Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.