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

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, ble_MBL, downstream of the bla_NDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRP_MBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the bla_NDM-1-ble_MBL operon 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 bla_NDM-1-ble_MBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-transferase (GST)-BRP_MBL, we demonstrated that BRP_MBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRP_MBL protein in order to sequester bleomycin and prevent DNA damage. BRP_MBL acts specifically on bleomycin-like molecules since cloning and expression of ble_MBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.