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