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In vivo impact of Met221 substitution in GOB metallo-?-lactamase.

ABSTRACT: Metallo-?-lactamases (M?Ls) represent one of the main mechanisms of bacterial resistance against ?-lactam antibiotics. The elucidation of their mechanism has been limited mostly by the structural diversity among their active sites. All M?Ls structurally characterized so far present a Cys or a Ser residue at position 221, which is critical for catalysis. GOB lactamases stand as an exception within this picture, possessing a Met residue in this location. We studied different mutants in this position, and we show that Met221 is essential for protein stability, most likely due to its involvement in a hydrophobic core. In contrast to other known M?Ls, residue 221 is not involved in metal binding or in catalysis in GOB enzymes, further highlighting the structural diversity of M?Ls. We also demonstrate the usefulness of protein periplasmic profiles to assess the contribution of protein stability to antibiotic resistance.

SUBMITTER: Moran-Barrio J

PROVIDER: S-EPMC3318309 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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In vivo impact of Met221 substitution in GOB metallo-β-lactamase.

Morán-Barrio Jorgelina J Lisa María-Natalia MN Vila Alejandro J AJ

Antimicrobial agents and chemotherapy 20120117 4

Metallo-β-lactamases (MβLs) represent one of the main mechanisms of bacterial resistance against β-lactam antibiotics. The elucidation of their mechanism has been limited mostly by the structural diversity among their active sites. All MβLs structurally characterized so far present a Cys or a Ser residue at position 221, which is critical for catalysis. GOB lactamases stand as an exception within this picture, possessing a Met residue in this location. We studied different mutants in this positi ...[more]

PMID: 22252824

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Project description:Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Dataset Information

In vivo impact of Met221 substitution in GOB metallo-?-lactamase.

Publications

In vivo impact of Met221 substitution in GOB metallo-β-lactamase.

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