Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.本课题组此前已报道从肺炎克雷伯菌（Klebsiella pneumoniae）MGH78578中克隆耐药相关基因的研究工作。其中，我们鉴定出一段编码MATE型多药外排泵（MATE-type multidrug efflux pump）的推定基因，并将其命名为ketM。携带ketM质粒的大肠杆菌（Escherichia coli）KAM32，对诺氟沙星、环丙沙星、头孢噻肟、吖啶黄、Hoechst 33342及4',6-二脒基-2-苯基吲哚（DAPI）的最低抑菌浓度（minimum inhibitory concentrations, MIC）均有所升高。在携带ketM质粒的大肠杆菌KAM32菌株中，可观测到DAPI的主动外排现象。 我们在肺炎克雷伯菌菌体中检测到了ketM的mRNA表达，随后对肺炎克雷伯菌ATCC10031中的ketM基因进行了敲除。然而，亲本菌株ATCC10031与ketM敲除株SKYM的耐药性水平未出现显著变化。因此我们认为，KetM属于多药外排泵，但并未对肺炎克雷伯菌的抗菌药物固有耐药性产生显著贡献。 MATE型转运蛋白（MATE-type transporters）属于次级转运蛋白，因此我们探究了KetM的偶联阳离子类型。当添加乳酸以产生质子动力势（proton motive force, PMF）时，可观测到KetM介导的DAPI外排，表明KetM通过质子动力势完成底物外排。但在不含乳酸的反应体系中添加氯化钠时，同样可观测到KetM介导的微弱DAPI外排现象。该结果提示，KetM可能同时利用质子动力势与钠动力势完成底物外排功能。