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18 results on '"Enveloppes Bactériennes et Antibiotiques ( ENVBAC )"'

1. Structural and biochemical characterization of the -N-acetylglucosaminidase from Thermotoga maritima: Toward rationalization of mechanistic knowledge in the GH73 family

Author

Didier Nurizzo, Mireille Hervé, Florence Vincent, Alexandra Lipski, Yves Bourne, Dominique Mengin-Lecreulx, Vincent Lombard, Bases moléculaires et structurales des systèmes infectieux (BMSSI), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-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), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), European Synchrotron Radiation Facility (ESRF), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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)-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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut de biologie et chimie des protéines [Lyon] (IBCP), Architecture et fonction des Macromolécules Biologiques - UMR 6098 (AFMB), Université de Provence - Aix-Marseille 1-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Bases moléculaires et structurales des systèmes infectieux ( BMSSI ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire des Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Université Paris Sud, Orsay Cedex, France, Architecture et fonction des macromolécules biologiques ( AFMB ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA ), European Synchrotron Radiation Facility ( ESRF ), Enveloppes Bactériennes et Antibiotiques ( ENVBAC ), Département Microbiologie ( Dpt Microbio ), 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 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 ), 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)-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), and European Synchroton Radiation Facility [Grenoble] (ESRF)

Subjects
Molecular model, [SDV]Life Sciences [q-bio], Molecular Sequence Data, catalytic mechanism, peptidoglycan, Biochemistry, chemistry.chemical_compound, Bacterial Proteins, Catalytic Domain, Acetylglucosaminidase, Hydrolase, Thermotoga maritima, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Glycoside hydrolase, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Phylogeny, ComputingMilieux_MISCELLANEOUS, [ SDV ] Life Sciences [q-bio], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Mutagenesis, Active site, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, chemistry, biology.protein, β-N-acetylglucosaminidase, X-ray structure, Peptidoglycan, Lysozyme
Abstract

International audience; Members of the GH73 glycosidase family cleave the β-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/β fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/β fold of the family and displays β-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the β-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.

Published
2014

2. In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells

Author

Frédéric Schmidt, Claire Soudais, Mireille Hervé, Stéphanie Bessoles, Didier Blanot, Lionel Le Bourhis, Fatoumata Samassa, Olivier Lantz, Manal Sarkis, Dominique Mengin-Lecreulx, Immunité et cancer ( U932 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Enveloppes Bactériennes et Antibiotiques, Département Microbiologie ( Dpt Microbio ), 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 ) -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 ), Département de Chimie, Institut Curie, Unité Mixte de Recherche 176, Institut Curie, Enveloppes Bactériennes et Antibiotiques ( ENVBAC ), Immunité et cancer (U932), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie-Université Paris Descartes - Paris 5 (UPD5), Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), 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), 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 biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Chimie biologique des membranes et ciblage thérapeutique (CBMCT - UMR 3666 / U1143), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), 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 Curie [Paris], Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-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), and Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Gram-negative bacteria, Riboflavin, [SDV]Life Sciences [q-bio], Immunology, Cell, Mice, Transgenic, In Vitro Techniques, Ligands, Lymphocyte Activation, medicine.disease_cause, Minor Histocompatibility Antigens, Mice, 03 medical and health sciences, 0302 clinical medicine, In vivo, Escherichia coli, medicine, Animals, [CHIM]Chemical Sciences, Immunology and Allergy, Escherichia coli Infections, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Mucous Membrane, biology, [ SDV ] Life Sciences [q-bio], Histocompatibility Antigens Class I, Flow Cytometry, biology.organism_classification, In vitro, Mice, Inbred C57BL, Disease Models, Animal, medicine.anatomical_structure, Biochemistry, Polyclonal antibodies, biology.protein, Natural Killer T-Cells, Cell activation, Bacteria, 030215 immunology
Abstract

Mucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity.

Published
2015

3. Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?

Author

Delphine Patin, Dominique Mengin-Lecreulx, Thierry Touzé, Aurélie Barnéoud-Arnoulet, Roland Lloubès, Ahmed Bouhss, Didier Blanot, Michel Arthur, Hélène Barreteau, Denis Duché, Emmanuelle Sacco, Meriem El Ghachi, Institut de Biologie Intégrative de la Cellule (I2BC), 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, Enveloppes Bactériennes et Antibiotiques, Département Microbiologie (Dpt Microbio), 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), 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 biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-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 d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Enveloppes Bactériennes et Antibiotiques (ENVBAC), 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)-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), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), 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 ), Département Microbiologie ( Dpt Microbio ), 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 ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), 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 d'ingénierie des systèmes macromoléculaires ( LISM ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), and Enveloppes Bactériennes et Antibiotiques ( ENVBAC )

Subjects
Models, Molecular, Isomerase activity, Protein Conformation, Colicins, Peptidoglycan, Biology, medicine.disease_cause, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, Bacteriocin, Bacteriocins, Antibiosis, medicine, Escherichia coli, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, 0303 health sciences, Lipid II, 030306 microbiology, Periplasmic space, Uridine Diphosphate N-Acetylmuramic Acid, Anti-Bacterial Agents, chemistry, Colicin
Abstract

International audience; Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis-trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA-TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolated catalytic domain. Genes encoding proteins with similarity to the catalytic domain of ColM were identified in pathogenic strains of Pseudomonas and other genera. ColM acts on several structures of lipid II representative of the diversity of peptidoglycan chemotypes. All together, these data open the way to the potential use of ColM-related bacteriocins as broad spectrum antibacterial agents.

Published
2012

4. Characterization of colicin M and its orthologs targeting bacterial cell wall peptidoglycan biosynthesis

Author

Delphine Patin, Meriem El Ghachi, Ahmed Bouhss, Michel Arthur, Mark A. Brooks, Thierry Touzé, Dominique Mengin-Lecreulx, Hélène Barreteau, Denis Duché, Emmanuelle Sacco, Fabien Gérard, Roland Lloubès, Aurélie Barnéoud-Arnoulet, Herman van Tilbeurgh, Didier Blanot, Enveloppes Bactériennes et Antibiotiques, Département Microbiologie (Dpt Microbio), 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 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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Université Paris Descartes - Paris 5 (UPD5), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), 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), 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), Département Microbiologie ( Dpt Microbio ), 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 ) -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 ), 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-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'ingénierie des systèmes macromoléculaires ( LISM ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -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 ), Université Paris Descartes - Paris 5 ( UPD5 ), and Enveloppes Bactériennes et Antibiotiques ( ENVBAC )

Subjects
Microbiology (medical), Models, Molecular, Lysis, Immunology, Colicins, Peptidoglycan, Biology, medicine.disease_cause, Microbiology, Bacterial cell structure, Substrate Specificity, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Bacteriocin, Bacteriocins, Cell Wall, Pseudomonas, medicine, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, 030306 microbiology, Uridine Diphosphate N-Acetylmuramic Acid, Protein Structure, Tertiary, Enzyme, chemistry, Biochemistry, Colicin
Abstract

International audience; For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.

Published
2012

5. Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

Author

Martine Fourgeaud, Dominique Mengin-Lecreulx, Sébastien Petit-Frère, Laurent Gutmann, Michel Arthur, Samuel Bellais, Jean-Luc Mainardi, Arul Marie, Lionel Dubost, Sophie Magnet, 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), Laboratoire de chimie et biochimie des substances naturelles, Muséum national d'Histoire naturelle (MNHN)-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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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 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), This work was supported by the European Community (COBRA, contract LSHM-CT-2003- 503335, 6th PCRD), the National Institute of Allergy and Infectious Diseases (Grant R01 AI45626), and the Fondation pour la Recherche Médicale., Lecerf, Maxime, 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 ), Laboratoire de Chimie et Biochimie des Substances Naturelles, Centre National de la Recherche Scientifique ( CNRS ), Hôpital Européen Georges Pompidou [APHP] ( HEGP ), Enveloppes Bactériennes et Antibiotiques ( ENVBAC ), Département Microbiologie ( Dpt Microbio ), 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 ) -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
MESH : Escherichia coli, MESH : Molecular Sequence Data, MESH: Peptidyl Transferases, Genomics and Proteomics, Mutant, MESH: Escherichia coli Proteins, MESH: Amino Acid Sequence, medicine.disease_cause, chemistry.chemical_compound, MESH : Peptidoglycan, 0303 health sciences, biology, MESH : Peptides, MESH: Escherichia coli, MESH: Peptides, MESH: Peptidoglycan, MESH : Amino Acid Sequence, MESH : Escherichia coli Proteins, Escherichia coli Proteins, MESH: Lipoproteins, Enterobacteriaceae, Biochemistry, Lipoproteins, Molecular Sequence Data, MESH : Lipoproteins, Peptidoglycan, Microbiology, 03 medical and health sciences, MESH : Peptidyl Transferases, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Bacteriology, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, MESH: Molecular Sequence Data, 030306 microbiology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, chemistry, Peptidyl Transferases, bacteria, Peptides, Bacteria, Enterococcus faecium, Lipoprotein
Abstract

The l , d -transpeptidase Ldt fm catalyzes peptidoglycan cross-linking in β-lactam-resistant mutant strains of Enterococcus faecium . Here, we show that in Escherichia coli Ldt fm homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the l , d -transpeptidation reaction.

Published
2007

6. A Francisella tularensis L,D‐carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence

Author

Briana Zellner, William T. Gunning, Brenden G. Tully, Jason F. Huntley, Robert Booth, Dominique Mengin-Lecreulx, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and 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)

Subjects
Carboxypeptidases A, Neutrophils, D-carboxypeptidase, [SDV]Life Sciences [q-bio], Virulence, peptidoglycan, Biology, Cell morphology, Microbiology, Bacterial cell structure, Tularemia, Mice, 03 medical and health sciences, chemistry.chemical_compound, Cell Wall, medicine, Animals, Amino Acid Sequence, Francisella tularensis, Molecular Biology, Cells, Cultured, 030304 developmental biology, Mice, Inbred C3H, 0303 health sciences, 030306 microbiology, Macrophages, Intracellular parasite, medicine.disease, biology.organism_classification, tularemia, 3. Good health, virulence, Disease Models, Animal, chemistry, Female, Peptidoglycan, Sequence Alignment, Intracellular
Abstract

SummaryFrancisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell envelope-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG – a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

Published
2021

7. Use of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts

Author

Madeleine Boulanger, Cédric Delvaux, Johann Far, Bernard Joris, Marjorie Dauvin, Loïc Quinton, Edwin De Pauw, Dominique Mengin-Lecreulx, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and 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)

Subjects
Cytoplasm, Spectrometry, Mass, Electrospray Ionization, [SDV]Life Sciences [q-bio], Peptide, Peptidoglycan, Tripeptide, Bacterial growth, 010402 general chemistry, 01 natural sciences, Bacterial cell structure, Analytical Chemistry, chemistry.chemical_compound, Capillary electrophoresis, chemistry.chemical_classification, biology, Chemistry, 010401 analytical chemistry, Electrophoresis, Capillary, biology.organism_classification, Cephalosporins, 0104 chemical sciences, Biochemistry, Peptides, Bacteria, Bacillus subtilis
Abstract

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

Published
2021

8. CbrA mediates colicin M resistance in Escherichia coli through modification of undecaprenyl-phosphate-linked peptidoglycan precursors

Author

Delphine Patin, Ahmed Bouhss, Dominique Mengin-Lecreulx, Didier Blanot, Hélène Barreteau, Thierry Touzé, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), Fondation pour la Recherche Médicale (DBF20160635737), and ANR-11-BSV3-0002,BACTOPRENYL,Elucidation du métabolisme de l'undécaprényl phosphate, un lipide essentiel pour la biosynthèse des polymères de la paroi bactérienne(2011)

Subjects
[SDV]Life Sciences [q-bio], Colicins, Biology, peptidoglycan, medicine.disease_cause, Microbiology, Bacterial cell structure, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Bacteriocin, Polyisoprenyl Phosphates, Drug Resistance, Bacterial, medicine, Escherichia coli, undecaprenyl-phosphate, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Lipid II, Flavoproteins, 030306 microbiology, Escherichia coli Proteins, CbrA, bacterial metabolism, lipid II, Uridine Diphosphate N-Acetylmuramic Acid, Anti-Bacterial Agents, Enzyme, chemistry, Biochemistry, Colicin, reductase, lipids (amino acids, peptides, and proteins), Peptidoglycan, colicin, Research Article, bacterial cell wall
Abstract

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

Published
2020

9. Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b

Author

Boes, Adrien, Brunel, Jean Michel, Derouaux, Adeline, Kerff, Frédéric, Bouhss, Ahmed, Touze, Thierry, Breukink, Eefjan, Terrak, Mohammed, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Brunel, Jean Michel, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Membranes et cibles thérapeutiques (MCT), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA), Centre d’Ingénierie des Protéines [Université de Liège] = Centre for Protein Engineering [University of Liège] (CIP), 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), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Department of Chemical Biology and Organic Chemistry, Utrecht University [Utrecht], Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Centre d'Ingénierie des Protéines, Université de Liège-Institut de Chimie B6, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and Department Biochemistry of Membranes

Subjects
0301 basic medicine, Microbiology (medical), Glycan, [SDV]Life Sciences [q-bio], Peptidoglycan, cationic aminosterols, 010402 general chemistry, 01 natural sciences, Microbiology, Biochemistry, Bacterial cell structure, Article, 03 medical and health sciences, chemistry.chemical_compound, Pharmacology, Toxicology and Pharmaceutics(all), squalamine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Glycosyltransferase, Pharmacology (medical), General Pharmacology, Toxicology and Pharmaceutics, chemistry.chemical_classification, Pharmacology, Squalamine, biology, Lipid II, lcsh:RM1-950, Peptidoglycan glycosyltransferase activity, 0104 chemical sciences, 030104 developmental biology, Enzyme, Toxicology and Pharmaceutics(all), lcsh:Therapeutics. Pharmacology, Infectious Diseases, PBP1b, chemistry, Cationic aminosterols, biology.protein, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases
Abstract

International audience; Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Published
2020

10. The C-terminal domain of Corynebacterium glutamicum mycoloyltransferase A is composed of five repeated motifs involved in cell wall binding and stability

Author

Christiane Dietrich, Eric Girard, Muriel Masi, Maryelle Tropis, Florence Constantinesco-Becker, Mamadou Daffé, Nathalie Dautin, Herman van Tilbeurgh, Nicolas Bayan, Inès Li de la Sierra-Gallay, 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), Biologie Moléculaire des Corynébactéries et Mycobactéries (CORYNE), Département Microbiologie (Dpt Microbio), 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), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), Département Biochimie, Biophysique et Biologie Structurale (B3S), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)

Subjects
Protein Conformation, [SDV]Life Sciences [q-bio], mycolyltransferase, Peptidoglycan, Biology, Crystallography, X-Ray, Microbiology, Galactans, Corynebacterium glutamicum, Mycobacterium, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Protein Domains, Cell Wall, mycolic acid, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, C-terminus, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], arabinogalactan, Bacterial Outer Membrane, chemistry, Mycolic Acids, Cell envelope, Bacterial outer membrane, Sequence motif, Oligopeptides, Biogenesis, Acyltransferases
Abstract

International audience; The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N-terminal domain with esterase activity and a C-terminal extension containing a conserved repeated Leu-Gly-Phe-Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function. In this study, we determined the crystal structure of MytA and found that its C-terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N-terminal catalytic α/β-hydrolase domain. The LGFP motifs are composed of a 4-stranded β-fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand-binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C-terminal LGFP domain interacts with the cell wall peptidoglycan-arabinogalactan polymer. We also show that the C-terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope.

Published
2019

11. Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria

Author

Julia Vergalli, Bert van den Berg, James H. Naismith, Lucile Moynié, Anne Davin-Regli, Igor Bodrenko, Muriel Masi, Mathias Winterhalter, Silvia Acosta-Gutierrez, Matteo Ceccarelli, Jean-Marie Pagès, Membranes et cibles thérapeutiques (MCT), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA)

Subjects
Gram-negative bacteria, porins, [SDV]Life Sciences [q-bio], Microbiology, antibiotics, 03 medical and health sciences, Enterobacteriaceae, Drug Resistance, Bacterial, Inner membrane, 0303 health sciences, General Immunology and Microbiology, biology, 030306 microbiology, Cell Membrane, Biological membrane, Biological Transport, biology.organism_classification, Small molecule, General bacterial porin family, Anti-Bacterial Agents, diffusive transport, Infectious Diseases, Biophysics, Efflux, Bacterial outer membrane, Intracellular, Bacterial Outer Membrane Proteins
Abstract

International audience; Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.

Published
2019

12. Functional analysis of the three major PGRPLC isoforms in the midgut of the malaria mosquito Anopheles coluzzii

Author

Andre N. Pitaluga, Mathilde Gendrin, Faye H. Rodgers, Julia A. Cai, George K. Christophides, Dominique Mengin-Lecreulx, Imperial College London, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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 Pasteur de la Guyane, Réseau International des Instituts Pasteur (RIIP), Département Parasites et Insectes vecteurs - Department of Parasites and Insect Vectors, Institut Pasteur [Paris] (IP), BBSRC project BB/K009338/1 and Welcome Trust Investigator Award 107983/Z/15/Z., Department of Life Sciences, Imperial College London, London, United Kingdom, and Institut Pasteur [Paris]

Subjects
0106 biological sciences, [SDV]Life Sciences [q-bio], ved/biology.organism_classification_rank.species, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Immune system, Anopheles, Animals, Protein Isoforms, Model organism, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, Effector, ved/biology, fungi, Pattern recognition receptor, Midgut, biology.organism_classification, Transmembrane protein, 3. Good health, Cell biology, Gastrointestinal Tract, 010602 entomology, chemistry, Insect Science, Insect Proteins, Female, Peptidoglycan, Carrier Proteins
Abstract

International audience; Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

Published
2019

13. Impact of FiuA outer-membrane receptor polymorphism on the resistance of Pseudomonas aeruginosa towards peptidoglycan lipid II-targeting PaeM pyocins

Author

Dominique Mengin-Lecreulx, Libera Latino, Hélène Barreteau, Delphine Patin, Dimitri Chérier, Christine Pourcel, Thierry Touzé, 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 Microbiologie (Dpt Microbio), 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), 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), Département Biologie des Génomes (DBG), Séquence, Structure et Fonction des ARN (SSFA), and Fondation pour la Recherche Médicale (DBF20160635737)

Subjects
TonB, [SDV]Life Sciences [q-bio], Biology, peptidoglycan, medicine.disease_cause, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Bacteriocin, bacteriocin, receptor structure-function, medicine, Molecular Biology, 030304 developmental biology, 0303 health sciences, Lipid II, 030306 microbiology, Pseudomonas aeruginosa, lipid II, Periplasmic space, FiuA, biology.organism_classification, chemistry, Colicin, cell wall, Peptidoglycan, Bacterial outer membrane, colicin M, Bacteria, PaeM pyocin
Abstract

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

Published
2019

14. Bacterial Transferase MraY, a Source of Inspiration towards New Antibiotics

Author

Nicolas Pietrancosta, Sandrine Calvet-Vitale, Nathalie Evrard-Todeschi, Christine Gravier-Pelletier, Laurent Le Corre, Ahmed Bouhss, Mickaël J Fer, Samir Olatunji, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Modélisation et Immunologie pour la Thérapie (CBMIT), Université Paris Descartes - Paris 5 (UPD5), Institut de Biologie Intégrative de la Cellule (I2BC), 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, Trinity College Dublin, Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE), Centre d'Ingénierie des Protéines, Université de Liège-Institut de Chimie B6, Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), 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-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Systèmes glutamatergiques normaux et pathologiques = Normal and Pathologic Glutamatergic Neurons (NPS), Neuroscience Paris Seine (NPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Analyse, Interactions Moléculaires et Cellulaires (LBM-E2), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
nucleoside antibiotics, Research groups, medicine.drug_class, [SDV]Life Sciences [q-bio], Antibiotics, Transferases (Other Substituted Phosphate Groups), Computational biology, Peptidoglycan, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Biology, 01 natural sciences, Biochemistry, MraY, Structure-Activity Relationship, Antibiotic resistance, Bacterial Proteins, biological evaluations, Transferases, Drug Discovery, medicine, Transferase, Peptidoglycan biosynthesis, Enzyme Inhibitors, triazoles, ComputingMilieux_MISCELLANEOUS, Pharmacology, Binding Sites, Bacteria, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Organic Chemistry, 3. Good health, 0104 chemical sciences, Anti-Bacterial Agents, molecular modelling, Molecular Docking Simulation, 010404 medicinal & biomolecular chemistry, Docking (molecular), Drug Design, docking, Molecular Medicine, mutagenesis
Abstract

The bacterial resistance to antibiotics constitutes more than ever a severe public health problem. The enzymes involved in bacterial peptidoglycan biosynthesis are pertinent targets for developing new antibiotics, notably the MraY transferase that is not targeted by any marketed drug. Many research groups are currently working on the study or the inhibition of this enzyme. After a concise overview of the role, mechanism and inhibition of MraY, the structure–activity relationships of 5’-triazole-containing aminoribosyluridine inhibitors, we previously synthetized, will be presented. The recently published MraY X-ray structures allowed us to achieve a molecular virtual high-throughput screening of commercial databases and our in-house library resulting in the identification of promising compounds for the further development of new antibiotics.

Published
2018

15. Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc

Author

Shih Chieh Su, Yi Ting Kuo, Dominique Mengin-Lecreulx, Meng-Ru Ho, Ching Hao Teng, Manjula Reddy, Shiou-Ru Tzeng, Lu Chu Ke, Chia Yun Wu, Ming Yuan Su, Nilanjan Som, Chung-I Chang, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and 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)

Subjects
0301 basic medicine, animal structures, Science, [SDV]Life Sciences [q-bio], Lipoproteins, PDZ domain, PDZ Domains, General Physics and Astronomy, Peptidoglycan, Plasma protein binding, Biology, Protein degradation, Crystallography, X-Ray, Article, Protein Structure, Secondary, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, chemistry.chemical_compound, Endopeptidases, Escherichia coli, Amino Acid Sequence, lcsh:Science, Multidisciplinary, Sequence Homology, Amino Acid, Escherichia coli Proteins, C-terminus, Signal transducing adaptor protein, General Chemistry, Periplasmic space, Cell biology, Molecular Docking Simulation, Cysteine Endopeptidases, Tetratricopeptide, 030104 developmental biology, chemistry, Biochemistry, Mutation, Periplasm, Proteolysis, lcsh:Q, Protein Binding
Abstract

Peptidoglycan (PG) is a highly cross-linked, protective mesh-like sacculus that surrounds the bacterial cytoplasmic membrane. Expansion of PG is tightly coupled to growth of a bacterial cell and requires hydrolases to cleave the cross-links for insertion of nascent PG material. In Escherichia coli, a proteolytic system comprising the periplasmic PDZ-protease Prc and the lipoprotein adaptor NlpI contributes to PG enlargement by regulating cellular levels of MepS, a cross-link-specific hydrolase. Here, we demonstrate how NlpI binds Prc to facilitate the degradation of its substrate MepS by structural and mutational analyses. An NlpI homodimer binds two molecules of Prc and forms three-sided MepS-docking cradles using its tetratricopeptide repeats. Prc forms a monomeric bowl-shaped structure with a lid-like PDZ domain connected by a substrate-sensing hinge that recognizes the bound C terminus of the substrate. In summary, our study reveals mechanistic details of protein degradation by the PDZ-protease Prc bound to its cognate adaptor protein., MepS is a peptidoglycan (PG) cross-link specific hydrolase needed for cell wall expansion and its cellular levels must be tightly regulated. Here the authors present the structure of the MepS degrading protease Prc bound to its adaptor NlpI and propose a model how the NlpI-Prc complex mediates MepS degradation.

Published
2017

16. Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

Author

Delphine Patin, Didier Blanot, Dominique Mengin-Lecreulx, Sébastien Dementin, Andreja Kovač, Audrey Boniface, Mireille Hervé, Hélène Barreteau, Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), inconnu temporaire UPEMLV, Inconnu, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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 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), and Azzopardi, Laure

Subjects
Staphylococcus aureus, Lysine, Glycine, Peptide, Peptidoglycan, Biology, Diaminopimelic Acid, medicine.disease_cause, Biochemistry, Substrate Specificity, Microbiology, Ligases, 03 medical and health sciences, chemistry.chemical_compound, Adenosine Triphosphate, Bacterial Proteins, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Escherichia coli, Serine, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Pathogen, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Alanine, Genetic Complementation Test, 030302 biochemistry & molecular biology, Temperature, General Medicine, Hydrogen-Ion Concentration, Recombinant Proteins, carbohydrates (lipids), Kinetics, Enzyme, chemistry, Mutation, Oligopeptides
Abstract

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His6-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l -Ala, l -Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l -Ala. S. aureus MurE was very specific for l -lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l -lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms ( l -lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l -lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.

Published
2010

17. Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Author

Hélène Barreteau, Boubekeur Boussaid, Dominique Mengin-Lecreulx, Roland Lloubès, Denis Duché, Ahmed Bouhss, Fabien Gérard, Thierry Touzé, Didier Blanot, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Interactions cellulaires et moléculaires (ICM), Centre National de la Recherche Scientifique (CNRS)-IFR140-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
Models, Molecular, Protein Conformation, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Colicins, Peptidoglycan, Biology, Biochemistry, Microbiology, Models, Biological, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, Escherichia coli, Amino Acid Sequence, Molecular Biology, Protein maturation, Peptide sequence, 030304 developmental biology, DNA Primers, Sequence Deletion, 0303 health sciences, Binding Sites, Lipid II, Base Sequence, Sequence Homology, Amino Acid, 030306 microbiology, Escherichia coli Proteins, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Periplasmic space, Peptidylprolyl Isomerase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Recombinant Proteins, Protein Structure, Tertiary, chemistry, Amino Acid Substitution, Colicin, Mutagenesis, Site-Directed, Bacterial outer membrane
Abstract

International audience; Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Published
2010

18. Role of AmiA in the Morphological Transition of Helicobacter pylori and in Immune Escape

Author

Marie Christine Prevost, Dominique Mengin-Lecreulx, Chantal Ecobichon, Stephen E. Girardin, Nadège Cayet, Ivo G. Boneca, Catherine Chaput, Agnès Labigne, Catherine Werts, Stéphanie Guadagnini, Gomperts Boneca, Ivo, Pathogénie Bactérienne des Muqueuses, Institut Pasteur [Paris] (IP), Microscopie électronique (Plate-forme), Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunité Innée et Signalisation, Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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 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 Pasteur [Paris], and Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Amino Acid Motifs, Biochemistry, MESH: Amino Acid Motifs, chemistry.chemical_compound, Cell Wall, NOD1, Biology (General), MESH: Bacterial Proteins, Pathogen, MESH: Genetic Complementation Test, 0303 health sciences, biology, MESH: Peptidoglycan, Homo (human), MESH: Amoxicillin, MESH: Lipoproteins, Anti-Bacterial Agents, 3. Good health, In Vitro, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Epithelial Cells, Research Article, MESH: Mutation, QH301-705.5, Lipoproteins, Immunology, MESH: Carrier Proteins, Peptidoglycan, Microbiology, Cell Line, Bacterial genetics, 03 medical and health sciences, MESH: Cell Wall, Bacterial Proteins, MESH: Anti-Bacterial Agents, Virology, Genetics, Humans, MESH: Cell Shape, Secretion, Cell Shape, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Molecular Biology, 030304 developmental biology, MESH: Humans, Helicobacter pylori, 030306 microbiology, Genetic Complementation Test, Amoxicillin, Epithelial Cells, RC581-607, biology.organism_classification, In vitro, MESH: Cell Line, Eubacteria, chemistry, Cell culture, Mutation, MESH: Helicobacter pylori, Parasitology, Immunologic diseases. Allergy, Carrier Proteins
Abstract

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection., Synopsis Helicobacter pylori is a human pathogen responsible for gastric diseases such as ulcers and gastric cancers. Despite the host's vigorous immune response, H. pylori is capable of persisting for decades in its human host. H. pylori is found in biopsies in two distinct forms, a spiral rod form and a coccoid form. Chaput et al. investigated the molecular mechanisms leading to the transition of H. pylori from a spiral rod–shaped organism to a coccoid organism. The morphological transition is accompanied by modifications of the bacterial cell wall peptidoglycan. The authors have identified the AmiA protein as essential for this morphological transition and modification of the cell wall peptidoglycan. Additionally, the authors show that the cell wall modifications and morphological transition allow these coccoid forms to escape detection by the immune system and therefore could participate in the persistence of H. pylori infection during the lifetime of its human host.

Published
2006

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