title: The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.
abstract: Nosocomial infections with Enterococcus faecalis are 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 of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted 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 of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-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 in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. 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.
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
authors.forename: Lea M
authors.initials: LM
authors.lastname: Hürlimann
authors.affiliation: Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Valentina
authors.initials: V
authors.lastname: Corradi
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Hohl
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
authors.forename: Guido V
authors.initials: GV
authors.lastname: Bloemberg
authors.affiliation: Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: D Peter
authors.initials: DP
authors.lastname: Tieleman
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland m.seeger@imm.uzh.ch.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Seeger
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000378:metabolism; D003630:Daunorubicin / Q000737:chemistry / Q000378:metabolism; D004317:Doxorubicin / Q000737:chemistry / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D024841:Fluoroquinolones / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D056426:Genetic Loci; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019076:Transgenes
chemical_list: D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001562:Benzimidazoles; D024841:Fluoroquinolones; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride; D003630:Daunorubicin