CONICET | Buscador de Institutos y Recursos Humanos

Background. The metallo-beta-lactamase(MBL) NDM-1 is an efficient carbapenemase with widespread dissemination, having been detected in more than 70 countries worldwide. Up to date, 16 natural variants of this enzyme were reported in clinical settings, with 1 to 5 amino acid substitutions when compared to NDM-1. Previous studies indicated no major differences in protein structure, catalytic efficiency or stability among alleles, and thus it is unclear whether these substitutions confer an evolutionary advantage to the MBL. However, the Zn(II) dependence of these MBLvariants was not assayed. Given that MBLs must tolerate Zn(II) starvation during infection, due to the host´s nutritional immunity response, we evaluated the effect of Zn(II) availability on the fitness of these variants. Methods. blaNDM alleles were engineered by site-directed mutagenesis, cloned in the pMBLe plasmid under an inducible pTac promoter, and transformed in Escherichia coli DH5α cells. Whole cell steady state expression levels for NDM variants were evaluated by immunoblotting. Minimum inhibitory concentrations (MICs) were determined by the agar macrodilution method, and the ability of NDM variants to confer resistance under limiting Zn(II) conditions was assayed by measuring cefotaxime MICs with addition of increasing amounts of metal chelator dipicolinic acid (DPA). Stability of NDM alleles under Zn(II) starvation was next evaluated by measuring NDM protein levels as a function of time after addition of 500 µM DPA to the growth medium, and by pulse proteolysis assays of the detergent-solubilized enzyme in urea. Results. No significant differences in beta-lactam MICs nor expression levels among strains expressing the different NDM variants were observed. Interestingly, most alleles displayed a greater ability to confer resistance under Zn(II) starvation conditions than NDM-1, particularly those possessing two amino acid substitutions. We have previously shown that Zn(II) depletion leads to rapid degradation of MBLs within the periplasmic space (1). Variants for which resistance was less dependent on Zn(II) availability displayed a higher stability under Zn(II) starvation, being degraded at a slower rate than NDM-1 upon addition of 500 µM DPA. Conclusions. Our results show that clinical evolution ofNDM has yielded variants with increased stability and ability to conferresistance upon Zn(II) deprivation. We propose that the selective pressure exerted by the host´s immune response has been critical in shaping the evolution of this major carbapenemase.References:(1) González et al., Nat Chem Bio,12, 516522(2016)