Characterization of BRP MBL , the Bleomycin Resistance Protein Associated with the Carbapenemase NDM

Abstract

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, 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.