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1. Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

Author

Yucheng Liang, Jean-Emmanuel Hugonnet, Filippo Rusconi, and Michel Arthur

Subjects
peptidoglycan, braun lipoprotein, mass spectrometry, Medicine, Science, Biology (General), QH301-705.5
Abstract

Peptidoglycan (PG) is a giant macromolecule that completely surrounds bacterial cells and prevents lysis in hypo-osmotic environments. This net-like macromolecule is made of glycan strands linked to each other by two types of transpeptidases that form either 4→3 (PBPs) or 3→3 (LDTs) cross-links. Previously, we devised a heavy isotope-based PG full labeling method coupled to mass spectrometry to determine the mode of insertion of new subunits into the expanding PG network (Atze et al., 2022). We showed that PG polymerization operates according to different modes for the formation of the septum and of the lateral cell walls, as well as for bacterial growth in the presence or absence of β-lactams in engineered strains that can exclusively rely on LDTs for PG cross-linking when drugs are present. Here, we apply our method to the resolution of the kinetics of the reactions leading to the covalent tethering of the Braun lipoprotein (Lpp) to PG and the subsequent hydrolysis of that same covalent link. We find that Lpp and disaccharide-peptide subunits are independently incorporated into the expanding lateral cell walls. Newly synthesized septum PG appears to contain small amounts of tethered Lpp. LDTs did mediate intense shuffling of Lpp between PG stems leading to a dynamic equilibrium between the PG-tethered and free forms of Lpp.

Published
2024
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2. Genome-wide identification of genes required for alternative peptidoglycan cross-linking in Escherichia coli revealed unexpected impacts of β-lactams

Author

Henri Voedts, Sean P. Kennedy, Guennadi Sezonov, Michel Arthur, and Jean-Emmanuel Hugonnet

Subjects
Science
Abstract

β-lactam-induced bacterial killing is complex and not fully resolved. Authors carry out a genome-wide analysis, through penicillin-binding protein replacement, to identify genes essential for drug efficacy.

Published
2022
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3. Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

Author

Heiner Atze, Yucheng Liang, Jean-Emmanuel Hugonnet, Arnaud Gutierrez, Filippo Rusconi, and Michel Arthur

Subjects
peptidoglycan, cell wall, antibiotics, mass spectrometry, peptidoglycan polymerization, L,D-transpeptidase, Medicine, Science, Biology (General), QH301-705.5
Abstract

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

Published
2022
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4. Structural insight into YcbB-mediated beta-lactam resistance in Escherichia coli

Author

Nathanael A. Caveney, Guillermo Caballero, Henri Voedts, Ana Niciforovic, Liam J. Worrall, Marija Vuckovic, Matthieu Fonvielle, Jean-Emmanuel Hugonnet, Michel Arthur, and Natalie C. J. Strynadka

Subjects
Science
Abstract

In E. coli, alternate peptidoglycan crosslinking reactions carried out by the L,D-transpeptidase YcbB can lead to beta-lactam resistance. Here, Caveney et al. solve the crystal structure of YcbB and shed light into its mechanism of action and into YcbB-mediated antibiotic resistance.

Published
2019
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5. Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

Author

Landys Lopez Quezada, Robert Smith, Tania J. Lupoli, Zainab Edoo, Xiaojun Li, Ben Gold, Julia Roberts, Yan Ling, Sae Woong Park, Quyen Nguyen, Frank J. Schoenen, Kelin Li, Jean-Emmanuel Hugonnet, Michel Arthur, James C. Sacchettini, Carl Nathan, and Jeffrey Aubé

Subjects
M. tuberculosis, cephalosporin, non-replicating, β-lactams, ABPP, click chemistry, Microbiology, QR1-502
Abstract

As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.

Published
2020
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6. Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site

Author

Zainab Edoo, Michel Arthur, and Jean-Emmanuel Hugonnet

Subjects
Medicine, Science
Abstract

Abstract β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

Published
2017
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7. Methicillin-Susceptible, Vancomycin-Resistant Staphylococcus aureus, Brazil

Author

Diana Panesso, Paul Planet, Lorena Diaz, Jean-Emmanuel Hugonnet, Truc T. Tran, Apurva Narechania, Jose M. Munita, Sandra Rincon, Lina P. Carvajal, Jinnethe Reyes, Alejandra Londoño, Hannah Smith, Robert Sebra, Gintaras Deikus, George M. Weinstock, Barbara E. Murray, Flavia Rossi, Michel Arthur, and Cesar A. Arias

Subjects
Staphylococcus aureus, bacteria, antimicrobial resistance, drug resistance, methicillin susceptibility, vancomycin resistance, Medicine, Infectious and parasitic diseases, RC109-216
Abstract

We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.

Published
2015
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8. Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

Author

Jean-Emmanuel Hugonnet, Dominique Mengin-Lecreulx, Alejandro Monton, Tanneke den Blaauwen, Etienne Carbonnelle, Carole Veckerlé, Yves, V. Brun, Michael van Nieuwenhze, Christiane Bouchier, Kuyek Tu, Louis B Rice, and Michel Arthur

Subjects
β-lactam, PBP1b, PBP5, L,D-transpeptidase, mecillinam, (p)ppGpp, Medicine, Science, Biology (General), QH301-705.5
Abstract

The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB.

Published
2016
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9. Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity.

Author

Sébastien Triboulet, Vincent Dubée, Lauriane Lecoq, Catherine Bougault, Jean-Luc Mainardi, Louis B Rice, Mélanie Ethève-Quelquejeu, Laurent Gutmann, Arul Marie, Lionel Dubost, Jean-Emmanuel Hugonnet, Jean-Pierre Simorre, and Michel Arthur

Subjects
Medicine, Science
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.

Published
2013
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10. Polysaccharide II Surface Anchoring, the Achilles’ Heel of Clostridioides difficile

Author

Jeanne Malet-Villemagne, Liang Yucheng, Laurent Evanno, Sandrine Denis-Quanquin, Jean-Emmanuel Hugonnet, Michel Arthur, Claire Janoir, and Thomas Candela

Subjects
Microbiology (medical), Infectious Diseases, General Immunology and Microbiology, Ecology, Physiology, Genetics, Cell Biology
Abstract

Cell wall glycopolymers (CWGPs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. CWGP anchoring to peptidoglycan is important for growth and virulence.

Published
2023
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12. Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross‐linking enzymes in Escherichia coli

Author

Waldemar Vollmer, Henri Voedts, Adam Lodge, Jean-Emmanuel Hugonnet, Delphine Dorchêne, Michel Arthur, Université Paris Cité, Equipe HAL, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Régulation de la synthèse du peptidoglycan chez Escherichia coli - - RegOPepS2019 - ANR-19-CE44-0007 - AAPG2019 - VALID, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Newcastle University [Newcastle], ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), and ANR-19-CE44-0007,RegOPepS,Régulation de la synthèse du peptidoglycan chez Escherichia coli(2019)

Subjects
Glycan, β-lactam, Stereochemistry, Peptidoglycan, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Catalysis, Gene Expression Regulation, Enzymologic, Mass Spectrometry, beta-Lactam Resistance, chemistry.chemical_compound, D-transpeptidase, Endopeptidases, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, polycyclic compounds, medicine, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, endopeptidase, chemistry.chemical_classification, General Immunology and Microbiology, biology, General Neuroscience, Escherichia coli Proteins, Hydrolysis, Membrane Proteins, Articles, Gene Expression Regulation, Bacterial, biology.organism_classification, Endopeptidase, Anti-Bacterial Agents, Enzyme Activation, Enzyme, Biochemistry, Polymerization, chemistry, Cytoplasm, Mutation, Peptidyl Transferases, biology.protein, Bacteria, Macromolecule
Abstract

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Published
2021
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13. Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

Author

Mélanie Etheve-Quelquejeu, Fabrice Compain, Jean-Emmanuel Hugonnet, Michel Arthur, Adrian Dupuis, Laetitia Sutterlin, Clément Ourghanlian, Vincent Dubée, Sébastien Triboulet, Heiner Atze, and Zainab Edoo

Subjects
0301 basic medicine, Stereochemistry, Acylation, Imine, 030106 microbiology, Reaction intermediate, beta-Lactams, Thioester, beta-Lactamases, Fluorescence spectroscopy, Hydrophobic effect, 03 medical and health sciences, chemistry.chemical_compound, Residue (chemistry), Catalytic Domain, Serine, polycyclic compounds, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Quenching (fluorescence), 030306 microbiology, Chemistry, Tryptophan, Mycobacterium tuberculosis, Mesomeric effect, 3. Good health, Spectrometry, Fluorescence, 030104 developmental biology, Infectious Diseases, Enzyme, Covalent bond, Peptidyl Transferases, Lactam
Abstract

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

Published
2019
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14. Structural insight into YcbB-mediated beta-lactam resistance in Escherichia coli

Author

Michel Arthur, G. Caballero, Natalie C. J. Strynadka, Nathanael A. Caveney, Ana P. Niciforovic, Liam J. Worrall, Jean-Emmanuel Hugonnet, Matthieu Fonvielle, Marija Vuckovic, and Henri Voedts

Subjects
0301 basic medicine, Acylation, Science, General Physics and Astronomy, Peptidoglycan, 02 engineering and technology, Molecular Dynamics Simulation, Crystallography, X-Ray, medicine.disease_cause, beta-Lactam Resistance, Article, General Biochemistry, Genetics and Molecular Biology, Bacterial cell structure, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Cell Wall, Antibiotics, In vivo, Catalytic Domain, Escherichia coli, medicine, Penicillin-Binding Proteins, Protein Interaction Maps, lcsh:Science, X-ray crystallography, Multidisciplinary, Escherichia coli Proteins, Substrate (chemistry), Meropenem, General Chemistry, 021001 nanoscience & nanotechnology, Recombinant Proteins, Anti-Bacterial Agents, 3. Good health, 030104 developmental biology, Amino Acid Substitution, chemistry, Mechanism of action, Peptidyl Transferases, Biophysics, lcsh:Q, medicine.symptom, 0210 nano-technology
Abstract

The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli, the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance., In E. coli, alternate peptidoglycan crosslinking reactions carried out by the L,D-transpeptidase YcbB can lead to beta-lactam resistance. Here, Caveney et al. solve the crystal structure of YcbB and shed light into its mechanism of action and into YcbB-mediated antibiotic resistance.

Published
2019

15. Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

Author

Kelin Li, Yan Ling, Xiaojun Li, Frank J. Schoenen, Zainab Edoo, Landys Lopez Quezada, Michel Arthur, Julia Roberts, Sae Woong Park, Carl Nathan, Robert A. Smith, Jean Emmanuel Hugonnet, Jeffrey Aubé, Ben Gold, James C. Sacchettini, Tania J. Lupoli, Quyen Nguyen, Weill Medical College of Cornell University [New York], University of Kansas [Lawrence] (KU), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Texas A&M University [College Station], University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC), and HUGONNET, Jean-Emmanuel

Subjects
Microbiology (medical), Tuberculosis, Protein family, medicine.drug_class, Cephalosporin, Mutant, cephalosporin, lcsh:QR1-502, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microbiology, lcsh:Microbiology, law.invention, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, law, medicine, polycyclic compounds, M. tuberculosis, ABPP, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Activity-based proteomics, β-lactams, non-replicating, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biochemical phenomena, metabolism, and nutrition, respiratory system, biology.organism_classification, medicine.disease, bacterial infections and mycoses, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, chemistry, click chemistry, Recombinant DNA, Peptidoglycan
Abstract

International audience; As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb's survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.

Published
2020
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16. Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating

Author

Landys, Lopez Quezada, Robert, Smith, Tania J, Lupoli, Zainab, Edoo, Xiaojun, Li, Ben, Gold, Julia, Roberts, Yan, Ling, Sae Woong, Park, Quyen, Nguyen, Frank J, Schoenen, Kelin, Li, Jean-Emmanuel, Hugonnet, Michel, Arthur, James C, Sacchettini, Carl, Nathan, and Jeffrey, Aubé

Subjects
cephalosporin, click chemistry, polycyclic compounds, M. tuberculosis, β-lactams, non-replicating, ABPP, biochemical phenomena, metabolism, and nutrition, respiratory system, bacterial infections and mycoses, Microbiology, Original Research
Abstract

As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.

Published
2019

17. Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

Author

Michael S. VanNieuwenhze, Waldemar Vollmer, Alessandra M. Martorana, Michel Arthur, Manuel Pazos, Alessandra Polissi, Jean-Emmanuel Hugonnet, Zainab Edoo, and Katharina Peters

Subjects
0301 basic medicine, Penicillin binding proteins, 030106 microbiology, Enterococcus faecium, Microbial Sensitivity Tests, Peptidoglycan, medicine.disease_cause, beta-Lactams, Bacterial cell structure, beta-Lactam Resistance, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, Minimum inhibitory concentration, Cell Wall, medicine, Escherichia coli, Penicillin-Binding Proteins, Sodium dodecyl sulfate, Copper chloride, Multidisciplinary, Biological Sciences, Aminoacyltransferases, Anti-Bacterial Agents, Trace Elements, 030104 developmental biology, chemistry, Biochemistry, Cell envelope, Copper
Abstract

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4-3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by utilizing β-lactam-insensitive LD-transpeptidases (LDTs) to produce exclusively 3-3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3-3 cross-links in PG and displayed reduced covalent attachment of Braun's lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

Published
2018

18. Critical impact of peptidoglycan precursor amidation on the activity of l,d -transpeptidases from $Enterococcus\ faecium$ and $Mycobacterium\ tuberculosis$

Author

Matthieu Fonvielle, Emmanuelle Braud, Laura Iannazzo, Jean-Emmanuel Hugonnet, Saidbakhrom Saidjalolov, Delphine Patin, Dominique Mengin-Lecreulx, Dirk Schnappinger, Sabine Ehrt, Flora Ngadjeua, Mélanie Etheve-Quelquejeu, Michel Arthur, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Weill Medical College of Cornell University [New York], Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université - Faculté de Médecine (SU FM), Sorbonne Université (SU), Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), BRAUD, Emmanuelle, École pratique des hautes études (EPHE), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, and Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE)

Subjects
0301 basic medicine, 030106 microbiology, Enterococcus faecium, Peptide, Peptidoglycan, medicine.disease_cause, beta-Lactams, Catalysis, Bacterial cell structure, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Cell Wall, [CHIM] Chemical Sciences, medicine, [CHIM]Chemical Sciences, Transaminases, Glutamine amidotransferase, chemistry.chemical_classification, biology, Organic Chemistry, Pathogenic bacteria, General Chemistry, biology.organism_classification, 3. Good health, 030104 developmental biology, chemistry, Biochemistry, Peptidyl Transferases
Abstract

International audience; The bacterial cell wall peptidoglycan contains unusual Land D amino acids assembled in branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by L,D-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the L,D-transpeptidase Ldt$_{Mt2}$ from M. tuberculosis. Accordingly, construction of a conditional mutant established the essentiality of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Published
2018
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19. Synthesis of Avibactam Derivatives and Activity on β-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria

Author

Flavie Bouchet, Mélanie Etheve-Quelquejeu, Inès Li de la Sierra Gallay, Michel Arthur, Fabrice Compain, Jean-Luc Mainardi, Jean-Emmanuel Hugonnet, Matthieu Fonvielle, Laura Iannazzo, Herman van Tilbeurgh, Zainab Edoo, Eva Le Run, Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Service de Microbiologie [CHU GeorgesPompidou], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Sorbonne Université (SU), Université Sorbonne Paris Cité (USPC), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Métabolisme et physiologie rénale (CRC - UMR_S 1138), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Sorbonne Université - Faculté de Médecine (SU FM), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Centre National de la Recherche Scientifique (CNRS)-Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École Pratique des Hautes Études (EPHE), ANR-17-CE18-0010,MYCWall,Développement d'inhibiteurs spécifiques de la synthèse de la paroi des mycobactéries(2017), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Sorbonne Universités, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), and Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)

Subjects
0301 basic medicine, Avibactam, [SDV]Life Sciences [q-bio], 030106 microbiology, Triazole, Peptidoglycan, Catalysis, beta-Lactamases, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, Minimum inhibitory concentration, Beta-Lactamase Inhibitors, Polymerase, chemistry.chemical_classification, d-transpeptidases, biology, Mycobacterium abscessus, Organic Chemistry, General Chemistry, biology.organism_classification, β-lactamase, 3. Good health, Enzyme, chemistry, Biochemistry, biology.protein, beta-Lactamase Inhibitors, Azabicyclo Compounds
Abstract

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 μg mL-1 for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Published
2018
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20. Impact of β-Lactamase Inhibition on the Activity of Ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus

Author

Vincent Dubée, Jean-Emmanuel Hugonnet, Mélanie Cortes, Michel Arthur, Daria Soroka, Laurent Gutmann, Anne-Laure Lefebvre, Jean-Luc Mainardi, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Dubee, Vincent, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP)

Subjects
Tuberculosis, Mycobacterium abscessus, beta-Lactamases, Mycobacterium, Microbiology, Mycobacterium tuberculosis, Medicine, Pharmacology (medical), Mechanisms of Action: Physiological Effects, Pharmacology, chemistry.chemical_classification, biology, business.industry, bacterial infections and mycoses, biology.organism_classification, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cephalosporins, 3. Good health, Infectious Diseases, Enzyme, chemistry, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, beta-Lactamase Inhibitors, business, Azabicyclo Compounds
Abstract

The production of β-lactamases Bla Mab and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis , respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.

Published
2015
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21. Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic

Author

Lluis Ballell, Michel Arthur, Vincent Dubée, Jean-Emmanuel Hugonnet, Guillaume Cuinet, François Signorino-Gelo, Neeraj Dhar, John D. McKinney, and David Barros

Subjects
Tuberculosis, medicine.drug_class, Antibiotics, Antitubercular Agents, Biology, Pharmacology, beta-Lactams, Meropenem, Viable but nonculturable, Microbiology, Mycobacterium tuberculosis, chemistry.chemical_compound, Isoniazid, medicine, Experimental Therapeutics, Pharmacology (medical), Faropenem, medicine.disease, biology.organism_classification, Cytolysis, Infectious Diseases, chemistry, Peptidyl Transferases, medicine.drug
Abstract

Recent clinical studies indicate that meropenem, a β-lactam antibiotic, is a promising candidate for therapy of drug-resistant tuberculosis. However, meropenem is chemically unstable, requires frequent intravenous injection, and must be combined with a β-lactamase inhibitor (clavulanate) for optimal activity. Here, we report that faropenem, a stable and orally bioavailable β-lactam, efficiently kills Mycobacterium tuberculosis even in the absence of clavulanate. The target enzymes, l , d -transpeptidases, were inactivated 6- to 22-fold more efficiently by faropenem than by meropenem. Using a real-time assay based on quantitative time-lapse microscopy and microfluidics, we demonstrate the superiority of faropenem to the frontline antituberculosis drug isoniazid in its ability to induce the rapid cytolysis of single cells. Faropenem also showed superior activity against a cryptic subpopulation of nongrowing but metabolically active cells, which may correspond to the viable but nonculturable forms believed to be responsible for relapses following prolonged chemotherapy. These results identify faropenem to be a potential candidate for alternative therapy of drug-resistant tuberculosis.

Published
2015
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22. Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site

Author

Michel Arthur, Jean-Emmanuel Hugonnet, Zainab Edoo, HAL-UPMC, Gestionnaire, Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers ( CRC ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -École pratique des hautes études ( EPHE ) -Université Paris Diderot - Paris 7 ( UPD7 ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), and Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE)

Subjects
0301 basic medicine, Stereochemistry, Science, Acylation, 030106 microbiology, Enterococcus faecium, beta-Lactams, Bacterial cell structure, Article, Mass Spectrometry, Ring strain, 03 medical and health sciences, chemistry.chemical_compound, Residue (chemistry), Bacterial Proteins, Catalytic Domain, polycyclic compounds, Nitrocefin, Multidisciplinary, biology, Active site, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, biochemical phenomena, metabolism, and nutrition, Aminoacyltransferases, 3. Good health, Anti-Bacterial Agents, Cephalosporins, chemistry, Biochemistry, Cyclization, Spectrophotometry, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, biology.protein, Lactam, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Medicine, Peptidoglycan
Abstract

β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

Published
2017
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23. Peptidoglycan cross-linking activity of L,D-transpeptidases from Clostridium difficile and inactivation of theses enzymes by β-lactams

Author

Jean-Luc Mainardi, Zainab Edoo, Michel Arthur, Laetitia Sutterlin, Jean-Emmanuel Hugonnet, Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), HAL-UPMC, Gestionnaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Centre de Recherche des Cordeliers ( CRC ), Université Paris Diderot - Paris 7 ( UPD7 ) -École pratique des hautes études ( EPHE ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), and Hôpital Européen Georges Pompidou [APHP] ( HEGP )

Subjects
0301 basic medicine, Imipenem, medicine.drug_class, β-lactam, Acylation, 030106 microbiology, Cephalosporin, D-transpeptidases, Peptidoglycan, Meropenem, Mass Spectrometry, Microbiology, peptidoglycan 12, 03 medical and health sciences, chemistry.chemical_compound, [ SDV.MP ] Life Sciences [q-bio]/Microbiology and Parasitology, Mechanisms of Resistance, Ampicillin, polycyclic compounds, medicine, Pharmacology (medical), [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, ComputingMilieux_MISCELLANEOUS, Pharmacology, Clostridioides difficile, Chemistry, Hydrolysis, Clostridium difficile, biochemical phenomena, metabolism, and nutrition, Anti-Bacterial Agents, Cephalosporins, 3. Good health, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Peptidyl Transferases, Ceftriaxone, carbapenems, Ertapenem, medicine.drug
Abstract

In most bacteria, the essential targets of β-lactam antibiotics are the d , d -transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l , d -transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l , d -transpeptidase paralogues Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 , which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt Cd2 and Ldt Cd3 catalyzed the formation of 3→3 cross-links ( l , d -transpeptidase activity), the hydrolysis of the C-terminal d -Ala residue of the disaccharide-tetrapeptide substrate ( l , d -carboxypeptidase activity), and the exchange of the C-terminal d -Ala for d -Met. Ldt Cd1 displayed only l , d -carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt Cd1 and Ldt Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt Cd1 and Ldt Cd2 was higher for the carbapenems (480 to 6,600 M −1 s −1 ) than for ampicillin and ceftriaxone (3.9 to 82 M −1 s −1 ). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt Cd1 and Ldt Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt Cd1 and Ldt Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.

Published
2017
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24. Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

Author

Lionel Dubost, Arul Marie, Jean-Luc Mainardi, Jean-Emmanuel Hugonnet, Noriyasu Shikura, Louis B. Rice, Carole Veckerlé, Michel Arthur, and Nabila Haddache

Subjects
Pharmacology, Tetrapeptide, biology, Stereochemistry, Streptomyces coelicolor, Glycopeptides, Peptidoglycan, Periplasmic space, biology.organism_classification, Pentapeptide repeat, Glycopeptide, Anti-Bacterial Agents, chemistry.chemical_compound, Infectious Diseases, Biochemistry, chemistry, Mechanisms of Resistance, Actinomycetales, Drug Resistance, Bacterial, Gene cluster, bacteria, Pharmacology (medical)
Abstract

Synthesis of peptidoglycan precursors ending in d -lactate ( d -Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l , d -transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d -Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d -Lac into cytoplasmic precursors. This was due to a d , d -transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d -Lac for d -Ala and Gly. The contribution of l , d -transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo- d , d -carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d -Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d , d -transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l , d -transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo- d , d -carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l , d -transpeptidases merely act as a tolerance mechanism in this bacterium.

Published
2014
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25. Characterization of broad-spectrum Mycobacterium abscessus class A beta-lactamase

Author

Olivia Soulier-Escrihuela, Laurent Gutmann, Michel Arthur, Jean-Luc Mainardi, Jean-Emmanuel Hugonnet, Daria Soroka, Vincent Dubée, and Guillaume Cuinet

Subjects
Microbiology (medical), Imipenem, Biology, medicine.disease_cause, Tazobactam, beta-Lactam Resistance, beta-Lactamases, Mycobacterium, Substrate Specificity, Clavulanic acid, polycyclic compounds, medicine, Pharmacology (medical), Enzyme kinetics, Cefoxitin, Escherichia coli, Pharmacology, chemistry.chemical_classification, Hydrolysis, Sulbactam, biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses, 3. Good health, Kinetics, Infectious Diseases, Enzyme, chemistry, Biochemistry, bacteria, medicine.drug
Abstract

Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC₅₀ of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other β-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A β-lactamase (Bla(Mab)) and investigated its role in β-lactam resistance.Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various β-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of β-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC.Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K(m) = 6.7 M(-1) s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K(m) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a β-lactamase inhibitor. β-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters.M. abscessus produces a clavulanate-insensitive broad-spectrum β-lactamase that limits the in vivo efficacy of β-lactams.

Published
2014
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26. Combinations of β-Lactam Antibiotics Currently in Clinical Trials Are Efficacious in a DHP-I-Deficient Mouse Model of Tuberculosis Infection

Author

Joaquín Rullas, David Barros-Aguirre, Michel Arthur, Joël Lelièvre, Lluis Ballell, Andreas H. Diacon, Adolfo García-Pérez, Iñigo Angulo-Barturen, Jean-Emmanuel Hugonnet, John D. McKinney, and Neeraj Dhar

Subjects
Dipeptidases, Tuberculosis, medicine.drug_class, Antibiotics, Microbial Sensitivity Tests, Pharmacology, GPI-Linked Proteins, beta-Lactams, Staphylococcal infections, Mycobacterium tuberculosis, Mice, Pharmacotherapy, Blood drug, Animals, Medicine, Pharmacology (medical), Lung, Respiratory Tract Infections, Respiratory tract infections, biology, business.industry, Staphylococcal Infections, biology.organism_classification, medicine.disease, Anti-Bacterial Agents, 3. Good health, Mice, Inbred C57BL, Clinical trial, Disease Models, Animal, Infectious Diseases, Immunology, Drug Therapy, Combination, business
Abstract

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC ( T >MIC ) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Published
2015
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27. In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by <scp>l</scp> , <scp>d</scp> -Transpeptidases and Inactivation of These Enzymes by Carbapenems

Author

Mathilde Cordillot, Lionel Dubost, Jean-Luc Mainardi, Michel Arthur, Vincent Dubée, Sébastien Triboulet, Jean-Emmanuel Hugonnet, and Arul Marie

Subjects
Ertapenem, Carbapenem, Imipenem, Antitubercular Agents, Gene Expression, Peptidoglycan, beta-Lactams, beta-Lactamases, Microbiology, Mycobacterium tuberculosis, Acylation, chemistry.chemical_compound, Bacterial Proteins, Cell Wall, polycyclic compounds, Escherichia coli, medicine, Pharmacology (medical), Mechanisms of Action: Physiological Effects, Beta-Lactamase Inhibitors, Clavulanic Acid, Enzyme Assays, Pharmacology, biology, Doripenem, Meropenem, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Recombinant Proteins, Kinetics, Infectious Diseases, Carbapenems, chemistry, Biochemistry, Peptidyl Transferases, Thienamycins, beta-Lactamase Inhibitors, medicine.drug
Abstract

The Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by l , d -transpeptidases (LDTs), which are efficiently inactivated by a single β-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the β-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five l , d -transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), β-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the cross-linking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli . Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the β-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.

Published
2013
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28. Chemical shift perturbations induced by the acylation of Enterococcus faecium l,d-transpeptidase catalytic cysteine with ertapenem

Author

Jean-Pierre Simorre, Vincent Dubée, Lauriane Lecoq, Sébastien Triboulet, Catherine M. Bougault, Michel Arthur, and Jean-Emmanuel Hugonnet

Subjects
Ertapenem, Carbapenem, Acylation, Enterococcus faecium, Molecular Sequence Data, beta-Lactams, Biochemistry, Microbiology, Mycobacterium tuberculosis, chemistry.chemical_compound, Structural Biology, Catalytic Domain, polycyclic compounds, medicine, Amino Acid Sequence, Cysteine, Nuclear Magnetic Resonance, Biomolecular, chemistry.chemical_classification, biology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Enzyme, chemistry, Peptidyl Transferases, Peptidoglycan, Bacteria, medicine.drug
Abstract

Penicillin-binding proteins were long considered as the only peptidoglycan cross-linking enzymes and one of the main targets of β-lactam antibiotics. A new class of transpeptidases, the l,d-transpeptidases, has emerged in the last decade. In most Gram-negative and Gram-positive bacteria, these enzymes generally have nonessential roles in peptidoglycan synthesis. In some clostridiae and mycobacteria, such as Mycobacterium tuberculosis, they are nevertheless responsible for the major peptidoglycan cross-linking pathway. l,d-Transpeptidases are thus considered as appealing new targets for the development of innovative therapeutic approaches. Carbapenems are currently investigated in this perspective as they are active on extensively drug-resistant M. tuberculosis and represent the only β-lactam class inhibiting l,d-transpeptidases. The molecular basis of the enzyme selectivity for carbapenems nevertheless remains an open question. Here we present the backbone and side-chain 1H, 13C, 15N NMR assignments of the catalytic domain of Enterococcus faecium l,d-transpeptidase before and after acylation with the carbapenem ertapenem, as a prerequisite for further structural and functional studies.

Published
2013
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29. Structure of Enterococcus faecium<scp>l</scp>,<scp>d</scp>-Transpeptidase Acylated by Ertapenem Provides Insight into the Inactivation Mechanism

Author

Jean-Emmanuel Hugonnet, Sébastien Triboulet, Catherine M. Bougault, Lauriane Lecoq, Jean-Pierre Simorre, Michel Arthur, and Vincent Dubée

Subjects
Ertapenem, Models, Molecular, Carbapenem, Protein Conformation, Stereochemistry, Enterococcus faecium, Biology, beta-Lactams, Biochemistry, Article, Bacterial cell structure, Acylation, chemistry.chemical_compound, polycyclic compounds, medicine, Humans, Gram-Positive Bacterial Infections, chemistry.chemical_classification, Acetylation, General Medicine, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Anti-Bacterial Agents, Enzyme, Enterococcus, chemistry, Peptidyl Transferases, bacteria, Molecular Medicine, Peptidoglycan, medicine.drug
Abstract

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a biopolymer highly cross-linked through d,d-transpeptidation. Peptidoglycan cross-linking is catalyzed by penicillin-binding proteins (PBPs) that are the essential target of β-lactam antibiotics. PBPs are functionally replaced by l,d-transpeptidases (Ldts) in ampicillin-resistant mutants of Enterococcus faecium and in wild-type Mycobacterium tuberculosis. Ldts are inhibited in vivo by a single class of β-lactams, the carbapenems, which act as a suicide substrate. We present here the first structure of a carbapenem-acylated l,d-transpeptidase, E. faecium Ldtfm acylated by ertapenem, which revealed key contacts between the carbapenem core and residues of the catalytic cavity of the enzyme. Significant reorganization of the antibiotic conformation occurs upon enzyme acylation. These results, together with the analysis of protein-to-carbapenem proton transfers, provide new insights into the mechanism of Ldt acylation by carbapenems.

Published
2013
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30. Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

Author

Christiane Bouchier, Jean-Emmanuel Hugonnet, Dominique Mengin-Lecreulx, Alejandro Monton, Carole Veckerlé, Tanneke den Blaauwen, Louis B. Rice, Michael S. Van Nieuwenhze, E. Carbonnelle, V. Brun Yves, Kuyek Tu, Michel Arthur, Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Bacterial Cell Biology Department [Amsterdam], University of Amsterdam [Amsterdam] (UvA), Indiana University, Indiana University System, Brown University, Bacterial Cell Biology & Physiology (SILS, FNWI), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers ( CRC ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -École pratique des hautes études ( EPHE ) -Université Paris Diderot - Paris 7 ( UPD7 ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), University of Amsterdam [Amsterdam] ( UvA ), Université Paris Diderot - Paris 7 ( UPD7 ) -École pratique des hautes études ( EPHE ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Peptidyl transferase, Multidrug tolerance, QH301-705.5, β-lactam, Science, infectious disease, [SDV]Life Sciences [q-bio], 030106 microbiology, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Bacterial cell structure, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, D-transpeptidase, medicine, PBP5, Biology (General), Guanosine pentaphosphate, Escherichia coli, chemistry.chemical_classification, (p)ppGpp, [ SDV ] Life Sciences [q-bio], General Immunology and Microbiology, biology, General Neuroscience, microbiology, E. coli, L,D-transpeptidase, mecillinam, General Medicine, 3. Good health, 030104 developmental biology, Enzyme, PBP1b, Biochemistry, chemistry, biology.protein, Medicine, Peptidoglycan, Alarmone
Abstract

International audience; The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB.

Published
2016
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31. Author response: Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

Author

Tanneke den Blaauwen, Dominique Mengin-Lecreulx, Michael S. Van Nieuwenhze, Jean-Emmanuel Hugonnet, Christiane Bouchier, Michel Arthur, Louis B. Rice, V. Brun Yves, Alejandro Monton, Carole Veckerlé, Kuyek Tu, and E. Carbonnelle

Subjects
0301 basic medicine, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Chemistry, medicine, Lactam, Peptidoglycan, medicine.disease_cause, Escherichia coli, Microbiology
Published
2016
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32. Inhibition of β-lactamases of mycobacteria by avibactam and clavulanate

Author

Clément Ourghanlian, Jean-Emmanuel Hugonnet, Fabrice Compain, Michel Arthur, Daria Soroka, Vincent Dubée, Jean-Luc Mainardi, and Marion Fichini

Subjects
0301 basic medicine, Microbiology (medical), medicine.drug_class, Avibactam, 030106 microbiology, Mycobacterium abscessus, medicine.disease_cause, Monoclonal antibody, Mass Spectrometry, beta-Lactamases, Microbiology, Mycobacterium, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, Enzyme Stability, medicine, Escherichia coli, Pharmacology (medical), Enzyme kinetics, Clavulanic Acid, Pharmacology, biology, Chemistry, Amoxicillin, biology.organism_classification, 030104 developmental biology, Infectious Diseases, Amino Acid Substitution, Mutant Proteins, Antibacterial activity, beta-Lactamase Inhibitors, Azabicyclo Compounds, medicine.drug
Abstract

Objectives Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A β-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of β-lactamases from slowly and rapidly growing mycobacteria. Methods Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on β-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the β-lactamases in Escherichia coli . Results The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the β-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa. Conclusions G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the β-lactamase inhibitors, but not to both of them.

Published
2016

33. Kinetic Analysis of Enterococcus faecium <scp>l</scp> , <scp>d</scp> -Transpeptidase Inactivation by Carbapenems

Author

Louis B. Rice, Hélène Fief, Jean-Emmanuel Hugonnet, Matthieu Sollogoub, Jean-Luc Mainardi, Vincent Dubée, Sébastien Triboulet, Mélanie Etheve-Quelquejeu, Michel Arthur, and Laurent Gutmann

Subjects
Stereochemistry, Enterococcus faecium, Mutant, Kinetics, Kinetic analysis, 03 medical and health sciences, Hydrolysis, Amp resistance, polycyclic compounds, Pharmacology (medical), 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, biology, 030306 microbiology, Chemistry, Biosynthesis, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, 3. Good health, Enzyme Activation, Infectious Diseases, Enzyme, Carbapenems, Biochemistry, chemistry, Peptidyl Transferases, bacteria
Abstract

Bypass of classical penicillin-binding proteins by the l , d -transpeptidase of Enterococcus faecium (Ldt fm ) leads to high-level ampicillin resistance in E. faecium mutants, whereas carbapenems remain the lone highly active β-lactams. Kinetics of Ldt fm inactivation was determined for four commercial carbapenems and a derivative obtained by introducing a minimal ethyl group at position 2. We show that the bulky side chains of commercial carbapenems have both positive and negative effects in preventing hydrolysis of the acyl enzyme and impairing drug binding.

Published
2012
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34. Dynamics Induced by β-Lactam Antibiotics in the Active Site of Bacillus subtilis l,d-Transpeptidase

Author

Ombeline Pessey, Jean-Pierre Simorre, Catherine M. Bougault, Jean-Emmanuel Hugonnet, Michel Arthur, Carole Veckerlé, and Lauriane Lecoq

Subjects
Steric effects, Stereochemistry, Bacillus subtilis, Molecular Dynamics Simulation, beta-Lactams, Substrate Specificity, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Catalytic Domain, polycyclic compounds, Transferase, Cysteine, Sulfhydryl Compounds, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, chemistry.chemical_classification, biology, Active site, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Anti-Bacterial Agents, Enzyme, chemistry, Nitrobenzoates, Peptidyl Transferases, biology.protein, Lactam, Peptidoglycan
Abstract

Summary β-lactams inhibit peptidoglycan polymerization by acting as suicide substrates of essential d,d-transpeptidases. Bypass of these enzymes by unrelated l,d-transpeptidases results in β-lactam resistance, although carbapenems remain unexpectedly active. To gain insight into carbapenem specificity of l,d-transpeptidases (Ldts), we solved the nuclear magnetic resonance (NMR) structures of apo and imipenem-acylated Bacillus subtilis Ldt and show that the cysteine nucleophile is present as a neutral imidazole-sulfhydryl pair in the substrate-free enzyme. NMR relaxation dispersion does not reveal any preexisting conformational exchange in the apoenzyme, and change in flexibility is not observed upon noncovalent binding of β-lactams (K D > 37.5 mM). In contrast, covalent modification of active cysteine by both carbapenems and 2-nitro-5-thiobenzoate induces backbone flexibility that does not result from disruption of the imidazole-sulfhydryl proton interaction or steric hindrance. The chemical step of the reaction determines enzyme specificity since no differences in drug affinity were observed.

Published
2012
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35. Inactivation of Mycobacterium tuberculosis L,D-Transpeptidase Ldt(Mt1) by Carbapenems and Cephalosporins

Author

Mélanie Etheve-Quelquejeu, Jean-Emmanuel Hugonnet, Arul Marie, Michel Arthur, Vincent Dubée, Sébastien Triboulet, Laurent Gutmann, Jean-Luc Mainardi, and Lionel Dubost

Subjects
Pharmacology, 0303 health sciences, Imipenem, Carbapenem, Cefotaxime, 030306 microbiology, medicine.drug_class, Stereochemistry, Cephalosporin, Meropenem, Acylation, 03 medical and health sciences, chemistry.chemical_compound, Infectious Diseases, chemistry, medicine, Doripenem, Pharmacology (medical), Ertapenem, 030304 developmental biology, medicine.drug
Abstract

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l , d -transpeptidases that replace 4→3 cross-links formed by the d , d -transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l , d -transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis . Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l , d -transpeptidase Ldt Mt1 by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt Mt1 , which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt Mt1 , with a binding rate constant of 0.08 μM −1 min −1 . The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (−1 ) in comparison to the acylation rate constant (3.1 min −1 ). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10 −3 min −1 . Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt Mt1 inactivator. Cephalosporins also formed covalent adducts with Ldt Mt1 , although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis .

Published
2012
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36. Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

Author

Nathalie Josseaume, Lionel Dubost, Claudine Mayer, Jean-Marc Valery, Jean-Emmanuel Hugonnet, Stéphane Mesnage, Mélanie Etheve-Quelquejeu, Maryline Chemama, Michel Arthur, Antoine P. Maillard, Matthieu Fonvielle, Patricia Busca, Arul Marie, Régis Villet, Structure bactérienne et résistance aux antibiotiques (CRC - Inserm U1138), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de chimie et biochimie des substances naturelles, Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Molécules de Communication et Adaptation des Micro-Organismes (MCAM), Centre National de la Recherche Scientifique (CNRS)-Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Krebs Inst, University of Sheffield [Sheffield], Université Paris Diderot - Paris 7 (UPD7), Synthèse, Structure et Fonction de Molécules Bioactives (SSFMB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École Pratique des Hautes Études (EPHE), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Chimie et Biochimie des Substances Naturelles, Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Base pair, [SDV]Life Sciences [q-bio], Molecular Sequence Data, RNA, Transfer, Ala, Peptidoglycan, Wobble base pair, MESH: Base Sequence, Biology, 03 medical and health sciences, chemistry.chemical_compound, MESH: Cell Wall, Cell Wall, Genetics, Transferase, Nucleotide, Peptide Synthases, MESH: RNA, Transfer, Ala, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, MESH: Molecular Sequence Data, Bacteria, Base Sequence, Nucleic Acid Enzymes, MESH: Peptidoglycan, 030306 microbiology, Mutagenesis, MESH: Peptide Synthases, Amino acid, MESH: Bacteria, MESH: Mutagenesis, Site-Directed, chemistry, Biochemistry, Transfer RNA, Mutagenesis, Site-Directed, MESH: Uridine Diphosphate N-Acetylmuramic Acid, MESH: Models, Molecular
Abstract

International audience; The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

Published
2007
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37. Irreversible Inhibition of the Mycobacterium tuberculosis β-Lactamase by Clavulanate

Author

Jean Emmanuel Hugonnet and John S. Blanchard

Subjects
Spectrometry, Mass, Electrospray Ionization, Imipenem, medicine.drug_class, Antibiotics, Cephalosporinase activity, Biology, Biochemistry, Tazobactam, Meropenem, Article, Microbiology, Mycobacterium tuberculosis, polycyclic compounds, medicine, Nitrocefin, Enzyme Inhibitors, Clavulanic Acid, Fourier Analysis, Hydrolysis, Sulbactam, biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses, biology.organism_classification, bacteria, beta-Lactamase Inhibitors, medicine.drug
Abstract

Members of the beta-lactam class of antibiotics, which inhibit the bacterial d,d-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A beta-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum beta-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved beta-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.

Published
2007
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38. Hydrolysis of clavulanate by Mycobacterium tuberculosis β-lactamase BlaC harboring a canonical SDN motif

Author

David Barros, Daria Soroka, Sébastien Triboulet, Michel Arthur, Lluis Ballell, Jean-Emmanuel Hugonnet, Jean-Luc Mainardi, Herman van Tilbeurgh, Fabrice Compain, Vincent Dubée, Inès Li de la Sierra-Gallay, Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Diseases of the Developing World [Tres Cantos, Madrid] (DDW), Tres Cantos Open Lab Foundation [London, UK] (TCOLF)-GlaxoSmithKline [Headquarters, London, UK] (GSK), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Diseases of the Developing World, GlaxoSmithKline, Madrid, ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers ( CRC ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -École pratique des hautes études ( EPHE ) -Université Paris Diderot - Paris 7 ( UPD7 ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Fonction et Architecture des Assemblages Macromoléculaires ( FAAM ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), ANR-10-INSB-05-01,FRISBI,French Infrastructure for Integrated Structural Biology, Université Paris Diderot - Paris 7 ( UPD7 ) -École pratique des hautes études ( EPHE ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), and Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS )

Subjects
Decarboxylation, [SDV]Life Sciences [q-bio], Microbial Sensitivity Tests, Biology, Mycobacterium abscessus, beta-Lactams, beta-Lactam Resistance, beta-Lactamases, Microbiology, Acylation, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, Hydrolysis, Mechanisms of Resistance, Clavulanic acid, Hydrolase, medicine, Pharmacology (medical), Clavulanic Acid, 030304 developmental biology, Pharmacology, 0303 health sciences, [ SDV ] Life Sciences [q-bio], 030306 microbiology, Faropenem, biology.organism_classification, 3. Good health, Infectious Diseases, Biochemistry, chemistry, medicine.drug
Abstract

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase Bla MAb of Mycobacterium abscessus , which efficiently hydrolyzes clavulanate. Comparison of Bla MAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G 132 N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10 4 -fold increase in k cat (0.41 s −1 ), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G 132 N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G 132 N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

Published
2015
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39. Acyl acceptor recognition by Enterococcus faecium l,d-transpeptidase Ldtfm

Author

Jean-Emmanuel Hugonnet, Sébastien Triboulet, Cédric Laguri, Jean-Pierre Simorre, Michel Arthur, Catherine M. Bougault, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), U1138 equipe 12, Centre de Recherche Saint-Antoine (CR Saint-Antoine), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Laboratoire de Chimie et Biologie des Métaux ( LCBM - UMR 5249 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Centre de Recherche Saint-Antoine ( CR Saint-Antoine ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut de biologie structurale ( IBS - UMR 5075 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -IFR58-Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris Descartes - Paris 5 ( UPD5 ), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Ertapenem, Models, Molecular, Stereochemistry, Acylation, Enterococcus faecium, Peptidoglycan, beta-Lactams, Microbiology, Article, Substrate Specificity, chemistry.chemical_compound, Bacterial Proteins, Catalytic Domain, Binding site, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Binding Sites, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Active site, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Acceptor, Anti-Bacterial Agents, 3. Good health, Molecular Docking Simulation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, chemistry, Docking (molecular), Peptidyl Transferases, Mutagenesis, Site-Directed, biology.protein, Cysteine, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

Summary In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by l,d-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l,d-transpeptidase Ldtfm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldtfm that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldtfm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams.

Published
2015

40. Mutation landscape of acquired cross-resistance to glycopeptide and β-lactam antibiotics in Enterococcus faecium

Author

Nathalie Josseaume, Jean-Emmanuel Hugonnet, Jean-Luc Mainardi, Louis B. Rice, Mélanie Cortes, Michel Arthur, Christiane Bouchier, Vincent Dubée, and Emmanuelle Sacco

Subjects
Pharmacology, Genetics, Silent mutation, Mutation rate, biology, Mutant, Enterococcus faecium, Glycopeptides, biology.organism_classification, beta-Lactams, Glycopeptide, Anti-Bacterial Agents, Infectious Diseases, Mechanisms of Resistance, Drug Resistance, Multiple, Bacterial, Mutation, Pharmacology (medical), Transversion, Gene, Neutral mutation
Abstract

Bypass of the d , d -transpeptidase activity of penicillin-binding proteins by an l , d -transpeptidase (Ldt fm ) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldt fm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ 2 = 5.0; P < 0.05) and silent mutations were underrepresented (χ 2 = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldt fm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l , d -transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism.

Published
2015

41. Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance in Enterococcus faecalis

Author

Jean-Emmanuel Hugonnet, Jean-Paul Brouard, Lionnel Dubost, Michel Arthur, Nathalie Josseaume, Heidi Segal, Ana Arbeloa, Dominique Mengin-Lecreulx, and Laurent Gutmann

Subjects
Penicillin binding proteins, Physiology and Metabolism, Mutant, Oligosaccharides, Microbial Sensitivity Tests, Peptidoglycan, Muramoylpentapeptide Carboxypeptidase, Biology, Microbiology, beta-Lactam Resistance, Enterococcus faecalis, chemistry.chemical_compound, Bacterial Proteins, Glycosyltransferase, Penicillin-Binding Proteins, Enzyme Inhibitors, Molecular Biology, Glycosyltransferases, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Complementation, Hexosyltransferases, chemistry, Biochemistry, Peptidyl Transferases, biology.protein, Heterologous expression, Carrier Proteins, Gene Deletion, Enterococcus faecium
Abstract

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis , class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 ( ponA , pbpF , and pbpZ ). Among mutants with single or double deletions, only JH2-2 Δ ponA Δ pbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus . Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis , and heterospecific complementation was observed with an ortholog from E. faecium . Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Published
2004
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42. The CroRS Two-Component Regulatory System Is Requiredfor Intrinsic β-Lactam Resistance in Enterococcusfaecalis

Author

Jean-Emmanuel Hugonnet, Lionnel Dubost, Michel Arthur, Yannick Comenge, Jean-Paul Brouard, Richard Quintiliani, and Ling Li

Subjects
Cytoplasm, Molecular Sequence Data, Mutant, Peptidoglycan, Biology, Microbiology, beta-Lactam Resistance, Enterococcus faecalis, chemistry.chemical_compound, Bacterial Proteins, medicine, RNA, Messenger, Promoter Regions, Genetic, Molecular Biology, Gene, Regulator gene, Ceftriaxone, Chromosome Mapping, biology.organism_classification, Two-component regulatory system, Anti-Bacterial Agents, Response regulator, Carbohydrate Sequence, chemistry, Gene Deletion, Signal Transduction, medicine.drug
Abstract

Enterococcus faecalis produces a specific penicillin-binding protein (PBP5) that mediates high-level resistance to the cephalosporin class ofβ -lactam antibiotics. Deletion of a locus encoding a previously uncharacterized two-component regulatory system of E. faecalis ( croRS ) led to a 4,000-fold reduction in the MIC of the expanded-spectrum cephalosporin ceftriaxone. The cytoplasmic domain of the sensor kinase (CroS) was purified and shown to catalyze ATP-dependent autophosphorylation followed by transfer of the phosphate to the mated response regulator (CroR). The croR and croS genes were cotranscribed from a promoter ( croRp ) located in the rrnC-croR intergenic region. A putative seryl-tRNA synthetase gene ( serS ) located immediately downstream from croS did not appear to be a target of CroRS regulation or to play a role in ceftriaxone resistance. A plasmid-borne croRp-lacZ fusion was trans -activated by the CroRS system in response to the presence of ceftriaxone in the culture medium. The fusion was also induced by representatives of other classes of β-lactam antibiotics and by inhibitors of early and late steps of peptidoglycan synthesis. The croRS null mutant produced PBP5, and expression of an additional copy of pbp5 under the control of a heterologous promoter did not restore ceftriaxone resistance. Deletion of croRS was not associated with any defect in the synthesis of the nucleotide precursor UDP-MurNAc-pentapeptide or of the d -Ala 4 → l -Ala- l -Ala-Lys 3 peptidoglycan cross-bridge. Thus, the croRS mutant was susceptible to ceftriaxone despite the production of PBP5 and the synthesis of wild-type peptidoglycan precursors. These observations constitute the first description of regulatory genes essential for PBP5-mediated β-lactam resistance in enterococci.

Published
2003
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43. Synthesis of the l-Alanyl-l-alanine Cross-bridge of Enterococcus faecalis Peptidoglycan

Author

Dominique Mengin-Lecreulx, Anatoly Severin, Nathalie Josseaume, David M. Shlaes, Ahmed Bouhss, Keiko Tabei, Jean van Heijenoort, Michel Arthur, and Jean-Emmanuel Hugonnet

Subjects
chemistry.chemical_classification, Alanine, biology, Strain (chemistry), Mutant, Dipeptides, Peptidoglycan, Cell Biology, biology.organism_classification, Biochemistry, Enterococcus faecalis, chemistry.chemical_compound, Residue (chemistry), Enzyme, chemistry, Side chain, Molecular Biology
Abstract

The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(2) side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-d-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(2))-d-Ala-d-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two l-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the dd-transpeptidases in the cross-linking reaction. However, the bppA2 deletion impaired expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.

Published
2002
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44. Beta-Lactamase inhibition by avibactam in Mycobacterium abscessus

Author

Jean-Luc Mainardi, Mélanie Cortes, Jean-Louis Herrmann, Audrey Bernut, Jean-Emmanuel Hugonnet, Laurent Kremer, Michel Arthur, Vincent Dubée, Jean-Louis Gaillard, Delphine Dorchêne, Tiffany Lesne, Laurent Gutmann, Anne-Laure Lefebvre, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), LPHI - Laboratory of Pathogen Host Interactions (LPHI), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Hôpital Raymond Poincaré [AP-HP], Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Dynamique des interactions membranaires normales et pathologiques (DIMNP), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Dubee, Vincent

Subjects
Microbiology (medical), Imipenem, Avibactam, Ceftazidime, Mycobacterium abscessus, beta-Lactamases, Microbiology, Cell Line, Mycobacterium, cystic fibrosis, chemistry.chemical_compound, Non-tuberculous mycobacteria, medicine, Animals, Humans, Beta-lactams antibiotic, Pharmacology (medical), Cefoxitin, Beta-Lactamase Inhibitors, Zebrafish, Pharmacology, Mycobacterium Infections, biology, business.industry, Macrophages, Amoxicillin, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, bacterial infections and mycoses, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Anti-Bacterial Agents, Antibiotic discovery, Infectious Diseases, Treatment Outcome, chemistry, Models, Animal, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, business, beta-Lactamase Inhibitors, Azabicyclo Compounds, Mycobacterium abscessus complex, medicine.drug
Abstract

Objectives Two β-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum β-lactamase, BlaMab, indicating that the combination of β-lactams with a BlaMab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of BlaMab production on the efficacy of β-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of β-lactams intracellularly and in an animal model. Methods We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-β-lactam β-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding BlaMab to assess the extent of BlaMab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection. Results We showed that avibactam efficiently inactivated BlaMab via the reversible formation of a covalent adduct. An inhibition of BlaMab by avibactam was observed in both infected macrophages and zebrafish. Conclusions Our data identify avibactam 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.

Published
2014
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46. Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity

Author

Louis B. Rice, Jean-Luc Mainardi, Arul Marie, Sébastien Triboulet, Mélanie Etheve-Quelquejeu, Vincent Dubée, Jean-Pierre Simorre, Michel Arthur, Laurent Gutmann, Lionel Dubost, Catherine M. Bougault, Jean-Emmanuel Hugonnet, Lauriane Lecoq, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Bacterial Diseases, Imipenem, MESH: Peptidyl Transferases, Acylation, Enterococcus faecium, lcsh:Medicine, Pathogenesis, Substrate Specificity, MESH: Recombinant Proteins, chemistry.chemical_compound, MESH: Structure-Activity Relationship, Tetrahedral carbonyl addition compound, MESH : Structure-Activity Relationship, MESH : Ampicillin, polycyclic compounds, MESH : Bacterial Proteins, MESH: Enterococcus faecium, Bacterial Physiology, MESH : Enterococcus faecium, lcsh:Science, MESH: Bacterial Proteins, MESH: Imipenem, 0303 health sciences, Multidisciplinary, MESH : Substrate Specificity, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Kinetics, Multi-Drug-Resistant Tuberculosis, Ceftriaxone, Streptococci, Recombinant Proteins, Bacterial Pathogens, Bacterial Biochemistry, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Infectious Diseases, Biochemistry, Lactam, Medicine, MESH : Kinetics, Antibacterial activity, Ertapenem, MESH : Imipenem, Research Article, medicine.drug, Drugs and Devices, MESH : Recombinant Proteins, Stereochemistry, Microbiology, beta-Lactam Resistance, Structure-Activity Relationship, 03 medical and health sciences, MESH : beta-Lactam Resistance, Bacterial Proteins, MESH : Peptidyl Transferases, medicine, Tuberculosis, Pharmacokinetics, Biology, Microbial Pathogens, MESH : Acylation, 030304 developmental biology, Gram Positive, MESH: Acylation, 030306 microbiology, lcsh:R, Bacteriology, biochemical phenomena, metabolism, and nutrition, MESH: beta-Lactam Resistance, MESH: Ceftriaxone, Kinetics, chemistry, Peptidyl Transferases, Ampicillin, lcsh:Q, MESH: Substrate Specificity, Peptidoglycan, MESH: Ampicillin, Penam, MESH : Ceftriaxone, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

International audience; 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.

Published
2013
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47. Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm)

Author

Jure Stojan, Matjaž Brvar, Ana Kroflič, Victoria J. Savage, Jean-Luc Mainardi, Anamarija Zega, Vincent Dubbée, Alex J. O'Neill, Tom Solmajer, Didier Blanot, Andrej Perdih, Michel Arthur, Jean-Emmanuel Hugonnet, Marija Bešter-Rogač, Sophie Magnet, and Veronika Škedelj

Subjects
Pharmacology, chemistry.chemical_classification, Biotin carboxylase, Models, Molecular, DNA ligase, Virtual screening, Dose-Response Relationship, Drug, Molecular Structure, Stereochemistry, Organic Chemistry, D-Aspartic Acid, Enterococcus faecium, Isothermal titration calorimetry, General Medicine, Ligand (biochemistry), Ligases, Structure-Activity Relationship, Enzyme, chemistry, Biochemistry, Drug Discovery, Binding site, Pharmacophore, Enzyme Inhibitors
Abstract

The d -aspartate ligase of Enterococcus faecium (Asl fm ) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Asl fm , we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12 , inhibits Asl fm with a K i value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Asl fm inhibitors with 7-amino-9 H -purine ( 18 ) and 7-amino-1 H -pyrazolo[3,4- d ]pyrimidine ( 30 and 34 ) scaffolds, and also with K i values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6 , 12 , 18 , 30 and 34 represent the first inhibitors of Asl fm reported to date, and are an important step forward in combating infections due to E. faecium .

Published
2013

48. l,d-Transpeptidase (Enterococcus)

Author

Michel Arthur, Jean-Luc Mainardi, and Jean-Emmanuel Hugonnet

Subjects
chemistry.chemical_classification, biology, education, Proteolytic enzymes, biology.organism_classification, humanities, Structural chemistry, body regions, surgical procedures, operative, Enzyme, chemistry, Biochemistry, Enterococcus, health care economics and organizations
Abstract

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 552 is l , d -Transpeptidase ( Enterococcus ).

Published
2013
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49. Contributors

Author

Catherine Anne Abbott, Carmela R. Abraham, Hideki Adachi, Osao Adachi, Zach Adam, Michael W.W. Adams, Michael J. Adang, Ibrahim M. Adham, Patrizia Aducci, David A. Agard, Alexey A. Agranovsky, Tetsuya Akamatsu, Yoshinori Akiyama, Reidar Albrechtsen, Alí Alejo, Sean M. Amberg, Alexander Y. Amerik, Piti Amparyup, Felipe Andrade, Germán Andrés, Daniel M. Andrews, Robert K. Andrews, Toni M. Antalis, Colin S. Anthony, Naoya Aoki, Suneel S. Apte, Kazunari Arima, Gérard Arlaud, Raghuvir Krishnaswamy Arni, Pascal Arnoux, Nathan N. Aronson, Michel Arthur, Yasuhisa Asano, Paolo Ascenzi, Marina T. Assakura, David S. Auld, Veridiana de Melo Rodrigues Ávila, Francesc X. Avilés, William M. Awad, Anand K. Bachhawat, Shan Bai, Teaster T. Baird, S. Paul Bajaj, Susan C. Baker, Agnieszka Banbula, Alan J. Barrett, Jemima Barrowman, John D. Bartlett, Jörg W. Bartsch, Nikola Baschuk, Isolda P. Baskova, Jyotsna Batra, Karl Bauer, Ulrich Baumann, Wolfgang Baumeister, Cédric Bauvois, Alex Bayés, Anne Beauvais, Christoph Becker-Pauly, Tadhg P. Begley, Miklós Békés, Robert Belas, Daniah Beleford, Teruhiko Beppu, Ernst M. Bergmann, Bruno A. Bernard, Dominique Bernard, Michael C. Berndt, Giovanna Berruti, Colin Berry, Greg P. Bertenshaw, Christian Betzel, Chetana Bhaskarla, Manoj Bhosale, Gabriele Bierbaum, B. Bjarnason Jón, Michael Blaber, Michael J. Blackman, Alexander Blinkovsky, Jef D. Boeke, Matthew Bogyo, Stefan Bohn, Guy Boileau, Mike Boland, Tové C. Bolken, Judith S. Bond, Jan Bondeson, Javier Bordallo, Claudia Borelli, Tiago O. Botelho, Richard R. Bott, David G. Bourne, Niels Bovenschen, Ralph A. Bradshaw, Klaus Breddam, Keith Brew, Paul J. Brindley, Diane L. Brinkman, Collette Britton, Jeff R. Broadbent, Anne Broadhurst, Dieter Brómme, Murray Broom, Jeremy S. Brown, Mark A. Brown, Iris Bruchhaus, Barbara A. Burleigh, Kristin E. Burns, James F. Burrows, Michael J. Butler, David J. Buttle, Chelsea M. Byrd, Tony Byun, Sandrine Cadel, Conor R. Caffrey, Santiago Cal, Javier Caldentey, Thomas Candela, Clemente Capasso, Daniel R. Capriogilio, Vincenzo Carginale, Adriana Karaoglanovic Carmona, Vern B. Carruthers, Francis J. Castellino, Joseph J. Catanese, Bruce Caterson, George H. Caughey, Naimh X. Cawley, Tim E. Cawston, Juan José Cazzulo, Jijie Chai, Karl X. Chai, Olga Meiri Chaim, L.S. Chang, Julie Chao, Marie-Pierre Chapot-Chartier, Jean-Louis Charli, Paulette Charlier, Karen J. Chave, Jian-Min Chen, Jinq-May Chen, Li-Mei Chen, Ya-Wen Chen, Yu-Yen Chen, Bernard Chevrier, Jean-François Chich, Jeremy Chien, Suneeta Chimalapati, Ki Joon Cho, Kwan Yong Choi, Woei-Jer Chuang, Chin Ha Chung, Ivy Yeuk Wah Chung, Christine Clamagirand, Ian M. Clark, Adrian K. Clarke, Nicola E. Clarke, Steven Gerard Clarke, Philippe Clauziat, Judith A. Clements, Catherine Coffinier, Paul Cohen, Alain Colige, Anne Collignon, Sean D. Colloms, Andreas Conzelmann, Graham H. Coombs, Jakki C. Cooney, Jonathan B. Cooper, Max D. Cooper, Nikki A. Copeland, Graeme S. Cottrell, Joseph T. Coyle, Charles S. Craik, John W.M. Creemers, Daniela Cretu, Jenifer Croce, Keith J. Cross, Rosario Cueva, Sheng Cui, Luis Cunha, Simon Cutting, Christophe d’Enfert, Hugues D’Orchymont, Björn Dahlbäck, Shujia Dai, Ross E. Dalbey, John P. Dalton, Pam M. Dando, R.M. Daniel, Sergei M. Danilov, Donna E. Davies, Heloisa S. De Araujo, Teresa De los Santos, Viviana de Luca, Ingrid De Meester, Ana Karina de Oliveira, Eduardo Brandt de Oliveira, Pedro Lagerblad De Oliveira, Sarah de Vos, Jeroen Declercq, Wim Declercq, Ala-Eddine Deghmane, Niek Dekker, Sonia Del Prete, Marina Del Rosal, Bernard Delmas, Robert DeLotto, Ilya V. Demidyuk, Mark R. Denison, Jan M. Deussing, Lakshmi A. Devi, Eleftherios P. Diamandis, Isabel Diaz, Araceli Díaz-Perales, Bauke W. Dijkstra, Yan Ding, Jack E. Dixon, Johannes Dodt, Terje Dokland, Iztok Dolenc, Ningzheng Dong, Tran Cat Dong, Ying Dong, Mitesh Dongre, Mark Donovan, Timothy M. Dore, Loretta Dorstyn, Hong Dou, Zhicheng Dou, Annette M. Dougall, Marcin Drag, Edward G. Dudley, Ben M. Dunn, Bruno Dupuy, Maria Conceicāo Duque-Magalhāes, M. Asunción Durá, Yves Eeckhout, Vincent Eijsink, Arthur Z. Eisen, Azza Eissa, Sandra Eklund, Ziad M. Eletr, Vincent Ellis, Wolfgang Engel, Ervin G. Erdös, Teresa Escalante, David A. Estell, Michael Etscheid, Herbert J. Evans, Roger D. Everett, Alex C. Faesen, Falk Fahrenholz, Miriam Fanjul-Fernández, Christopher J. Farady, Georges Feller, Hong Feng, Kurt M. Fenster, Claude Férec, Silvia Ferrari, Barbara Fingleton, Jed F. Fisher, Paula M. Fives-Taylor, Loren G. Fong, F. Forneris, Brian M. Forster, Friedrich Forster, Simon J. Foster, Thierry Foulon, Stephen I. Foundling, Jay William Fox, Bruno Franzetti, Alejandra P. Frasch, Hudson H. Freeze, Jean-Marie Frère, Teryl K. Frey, Beate Fricke, Lloyd D. Fricker, Rafael Fridman, Christopher J. Froelich, Camilla Fröhlich, Hsueh-Liang Fu, Cynthia N. Fuhrmann, Satoshi Fujimura, Hiroshi Fujiwara, Jun Fukushima, Keiichi Fukuyama, Robert S. Fuller, Martin Fusek, Christine Gaboriaud, Christian Gache, Oleksandr Gakh, Peter Gal, Junjun Gao, Adolfo García-Sastre, Donald L. Gardiner, John A. Gatehouse, G.M. Gaucher, Francis Gauthier, Jean-Marie Ghuysen, Wade Gibson, Jennifer Gillies, Elzbieta Glaser, Fabian Glaser, Michael H. Glickman, Peter Goettig, Colette Goffin, Eiichi Gohda, Alfred L. Goldberg, Daniel E. Goldberg, Gregory I. Goldberg, Nathan E. Goldfarb, F. Xavier Gomis-Rüth, B. Gopal, Alexander E. Gorbalenya, Stuart G. Gordon, Mark D. Gorrell, Friedrich Götz, Theodoros Goulas, Cécile Gouzy-Darmon, K. Govind, Lászlo Gráf, Robert R. Granados, Melissa Ann Gräwert, Douglas A. Gray, Thomas P. Graycar, Jonathan A. Green, Luiza Helena Gremski, Michael Groll, Tania Yu Gromova, P. Gros, Marvin J. Grubman, Amy M. Grunden, Ágústa Gudmundsdóttir, Micheline Guinand, Djamel Gully, Alla Gustchina, José María Gutiérrez, Byung Hak Ha, Jesper Z. Haeggström, James H. Hageman, Johanna Haiko, Stephan Hailfinger, Hans Michael Haitchi, Ji Seon Han, Chantal Hanquez, Minoru Harada, Ikuko Hara-Nishimura, Marianne Harboe, Torleif Härd, David A. Harris, Ulrich Hassiepen, Shoji Hata, Akira Hattori, Rong-Qiao He, Albert J.R. Heck, Dirk F. Hendricks, Bernhard Henrich, Patrick Henriet, Andrés Hernández-Arana, Irma Herrera-Camacho, Gerhard Heussipp, Toshihiko Hibino, P.M. Hicks, Bradley I. Hillman, B. Yukihiro Hiraoka, Jun Hiratake, Yohei Hizukuri, Heng-Chien Ho, Ngo Thi Hoa, Mark Hochstrasser, Kathryn M. Hodge, Theo Hofmann, Thomas Hohn, John R. Hoidal, Joachim-Volker Höltje, Koichi J. Homma, John F. Honek, Vivian Y.H. Hook, John D. Hooper, Nigel M. Hooper, Kazuo Hosoi, Christopher J. Howe, Dennis E. Hruby, James J.-D. Hseih, Chun-Chieh Hsu, Tony T. Huang, Tur-Fu Huang, Yoann Huet, Clare Hughes, Jean-Emmanuel Hugonnet, Adrienne L. Huston, Oumaïma Ibrahim-Granet, Eiji Ichishima, Yukio Ikehara, Tadashi Inagami, Jessica Ingram, R.E. Isaac, Grazia Isaya, Clara E. Isaza, Shin-ichi Ishii, Amandine Isnard, Kiyoshi Ito, Koreaki Ito, Yoshifumi Itoh, Xavier Iturrioz, Sadaaki Iwanaga, Ralph W. Jack, Mel C. Jackson, Michael N.G. James, Jiří Janata, Claire Janoir, Hanna Janska, Ken F. Jarrell, Mariusz Jaskolski, Sheila S. Jaswal, Ying Y. Jean, Dieter E. Jenne, Young Joo Jeon, Ping Jiang, John E. Johnson, Michael D. Johnson, James A. Johnston, Amanda Jones, Elizabeth W. Jones, Carine Joudiou, Luiz Juliano, Hea-Jin Jung, Ray Jupp, Todd F. Kagawa, Hubert Kalbacher, Yayoi Kamata, Shuichi Kaminogawa, Yoshiyuki Kamio, Makoto Kaneda, Sung Gyun Kang, Sung Hwan Kang, Mary Kania, Tomasz Kantyka, Nobuyuki Kanzawa, Abdulkarim Y. Karim, Takafumi Kasumi, Hiroaki Kataoka, Hardeep Kaur, Shun-Ichiro Kawabata, Mari Kawaguchi, John Kay, Murat Kaynar, Kenneth C. Keiler, R.M. Kelly, Nathaniel T. Kenton, Michael A. Kerr, Kristof Kersse, Jukka Kervinen, Benedikt M. Kessler, Efrat Kessler, Timo K. Khoronen, Simon Kidd, Marjolein Kikkert, Mogens Kilian, Do-Hyung Kim, Doyoun Kim, Eunice EunKyeong Kim, In Seop Kim, Jung-Gun Kim, Kyeong Kyu Kim, Kyung Hyun Kim, Matthew S. Kimber, Yukio Kimura, Heidrun Kirschke, Yoshiaki Kiso, Colin Kleanthous, Jürgen R. Klein, Michael Klemba, Beata Kmiec, Hideyuki Kobayashi, Hiroyuki Kodama, Gerald Koelsch, Jan Kok, P.E. Kolattukody, Fabrice A. Kolb, Harald Kolmar, Yumiko Komori, Jan Konvalinka, Brice Korkmaz, Sergey V. Kostrov, Hans-Georg Kräusslich, Gabi Krczal, Lawrence F. Kress, Magnüs Már Kristjánsson, Tomáš Kučera, Sayali S. Kukday, Hidehiko Kumagai, Sharad Kumar, Malika Kumarasiri, Takashi Kumazaki, Beate M. Kümmerer, Kouji Kuno, Markku Kurkinen, Eva Kutejová, Marie Kveiborg, Agnieszka Kwarciak, Liisa Laakkonen, Nikolaos E. Labrou, Gavin D. Laing, Gayle Lamppa, Thomas Langer, Richard A. Laursen, Richard A. Lawrenson, Matthew D. Layne, Bernard F. Le Bonniec, María C. Leal, Ronald M. Lechan, David H. Lee, Irene Lee, Jae Lee, Kye Joon Lee, Soohee Lee, Xiaobo Lei, Jonathan Leis, Ellen K. LeMosy, Thierry Lepage, Stephen H. Leppla, Adam Lesner, Ivan A.D. Lessard, Guy Lhomond, Huilin Li, Shu-Ming Li, Weiguo Li, Ta-Hsiu Liao, Robert C. Liddington, Toby Lieber, H.R. Lijnen, Christopher D. Lima, Chen-Yong Lin, Gang Lin, Ming T. Lin, Xinli Lin, Yee-Shin Lin, L.L. Lindsay, William N. Lipscomb, John W. Little, Ching-Chuan Liu, Chuan-ju Liu, Mark O. Lively, Nurit Livnat-Levanon, Per O. Ljungdahl, Catherine Llorens-Cortes, Peter Lobel, Y. Peng Loh, Jouko Lohi, G.P. Lomonossoff, Yvan Looze, Carlos López-Otin, Landys Lopez-Quezada, Alex Loukas, Long-Sheng Lu, Áke Lundwall, Liu-Ying Luo, Andrei Lupas, Dawn S. Luthe, Nicholas J. Lynch, Peter J. Lyons, Vivian L. MacKay, Jesica M. Levingston Macleod, Viktor Magdolen, Jean-Luc Mainardi, Kauko K. Mäkinen, Jeremy P. Mallari, Surya P. Manandhar, Fajga R. Mandelbaum, Anne M. Manicone, Johanna Mansfeld, Joseph Marcotrigiano, Michael Mares, Gemma Marfany, Francis S. Markland, Judith Marokházi, Hélène Marquis, Robert A. Marr, Enzo Martegani, Erik W. Martin, Manuel Martinez, L. Miguel Martins, Masato Maruyama, Masugi Maruyama, Sususmu Maruyama, Takeharu Masaki, Ramin Massoumi, Rency T. Mathew, Lynn M. Matrisian, Yoshihiro Matsuda, Osamu Matsushita, Marco Matuschek, Anna Matušková, Krisztina Matúz, Cornelia Mauch, Michael R. Maurizi, Lorenz M. Mayr, Dewey G. McCafferty, J. Ken McDonald, James H. McKerrow, David McMillan, Robert P. Mecham, Darshini P. Mehta, Chris Meisinger, Alan Mellors, Roger G. Melton, Jeffrey A. Melvin, Robert Ménard, Luis Menéndez-Arias, Milene C. Menezes, Andrew Mesecar, Stéphane Mesnage, Diane H. Meyer, Gregor Meyers, Susan Michaelis, Karolina Michalska, Wojciech P. Mielicki, Igor Mierau, Galina V. Mikoulinskaia, Charles G. Miller, Lydia K. Miller, John Mills, Kenneth V. Mills, Jinrong Min, Michel-Yves Mistou, Yoshio Misumi, Shin-ichi Miyoshi, Shigehiko Mizutani, Shahriar Mobashery, Satsuki Mochizuki, William L. Mock, Frank Möhrlen, Nathalie Moiré, Paul E. Monahan, Angela Moncada-Pazos, Véronique Monnet, Michel Monod, Cesare Montecucco, Laura Morelli, Sumiko Mori, Takashi Morita, James H. Morrissey, Richard J. Morse, John S. Mort, Uffe H. Mortensen, Rory E. Morty, Joel Moss, Hidemasa Motoshima, Jeremy C. Mottram, Ana M. Moura-da-Silva, Mary Beth Mudgett, Egbert Mundt, Kazuo Murakami, Mario Tyago Murakami, Kimiko MurakamiMurofoshi, Sawao Murao, Gillian Murphy, M.R.N. Murthy, Tatsushi Muta, Elmarie Myburgh, Nino Mzhavia, A.H.M. Nurun Nabi, Hideaki Nagase, Michael W. Nagle, Dorit K. Nägler, Rajesh R. Naik, Divya B. Nair, Toshiki Nakai, Yoshitaka Nakajima, Yukio Nakamura, Hitoshi Nakatogawa, Toru Nakayama, Natalia N. Nalivaeva, Dipankar Nandi, Maria Clara Leal Nascimento-Silva, Kim Nasmyth, Carl F. 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Published
2013
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50. Inactivation of Mycobacterium tuberculosis l,d-transpeptidase LdtMt₁ by carbapenems and cephalosporins

Author

Vincent, Dubée, Sébastien, Triboulet, Jean-Luc, Mainardi, Mélanie, Ethève-Quelquejeu, Laurent, Gutmann, Arul, Marie, Lionel, Dubost, Jean-Emmanuel, Hugonnet, and Michel, Arthur

Subjects
Carbapenems, Extensively Drug-Resistant Tuberculosis, Peptidyl Transferases, polycyclic compounds, Thienamycins, Meropenem, Microbial Sensitivity Tests, Mycobacterium tuberculosis, Peptidoglycan, Mechanisms of Action: Physiological Effects, Clavulanic Acid, Anti-Bacterial Agents, Cephalosporins
Abstract

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase Ldt(Mt1) by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt(Mt1), which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt(Mt1), with a binding rate constant of 0.08 μM(-1) min(-1). The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (0.1 min(-1)) in comparison to the acylation rate constant (3.1 min(-1)). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10(-3) min(-1). Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt(Mt1) inactivator. Cephalosporins also formed covalent adducts with Ldt(Mt1), although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

Published
2012

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