Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity.
Sébastien Triboulet,Sébastien Triboulet,Sébastien Triboulet,Vincent Dubée,Vincent Dubée,Vincent Dubée,Lauriane Lecoq,Lauriane Lecoq,Catherine M. Bougault,Catherine M. Bougault,Jean-Luc Mainardi,Louis B. Rice,Mélanie Etheve-Quelquejeu,Mélanie Etheve-Quelquejeu,Laurent Gutmann,Arul Marie,Lionel Dubost,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Jean-Pierre Simorre,Jean-Pierre Simorre,Michel Arthur,Michel Arthur,Michel Arthur +24 more
TLDR
It is shown that imipenem, ceftriaxone, and ampicillin acylate Ldtfm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl, which is critical for rapid L,D-transpeptidase inactivation and antibacterial activity.Abstract:
Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt(fm)) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt(fm) does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt(fm) by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt(fm) inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt(fm) acylation was followed by rupture of the C(5)-C(6) bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt(fm) blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.read more
Citations
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In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by l,d-Transpeptidases and Inactivation of These Enzymes by Carbapenems
Mathilde Cordillot,Mathilde Cordillot,Mathilde Cordillot,Vincent Dubée,Vincent Dubée,Vincent Dubée,Sébastien Triboulet,Sébastien Triboulet,Sébastien Triboulet,Lionel Dubost,Arul Marie,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Michel Arthur,Michel Arthur,Michel Arthur,Jean-Luc Mainardi +17 more
TL;DR: Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenems, indicating that modification of the carbapenems side chain could be used to optimize their antimycobacterial activity.
Journal ArticleDOI
Resistance to antibiotics targeted to the bacterial cell wall.
TL;DR: This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery and strategies toward the development of novel inhibitors that could overcome resistance.
Journal ArticleDOI
Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli
Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Dominique Mengin-Lecreulx,Alejandro Monton,Tanneke den Blaauwen,E. Carbonnelle,E. Carbonnelle,E. Carbonnelle,Carole Veckerlé,Carole Veckerlé,Carole Veckerlé,V. Brun Yves,Michael S. Van Nieuwenhze,Christiane Bouchier,Kuyek Tu,Kuyek Tu,Kuyek Tu,Louis B. Rice,Michel Arthur,Michel Arthur,Michel Arthur +21 more
TL;DR: Production of YcbB was sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance and identify a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities.
Journal ArticleDOI
β-Lactamase inhibition by avibactam in Mycobacterium abscessus
Vincent Dubée,Vincent Dubée,Vincent Dubée,Audrey Bernut,Audrey Bernut,Mélanie Cortes,Mélanie Cortes,Mélanie Cortes,Tiffany Lesne,Tiffany Lesne,Delphine Dorchêne,Delphine Dorchêne,Delphine Dorchêne,Anne-Laure Lefebvre,Anne-Laure Lefebvre,Anne-Laure Lefebvre,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Jean-Emmanuel Hugonnet,Laurent Gutmann,Jean-Luc Mainardi,Jean-Louis Herrmann,Jean-Louis Gaillard,Laurent Kremer,Laurent Kremer,Michel Arthur,Michel Arthur,Michel Arthur +27 more
TL;DR: Avibactam is identified as the first efficient inhibitor of BlaMab and strongly suggest that β-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.
Journal ArticleDOI
Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape.
Catherine Baranowski,Michael Welsh,Lok-To Sham,Lok-To Sham,Haig A. Eskandarian,Hoong Chuin Lim,Karen J. Kieser,Jeffrey C Wagner,John D. McKinney,Georg E. Fantner,Thomas R. Ioerger,Suzanne Walker,Thomas G. Bernhardt,Eric J. Rubin,E. Hesper Rego +14 more
TL;DR: It is found that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation, and knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.
References
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Journal ArticleDOI
The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis
TL;DR: An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.
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Journal ArticleDOI
Meropenem-Clavulanate Is Effective Against Extensively Drug-Resistant Mycobacterium tuberculosis
TL;DR: When meropenem was combined with the β-lactamase inhibitor clavulanate, potent activity against laboratory strains of M. tuberculosis was observed and could potentially be used to treat patients with currently untreatable disease.
Journal ArticleDOI
The Peptidoglycan of Stationary-Phase Mycobacterium tuberculosis Predominantly Contains Cross-Links Generated by l,d-Transpeptidation
Marie Lavollay,Michel Arthur,Michel Arthur,Michel Arthur,Martine Fourgeaud,Martine Fourgeaud,Martine Fourgeaud,Lionel Dubost,Arul Marie,Nicolas Veziris,Didier Blanot,Laurent Gutmann,Jean-Luc Mainardi +12 more
TL;DR: It is shown that a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3Cross- links generated by a transpePTidase of L,D specificity.
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