Effect of Zn(II) deprivation on MBL-mediated resistance

GONZÁLEZ LJ; BAHR G; BETHEL C; BONOMO RA; VILA AJ

Gran Canaria

Workshop; 12th Beta-Lactamase Meeting; 2014

University of Liége, Bélgica; Universities of Siena and Florence & Florence Careggi University Hospital, Italia; Veterans Affairs Medical Center, Cleveland, USA; Universidad de Las Palmas de Gran Canaria, España

SPM-1 is an MBL produced only by Psudomonas aeruginosa (Pa), while other MBLs are found in different bacteria. SPM-1 is unique among pathogen-associated MBLs in that in that it contains "atypical" second sphere residues (S84, G121) inside the protein. Our goal is to explore the role of these SS residues on catalytic activity and metal uptake in a native context. The blaSPM-1 gene from Pa 48-1997A was introduced into Pa PAO where codon randomization and selection of resistance-conferring mutants was performed. MICs and periplasmic activities revealed that WT combination (S84/G121), and mutant G121A, is advantageous only for hydrolyzing latest antipseudomonal beta-lactam antibiotics (cefepime and imipenem) with hydrolysis rates 2.3 to 100-fold higher than the rest of the mutants. Limiting Zn(II) availability affected MICs differentially, being WT between the most resistant variants to metal deprivation (MIC drop of 1 dilution), while G121A the most sensitive (MIC 64-fold lower). These results suggest that SS residues optimize the resistance profile of SPM-1 by modulating the substrate preference and Zn(II) binding features according to specific requirements in Pa. To further evaluate this hypothesis, we compared the resistance dependence with metal availability of SPM-1, NDM-1, VIM-2 and IMP-1 MBLs, expressed both in Pa PAO and E. coli, measuring MICs at different concentrations of supplemented metal chelator. The results revealed that SPM-1 is the MBL which most resists metal deprivation in both bacteria (SPM-1>IMP-1>NDM-1=VIM-2). In addition, resistance in Pa PAO was more sensitive to metal deprivation than E. coli. These results reinforce that SPM-1 metal binding ability must be optimized for conferring resistance at limiting concentrations of Zn(II) in the more sensitive Pa. These conditions are in fact typically encountered during bacterial infection, where the host immune system (neutrophils) secrete large amounts of Calprotectin, a small Zn(II) and Mn(II) binding protein, in the site of infection.