title: Characterization of BRPMBL, the Bleomycin Resistance Protein Associated with the Carbapenemase NDM.
abstract: The metallo-β-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene,bleMBL, downstream of theblaNDM-1gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of theblaNDM-1-bleMBLoperon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that theblaNDM-1-bleMBLoperon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathioneS-transferase (GST)-BRPMBL, we demonstrated that BRPMBLis able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation.In silicomodeling confirmed that the mechanism of action required the dimerization of the BRPMBLprotein in order to sequester bleomycin and prevent DNA damage. BRPMBLacts specifically on bleomycin-like molecules since cloning and expression ofbleMBLinStaphyloccoccus aureusdid not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.
authors.affiliation: Bacteriology-Hygiene unit, Assistance Publique/Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France laurent.dortet@aphp.fr.|EA7361 (Structure, dynamic, function and expression of broad spectrum β-lactamases), Paris-Sud University, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France.|Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-producing Enterobacteriaceae, Le Kremlin-Bicêtre, France.
authors.forename: Laurent
authors.identifier: 0000-0001-6596-7384
authors.initials: L
authors.lastname: Dortet
authors.affiliation: EA7361 (Structure, dynamic, function and expression of broad spectrum β-lactamases), Paris-Sud University, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Girlich
authors.affiliation: Bacteriology-Hygiene unit, Assistance Publique/Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France.
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Virlouvet
authors.affiliation: Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Department of Medicine, University of Fribourg, Switzerland.|INSERM European Unit (LEA Paris, IAME, France), University of Fribourg, Switzerland.
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirel
authors.affiliation: Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Department of Medicine, University of Fribourg, Switzerland.|INSERM European Unit (LEA Paris, IAME, France), University of Fribourg, Switzerland.|University hospital and University of Lausanne, Switzerland.
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
authors.affiliation: Institut de Chimie des Substances Naturelles, CNRS UPR 2301, LabEx LERMIT, Gif-sur-Yvette, France.
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Iorga
authors.affiliation: Bacteriology-Hygiene unit, Assistance Publique/Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France.|EA7361 (Structure, dynamic, function and expression of broad spectrum β-lactamases), Paris-Sud University, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France.|Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-producing Enterobacteriaceae, Le Kremlin-Bicêtre, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Naas
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D001761:Bleomycin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015378:Imipenem / Q000494:pharmacology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008958:Models, Molecular; D009876:Operon; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; C105427:Zeocin; D015378:Imipenem; D001618:beta-Lactamases; C063912:carbapenemase
keywords: antimicrobial resistance; cancer treatment; mechanism of action; structure