Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

洋葱伯克霍尔德菌J2315（Burkholderia cenocepacia J2315）是囊性纤维化（cystic fibrosis, CF）相关致病菌中极具临床挑战性的类群代表菌株。由于该菌对临床常用抗菌药物及消毒剂均具有高度耐药性，抗菌治疗难以根除其感染。RND外排泵（Resistance-Nodulation-Cell Division）被认为是革兰氏阴性菌多重耐药性的重要介导因子之一。鉴于洋葱伯克霍尔德菌中存在的16套RND外排系统（命名为RND-1至RND-16）的功能意义仅得到部分阐明，本研究旨在分析J2315菌株中RND-4与RND-9外排系统功能受损的突变株，并评估这两种外排泵在有毒物质外排过程中的作用。本研究通过基因芯片（microarray）分析技术，分别将单敲除RND-4、RND-9的突变株（分别命名为D4与D9）以及双敲除这两种外排泵的双重突变株（命名为D4-D9）的转录组与野生型洋葱伯克霍尔德菌的转录组进行比对。基因芯片数据通过实时荧光定量PCR（qRT-PCR）、表型实验以及表型芯片（Phenotype MicroArray）分析得到了验证。数据分析结果显示，RND-4对洋葱伯克霍尔德菌的抗生素耐药性具有显著贡献，而RND-9在此过程中仅发挥微弱作用。此外，双重突变株D4-D9的表型与基因表达谱均与单突变株D4高度相似。基因芯片数据分析表明，运动相关与趋化相关基因在D4及D4-D9突变株中均呈现上调表达；与之相反，在D9突变株中，此类基因集则呈现下调表达，或其表达水平与野生株J2315无显著差异。所有突变株的生物被膜（biofilm）生成能力均有所增强。综上，本研究结果表明，洋葱伯克霍尔德菌的RND外排泵的功能并不局限于耐药性调控，还可影响其他与致病过程密切相关的表型特征。