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.

耐药结节分割（RND）外排系统在革兰氏阴性细菌中普遍存在，并在抗菌素耐药性和其他不同的表型中起主要作用，但迄今为止，在厌氧里氏杆菌中对RND外排系统的知识了解甚少。根据序列注释，RA-GD菌株中的RIA_1117-RIA_1118-RIA_1119操纵子编码一个假定的三方RND外排系统。RIA_1117，RIA_1118和RIA_1119基因分别编码外部成员蛋白（OMP），内部膜泵蛋白（泵转运蛋白）和周质膜融合蛋白（MFP）。此外，RIA_1119蛋白被标注为MexE组件。在这项工作中，研究了RIA_1117-RIA_1118-RIA_1119蛋白的生物学功能。抗生素敏感性试验表明，RIA_1117，RIA_1118和RIA_1119基因的失活均提高了对丁胺卡那霉素，链霉素和SDS的敏感性。通过上述抗菌剂的诱导，RIA_1117和RIA_1118的转录水平qRT-PCR检测显着上调了这些基因，但RIA_1119基因的转录水平未见显着差异。CCCP抑制剂检测证实RIA_1117，RIA_1118和RIA_1119蛋白根据质子动力（PMF）介导丁胺卡那霉素，链霉素和SDS耐药性。斑点测定和链霉素积累测定证实RIA_1117，RIA_1118和RIA_1119蛋白有助于出口链霉素，而CCCP增加了链霉素的积累。此外，RIA_1117，RIA_1118和RIA_1119蛋白也与RA-GD菌株的适应性和毒力有关。这些结果表明RIA _ 1117 - RIA _ 1118 - RIA _1119操纵子编码一个RND外排系统，该系统对链霉素，丁胺卡那霉素和SDS具有底物特异性，并有助于RA-GD的生长和毒力。RIA_1117-RIA_1118-RIA_1119被指定为RaeE-RaeF - RopN外排系统。基于以上结果和结构分析，RIA_1117，RIA_1118和RIA_1119蛋白分别对应于RopN（OMP），RaeF（泵转运蛋白）和RaeE（MFP）。