RaeF-RopN efflux system. Based on the above results and structural analysis, RIA_1117, RIA_1118 and RIA_1119 proteins corresponded to RopN (OMP), RaeF (pump transporter) and RaeE (MFP), respectively.