ABSTRACTNosocomial infections withEnterococcus faecalisare an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters ofE. faecalisthat are annotated as drug efflux pumps. Deletion ofef0789-ef0790on the chromosome ofE. faecalisresulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context ofE. faecalis. In contrast, heterologous expression inLactococcus lactisrevealed that EfrAB, EfrCD, and the product ofef2226-ef2227(EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression inL. lactis. Since all seven transporters were purified as heterodimers after overexpression inL. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.
The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis
