CONICET | Buscador de Institutos y Recursos Humanos

Background NDM metallo-beta-lactamase(MBL) is a widespread carbapenemase identified in 2008. So far, 16 clinicalalleles have been detected, carrying 1 to 5 point mutations. No majordifferences regarding stability, catalytic efficiency or resistance profilewere reported among alleles and thus the role of these mutations remainsunclear. MBLs must endure low Zn(II) concentrations at infection sites, due tothe release of metal-chelating proteins by the host?s immune system. Here we have compared the effects of reducedZn(II) availability in NDM alleles to determine if these mutations can modulateenzyme fitness under such conditions. Methods NDM variantswere expressed using the pMBLe plasmid in E.coli DH5α cells. The effect of Zn(II) starvation on cefotaxime MICs valueswas determined by MIC assays in media supplemented with differentconcentrations of the metal chelator dipicolinic acid (DPA). Stability of NDMvariants under Zn(II) depletion was evaluated by measuring NDM protein levelsas a function of time after addition of 500 µM DPA to the growth medium. Enzymeinactivation by DPA was assayed by incubating spheroplasts expressing each allelewith different concentrations of DPA followed by determination of β-lactamaseactivity. Results While expressionlevels and MICs displayed no major differences among NDM alleles in Zn(II) richmedium, most variants presents a higher capability to confer resistance underZn(II) depletion than NDM-1. Furthermore, double mutants, all of which containthe M154L substitution, were more tolerant to metal starvation than single mutants.We have recently shown(1) that Zn(II) depletion leads to degradationof MBLs within the periplasmic space. We compared the stability of NDM variantsafter addition of 500 µM DPA, and observed that mutants with enhancedcapability to confer resistance under Zn(II) starvation were degraded at alower rate than NDM-1, particularly in alleles carrying the A233V and E152Kmutations. Finally, the presence of the M154L mutation protected the enzyme in DPAinactivation assays in spheroplasts, suggesting an increased Zn(II) bindingaffinity. Site-saturation experiments at position 154 reveal that this site istolerant to mutation, but the mutation M154L is the only one eliciting moreresistance to Zn(II) depletion. Conclusion Our resultsindicate that clinical NDM variants have been optimized to better endure Zn(II)depletion, with an increase in both ability to confer resistance and proteinstability under metal starvation. Substitution M154L, present in 7 out of 16alleles, seems to be instrumental in this role, through improvement of Zn(II)affinity of the enzyme. References(1) González etal., Nat Chem Bio,12, 516522 (2016)