L. M. HÜRLIMANN¹, M.A. SEEGER<sup>1

1</sup>Institute of Medical Microbiology, University of Zurich, 8006 Zürich, Switzerland

Increasing numbers of nosocomial infections caused by Enterococcus faecalis were reported and associated with serious diseases such as endocarditis, bacteremia, urinary tract infections and surgical wound infections. Interestingly, the contribution of drug efflux pumps to intrinsic resistance against antibiotics is poorly studied in E. faecalis. Recent studies on other Gram-positive bacteria have revealed heterodimeric ABC multidrug transporters to play an important role in multidrug efflux, including LmrCD of Lactococcus lactis and PatAB of Streptococcus pneumoniae. Here we investigated the complete set of seven heterodimeric ABC exporter homologs from E. faecalis, which are annotated as drug efflux pumps.
Unmarked gene deletion knockout of ef0789/ef0790 on the chromosome of E. faecalis resulted in increased susceptibility towards 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 of E. faecalis. In contrast, heterologous expression in L. lactis revealed EfrAB, EfrCD and the gene product of ef2226/ef2227 (EfrEF) to mediate efflux of fluorescent substrates and confer resistance towards multiple dyes and drugs including fluoroquinolones. Four out of seven transporters did not exhibit any measurable drug efflux activity even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, lacking drug efflux activity is unlikely attributed to poor expression or protein aggregation. Reconstitution of purified multidrug transporters EfrAB, EfrCD and EfrEF in proteoliposomes revealed a robust functional coupling between ATP hydrolysis and drug binding. Our analysis builds an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps are in fact incapable of drug transport and thus fulfil other physiological functions in the cell.