Molecular mechanisms governing stability of metallo-beta-lactamases during pathogenesis

GONZALEZ LJ; VILA AJ; GONZÁLEZ MM

Congreso; 13th Beta-Lactamase Meeting; 2017

BackgroundIn the last years, the number of carbapenem resistant bacteria has soared, principally due to the acquisition of zinc-dependent metallo-β-lactamases (MBLs). These enzymes are active in the periplasm of Gram negative bacteria, where they fold and bind the essential Zn(II) cofactor. During infection the host immune response withholds nutrient metal ions from microbial pathogens, which limits the Zn(II) necessary for MBL activity. Previously we showed that sequestration of Zn(II) ions provokes the degradation of periplasmic MBLs, rendering the bacteria susceptible to antibiotics. Degradation rates are variable among MBLs, ranging from a high stability (BcII and membrane-anchored NDM-1) to fast degradation rates (VIM-2 and soluble NDM-1 mutant). In the present work we aimed to unravel the molecular mechanisms by which MBLs are stabilized to evade the effects of metal deprivation.MethodsPure recombinant BcII, soluble NDM-1 and SPM-1 were used for measuring thermal stability, metal affinities and susceptibility to proteinase K in their metallated (holo) and apo forms. Backbone dynamics of 15N-BcII and soluble 15N-NDM-1 was assessed by NMR H/D exchange experiments. Degradation rates in vivo or in different cell fractions were followed by Western-blot. Escherichia coli knock-out mutants or variants of NDM-1 anchored to inner and outer-membranes were constructed for analysis. ResultsIn vitro studies revealed that degradation rates of soluble MBLs in vivo correlate with the thermal stability and susceptibility to proteolysis of the apo-enzymes, and not with their metal binding affinities. Apo NDM-1 is readily degraded while apo BcII is barely affected. H/D exchange experiments showed that the C-terminal region of NDM-1 becomes flexible upon metal loss. BcII, instead, remains rigid. Then we evaluated the role of membrane-anchoring in NDM-1 stabilization. Degradation rates of chimeric NDM-1 variants showed that specific anchoring to the outer membrane, rather than proximity to the membrane, augments NDM-1 stability upon Zn(II) limitation. Stability studies in periplasmic extracts and spheroplasts suggest that outer-membrane localization diminishes the access of apo NDM-1 to periplasmic proteases, probably due to the murein structure. Finally, we demonstrate that apo-MBLs aggregate as insoluble complexes within the periplasm if the periplasmic proteolytic activity is inhibited.ConclusionsMBLs can resist metal deprivation conditions encountered during pathogenesis by (a) membrane-anchoring to the outer membrane, which ?hides? the enzyme from proteases while preventing aggregation of the apo enzyme, or (b) by stiffening of the protein scaffold, which prevents degradation of the apo enzymes.