Characterization of BRPMBL, the Bleomycin Resistance Protein Associated with the Carbapenemase NDM

ABSTRACTThe 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 the blaNDM-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 the blaNDM-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 the blaNDM-1-bleMBLoperon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-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 of bleMBLin Staphyloccoccus aureusdid not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.