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Combinatorial active-site variants confer sustained clavulanate resistance in BlaC beta-lactamase from Mycobacterium tuberculosis

Egesborg, Philippe; Carlettini, Hélène; Volpato, Jordan P; Doucet, Nicolas (2014). Combinatorial active-site variants confer sustained clavulanate resistance in BlaC beta-lactamase from Mycobacterium tuberculosis Protein Science , vol. 24 , nº 4. p. 534-44. DOI: 10.1002/pro.2617.

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Résumé

Bacterial resistance to beta-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to beta-lactam treatment, primarily due to the chromosomally encoded BlaC beta-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant beta-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific beta-lactam-BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical beta-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual beta-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 mug/ml, 25 microM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in beta-lactamases, potentially casting a shadow on specific beta-lactam-BlaC inhibitor combination treatments against M. tuberculosis.

Type de document: Article
Mots-clés libres: Class A β-lactamases; Mycobacterium tuberculosis; antibiotic resistance; clavulanic acid; combinatorial mutagenesis; site-directed mutagenesis; β-lactam
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 19 sept. 2017 15:48
Dernière modification: 19 sept. 2017 15:48
URI: http://espace.inrs.ca/id/eprint/3035

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