Membrane Anchoring of Carbapenemase NDM-1 Favors Protein Stability

GONZÁLEZ LJ; BAHR G; BONOMO RA; VILA AJ

Washington DC

Congreso; Interscience Conference on Antimicrobial Agents and Chemotherapy 2014 (ICAAC 2014); 2014

American Society for Microbiology

In the last decade, the dissemination of genes encoding metallo-beta-lactamases (MBLs) across Gram-negative bacterial pathogens has constituted a growing clinical problem. These versatile enzymes are capable of hydrolyzing almost all beta-lactam antibiotics, and thus comprise a serious threat to the efficacy of the most widely used class of antibacterial drugs. The presence of Zn(II) in the active site of MBLs is essential for both substrate binding and catalysis, and it has been demonstrated that they are translocated to their site of action (the periplasmic space) as unfolded chains through the SecA/SecYEG system. As such, final folding and Zn(II) acquisition take place in the periplasmic space of the bacterial host, and MBLs have evolved to efficiently bind periplasmic Zn(II). Though zinc levels in the cytoplasm are tightly controlled, for most bacteria there are no known mechanisms for accumulation of this ion in the periplasm, and Zn(II) is thought to simply diffuse from the extracellular medium trough porins in the outer membrane. Therefore, the periplasmic levels of this metal ion available for MBLs would be highly dependent on its extracellular concentration. In this way, external Zn(II) scarcity could significantly hamper in-vivo functionality of these enzymes. Interestingly, the immune system of vertebrates is known to sequester Zn(II) and other metal ions as a response to infection in a phenomenon called nutritional immunity, where the infection sites become depleted of Zn(II). This metal restriction could have strong consequences on the resistance phenotype of pathogens harboring MBLs. In the present work we have analyzed how Zn(II) availability affects the in-vivo performance of clinically relevant MBLs. Genes coding for each MBL (NDM-1, VIM-2, SPM-1, BcII and IMP-1) were cloned under an IPTG-inducible promoter in the pMBLe plasmid, constructed in our lab. E. coli DH5a was used as the host strain for all assays. The effect of metal availability on in-vivo antibiotic resistance was analyzed through Minimum Inhibitory Concentration (MIC) determinations in LB-agar versus the beta-lactam antibiotic cefotaxime (CTX) for cells expressing each MBL. Zn(II) availability in the growth medium was increased by supplementation with 500 uM ZnSO4, and zinc depletion was attained by addition of various concentrations of the metal chelator Dipicolinic Acid (DPA). To determine the effect of metal depletion on protein levels, 1000 uM DPA was added to cultures that had previously been induced to express each MBL. Periplasm extractions were performed on aliquots taken at different time intervals after DPA addition (0 min, 10 min, 90 min, ON -overnight), and MBL protein levels were quantified by Western Blot. The determination of beta-lactam MICs under conditions of varying Zn(II) availability showed that, even when expressed in similar levels and in the same strain, MBLs exhibit different levels of susceptibility to zinc depletion. SPM-1, and to a lesser extent BcII, displayed the highest tolerance to reduced metal availability among the assayed enzymes. To further investigate the effects of zinc depletion on MBL in-vivo functionality, we quantified the lactamase protein levels present at different intervals of time after DPA addition. The extremely rapid decrease of periplasmic protein in the cases of VIM-2 and IMP-1 strongly suggests that upon the DPA-induced loss of Zn(II) from the active site, the MBL apoenzymes are promptly degraded in the periplasmic space. Supporting this hypothesis, we performed limited proteolysis studies with Proteinase K on purified MBLs, revealing that apo forms, compared to their holo counterparts, are more sensitive to proteolysis. In the current work we have demonstrated that, resulting from the lack of regulation of Zn(II) levels in the bacterial periplasm, the in-vivo functionality of MBLs is strongly dependent on the disponibility of this metal ion in the growth medium. Different MBLs display varying degrees of resistance to Zn(II) deprivation, and in conditions where the metal availability is low enough to generate the apo-enzymes these are rapidly degraded through proteolysis. Our results suggest that the development of strategies which reinforce the body´s natural metal sequestration defense could be helpful for the treatment of infections caused by MBL-producing pathogens.