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1. 5′-Methylene-triazole-substituted-aminoribosyl uridines as MraY inhibitors: synthesis, biological evaluation and molecular modeling

Author: Ahmed Bouhss, Nicolas Pietrancosta, Christine Gravier-Pelletier, Dominique Mengin-Lecreulx, Ana Maria Amoroso, Sandrine Calvet-Vitale, Laurent Le Corre, Bernard Joris, Mariana Patrão, Michael Fer, 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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Unité de Physiologie et Génétique Bactériennes, Centre d'Ingénierie des Protéines, Département des Sciences de la Vie, Université de Liège, 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 ), 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 ), 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 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-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, and 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
Subjects: Models, Molecular, Molecular model, Stereochemistry, [SDV]Life Sciences [q-bio], Triazole, Transferases (Other Substituted Phosphate Groups), Alkyne, Chemistry Techniques, Synthetic, Microbial Sensitivity Tests, 010402 general chemistry, 01 natural sciences, Biochemistry, chemistry.chemical_compound, Bacterial Proteins, Transferases, Catalytic Domain, Enzyme Inhibitors, Physical and Theoretical Chemistry, Methylene, Uridine, chemistry.chemical_classification, [ SDV ] Life Sciences [q-bio], 010405 organic chemistry, Organic Chemistry, Biological activity, Triazoles, Cycloaddition, Anti-Bacterial Agents, 0104 chemical sciences, chemistry, Azide, Antibacterial activity, Bacillus subtilis
Abstract: International audience; The straightforward synthesis of 5'-methylene-[1,4]-triazole-substituted aminoribosyl uridines is described. Two families of compounds were synthesized from a unique epoxide which was regioselectively opened by acetylide ions (for compounds II) or azide ions (for compounds III). Sequential diastereoselective glycosylation with a ribosyl fluoride derivative, Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) with various complementary azide and alkyne partners afforded the targeted compounds after final deprotection. The biological activity of the 16 resulting compounds together with that of 14 previously reported compounds I, lacking the 5' methylene group, was evaluated on the MraY transferase activity. Out of the 30 tested compounds, 18 compounds revealed MraY inhibition with IC50 ranging from 15 to 150 μM. A molecular modeling study was performed to rationalize the observed structure-activity relationships (SAR), which allowed us to correlate the activity of the most potent compounds with an interaction involving Leu191 of MraYAA. The antibacterial activity was also evaluated and seven compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 μg mL(-1), including the methicillin resistant Staphylococcus aureus (MRSA).
Published: 2015

2. 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

3. Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

Author: Andrej Perdih, Darko Kocjan, Simona Golic Grdadolnik, Stanislav Gobec, Hélène Barreteau, Kaja Pureber, Gerhard Wolber, Martina Hrast, Tomaz Solmajer, Institute of Pharmacy, Freie Universität Berlin, National Institute of Chemistry, Faculty of Pharmacy, University of Ljubljana, Centre of Excelence EN-FIST [Ljubljana], 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Freie Universität Berlin [Berlin], National Institute of Chemistry, Ljubljana, EN-FIST Centre of Excellence, 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 ), and Enveloppes Bactériennes et Antibiotiques ( ENVBAC )
Subjects: Stereochemistry, In silico, [SDV]Life Sciences [q-bio], Carboxylic Acids, Drug Evaluation, Preclinical, Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Ligases, 03 medical and health sciences, chemistry.chemical_compound, Structure-Activity Relationship, Non-competitive inhibition, Adenosine Triphosphate, Bacterial Proteins, Furan, Drug Discovery, Structure–activity relationship, Physical and Theoretical Chemistry, Binding site, Enzyme Inhibitors, Furans, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, DNA ligase, Binding Sites, [ SDV ] Life Sciences [q-bio], 0104 chemical sciences, Computer Science Applications, Anti-Bacterial Agents, Dicarboxylic acid, chemistry, Drug Design, Antibacterial activity
Abstract: International audience; Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.
Published: 2015

4. Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION

Author: Levefaudes, Marjorie, Patin, Delphine, de Sousa-d'Auria, Célia, Chami, Mohamed, Blanot, Didier, Hervé, Mireille, Arthur, Michel, Houssin, Christine, Mengin-Lecreulx, Dominique, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Center for Cellular Imaging and NanoAnalytics, Biozentrum [Basel, Suisse], University of Basel (Unibas)-University of Basel (Unibas), 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 ), 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 ), Biologie Moléculaire des Corynébactéries et Mycobactéries ( CORYNE ), 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 ), Enveloppes Bactériennes et Antibiotiques ( ENVBAC ), Biozentrum - University of Basel, 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 ), 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 ), 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), 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 Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), and 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)
Subjects: DAP amidation, [SDV]Life Sciences [q-bio], Blotting, Western, Molecular Sequence Data, Corynebacterium, peptidoglycan, Diaminopimelic Acid, Real-Time Polymerase Chain Reaction, Microbiology, antibiotics, gene knockout, Immunoenzyme Techniques, Corynebacteriales, Bacterial Proteins, Microscopy, Electron, Transmission, Amino Acid Sequence, RNA, Messenger, lysozyme, Cells, Cultured, Transaminases, Sequence Homology, Amino Acid, [ SDV ] Life Sciences [q-bio], Reverse Transcriptase Polymerase Chain Reaction, bacterial metabolism, glutaminase, Amides, humanities, Anti-Bacterial Agents, enzyme, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Mutation, cell wall, Muramidase
Abstract: International audience; A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.
Published: 2015

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux

Author: Vergalli, Julia, Atzori, Alessio, Pajovic, Jelena, Dumont, Estelle, Malloci, Giuliano, Masi, Muriel, Vargiu, Attilio, Winterhalter, Mathias, Réfrégiers, Matthieu, Ruggerone, Paolo, Pagès, Jean-Marie, 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), Università degli Studi di Cagliari = University of Cagliari (UniCa), University of Belgrade [Belgrade], Université Paris-Saclay, Jacobs University [Bremen], Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA), Universita degli Studi di Cagliari [Cagliari], rosa, emmanuelle, 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 Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA)-Aix Marseille Université (AMU)
Subjects: [SDE] Environmental Sciences, Binding Sites, Molecular Structure, [SDV]Life Sciences [q-bio], Escherichia coli Proteins, Biological Transport, Molecular Dynamics Simulation, Antimicrobial resistance, Article, Anti-Bacterial Agents, [SDV] Life Sciences [q-bio], Molecular Docking Simulation, Structure-Activity Relationship, Spectrometry, Fluorescence, lcsh:Biology (General), [SDE]Environmental Sciences, Drug Resistance, Bacterial, Escherichia coli, Molecular modelling, Multidrug Resistance-Associated Proteins, lcsh:QH301-705.5, Fluoroquinolones, Protein Binding
Abstract: With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps., To understand antibiotic resistance, Vergalli et al. quantify intracellular accumulation of 2 fluoroquinolones in E. coli cells expressing different levels of the AcrB multidrug efflux pumps. Their 3 study highlights the role of the molecular/structural dialogue between a drug family and the affinity 4 sites of membrane transporters.
Published: 2020

12. Anthranilic acid inhibitors of undecaprenyl pyrophosphate synthase (UppS), an essential enzyme for bacterial cell wall biosynthesis

Author: Matej Sova, Kaja Rožman, Marko Jukič, Hélène Barreteau, Stanislav Gobec, Faculty of Pharmacy, University of Ljubljana, 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), 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)-Institut de Biologie Intégrative de la Cellule (I2BC)
Subjects: Microbiology (medical), MICROBIO, bakterijska rezistenca, Stereochemistry, [SDV]Life Sciences [q-bio], undecaprenyl pyrophosphate synthase, Farnesyl pyrophosphate, Isopentenyl pyrophosphate, lcsh:QR1-502, mechanism, ENVBAC, antibacterial agents, Microbiology, lcsh:Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, inhibitors, Anthranilic acid, UppS, 030304 developmental biology, Original Research, chemistry.chemical_classification, 0303 health sciences, pharmacophore model, 030306 microbiology, ligands, celična stena, states, diphosphate synthase, Enzyme, chemistry, cell-wall, Docking (molecular), farmakomorfni model, Pharmacophore, Antibacterial activity, udc:615.015.8
Abstract: We report the successful implementation of virtual screening in the discovery of new inhibitors of undecaprenyl pyrophosphate synthase (UppS) from Escherichia coli. UppS is an essential enzyme in the biosynthesis of bacterial cell wall. It catalyzes the condensation of farnesyl pyrophosphate (FPP) with eight consecutive isopentenyl pyrophosphate units (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate. The latter serves as a lipid carrier for peptidoglycan synthesis, thus representing an important target in the antibacterial drug design. A pharmacophore model was designed on a known bisphosphonate BPH-629 and used to prepare an enriched compound library that was further docked into UppS conformational ensemble generated by molecular dynamics experiment. The docking resulted in three anthranilic acid derivatives with promising inhibitory activity against UppS. Compound 2 displayed high inhibitory potency (IC50 = 25 µM) and good antibacterial activity against E. coli BW25113 ΔtolC strain (MIC = 0.5 µg/mL).
Published: 2021

13. Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum

Author: Christiane Dietrich, Laila Sago, David Cornu, Célia de Sousa d’Auria, Niloofar Mohiman, Muriel Masi, Mohamed Chami, Cécile Labarre, Christophe Salmeron, Manuela Argentini, Nicolas Bayan, Christine Houssin, Nathalie Dautin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), 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), Institut de la Vision, Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), SICaPS (SICaPS), Département Plateforme (PF I2BC), Center for Cellular Imaging and NanoAnalytics, Biozentrum [Basel, Suisse], University of Basel (Unibas)-University of Basel (Unibas), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Observatoire océanologique de Banyuls (OOB), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Dautin, Nathalie, Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Phosphatidylglycerol, Signal peptide, 0303 health sciences, Glycosylation, cell envelope, 030306 microbiology, phosphatidylglycerol::prolipoprotein diacylglyceryl transferase, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Signal peptidase II, Translocon, Microbiology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Corynebacterium glutamicum, lipoproteins, 03 medical and health sciences, chemistry.chemical_compound, Secretory protein, chemistry, Biochemistry, lipids (amino acids, peptides, and proteins), [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell envelope, 030304 developmental biology
Abstract: Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified thelgtandlspAgenes fromCorynebacterium glutamicumand found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, aC. glutamicummaltose-binding lipoprotein, and LppX, aMycobacterium tuberculosislipoprotein. However, Lgt is not required for these proteins’ signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that inC. glutamicumthe association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.
Published: 2020

14. The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b

Author: Boes, Adrien, Kerff, Frédéric, Herman, Raphael, Touzé, Thierry, Breukink, Eefjan, Terrak, Mohammed, 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), Sub Membrane Biochemistry & Biophysics, and Membrane Biochemistry and Biophysics
Subjects: cell division, FtsN, cellular regulation, Escherichia coli Proteins, [SDV]Life Sciences [q-bio], penicillin-binding protein 1b (PBP1b), Membrane Proteins, lipid II, Peptidoglycan, Microbiology, Serine-Type D-Ala-D-Ala Carboxypeptidase, cell surface enzyme, PBP1b, eptidoglycan, Lipid II, Escherichia coli, Penicillin-Binding Proteins, cell wall, Peptidoglycan Glycosyltransferase, divisome, bacteria, Divisome, Protein Binding
Abstract: Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli , septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (L75 to Q93, known as E FtsN) was shown to be essential and sufficient for its functioning in vivo , but what exactly this sequence is doing remained unknown. Here, we show that E FtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with E FtsN, which demonstrates E FtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the E FtsN binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue Δ pon B- pon Ats strain, that lack PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that E FtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.
Published: 2020

15. 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

16. 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

17. Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

Author: Dominique Mengin-Lecreulx, Rodolphe Auger, Xudong Tian, Guillaume Manat, Frédéric Kerff, 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), Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Belgium Program InterUniversity Attraction Poles (IAP n° 7/44), 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: Models, Molecular, Thermotolerance, 0301 basic medicine, [SDV]Life Sciences [q-bio], Amino Acid Motifs, Phosphatase, Phosphatidate Phosphatase, lcsh:Medicine, Biochemistry, Microbiology, Article, Substrate Specificity, Gene Knockout Techniques, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Polyisoprenyl Phosphates, Biosynthesis, Catalytic triad, Escherichia coli, Penicillin-Binding Proteins, lcsh:Science, Multidisciplinary, Chemistry, Escherichia coli Proteins, Hydrolysis, Cell Membrane, Genetic Complementation Test, lcsh:R, Membrane Proteins, Phosphatidylglycerols, Lipid-phosphate phosphatase, Metabolic pathway, 030104 developmental biology, Membrane protein, lcsh:Q, Peptidoglycan Glycosyltransferase, Peptidoglycan, Cell envelope, Metabolic Networks and Pathways, 030217 neurology & neurosurgery
Abstract: Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.
Published: 2020

18. Synthesis and structure–activity relationship study of novel quinazolinone-based inhibitors of MurA

Author: Delphine Patin, Kaja Rožman, Martina Hrast, Matej Sova, Marko Jukič, Stanislav Gobec, Faculty of Pharmacy, University of Ljubljana, 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: [SDV]Life Sciences [q-bio], Clinical Biochemistry, Pharmaceutical Science, Microbial Sensitivity Tests, Peptidoglycan, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, Biochemistry, Structure-Activity Relationship, chemistry.chemical_compound, Mura, Drug Discovery, Escherichia coli, medicine, Humans, Structure–activity relationship, Enzyme Inhibitors, Molecular Biology, IC50, Quinazolinone, Escherichia coli Infections, MurA enzyme, Quinazolinones, chemistry.chemical_classification, Alkyl and Aryl Transferases, 010405 organic chemistry, Organic Chemistry, Antimicrobial, Anti-Bacterial Agents, 3. Good health, 0104 chemical sciences, Molecular Docking Simulation, Antibacterial agents, Enzyme, chemistry, Molecular Medicine
Abstract: MurA is an intracellular bacterial enzyme that is essential for peptidoglycan biosynthesis, and is therefore an important target for antibacterial drug discovery. We report the synthesis, in silico studies and extensive structure-activity relationships of a series of quinazolinone-based inhibitors of MurA from Escherichia coli. 3-Benzyloxyphenylquinazolinones showed promising inhibitory potencies against MurA, in the low micromolar range, with an IC50 of 8µM for the most potent derivative (58). Furthermore, furan-substituted quinazolinones (38, 46) showed promising antibacterial activities, with MICs from 1µg/mL to 8µg/mL, concomitant with their MurA inhibitory potencies. These data represent an important step towards the development of novel antimicrobial agents to combat increasing bacterial resistance.
Published: 2017

19. 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

20. H Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases

Author: Kaja Rožman, Delphine Patin, Stanislav Gobec, Matej Sova, Hélène Barreteau, Veronika Škedelj, Simona Golic Grdadolnik, Iza Ogris, Anamarija Zega, Martina Hrast, 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), Faculty of Pharmacy, University of Ljubljana, 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), 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)-Institut de Biologie Intégrative de la Cellule (I2BC)
Subjects: purification, [SDV]Life Sciences [q-bio], enzymes, design, Bacterial Mur (MurC-MurF) ligases, l-alanine, antibacterial agents, 01 natural sciences, Article, drugs, d-glutamate ligase, Ligases, Structure-Activity Relationship, Adenosine Triphosphate, Drug Discovery, Escherichia coli, NMR studies, Peptidoglycan biosynthesis, Enzyme Inhibitors, Pharmacology, chemistry.chemical_classification, Dose-Response Relationship, Drug, Molecular Structure, 010405 organic chemistry, Kinase, lcsh:RM1-950, cytoplasmic steps, published kinase inhibitor set, crystal-structure, General Medicine, Bacterial Mur (MurC–MurF) ligases, 0104 chemical sciences, 3. Good health, Anti-Bacterial Agents, 010404 medicinal & biomolecular chemistry, Kinetics, Enzyme, lcsh:Therapeutics. Pharmacology, chemistry, Biochemistry, efficiency, steady-state kinetics measurements, Intracellular, discovery
Abstract: International audience; The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 mu M). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.
Published: 2019

21. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM

Author: Rahul Kumar Singh, Delphine Patin, Dominique Mengin-Lecreulx, Charles Eldrid, Konstantinos Thalassinos, Tommaso Tosi, Charlotte Millership, Paul S. Freemont, Angelika Gründling, Fumiya Hoshiga, MRC Centre for Molecular Microbiology and Infection [Imperial College, London] (CMBI), Imperial College London, Institute of Structural and Molecular Biology (ISMB), 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), Department of Medicine, Imperial College London, London, United Kingdom, Medical Research Council (MRC), and Wellcome Trust
Subjects: Operon, Staphylococcus, [SDV]Life Sciences [q-bio], domain, medicine.disease_cause, Pathology and Laboratory Medicine, Biochemistry, Second Messenger Systems, cyclic dinucleotide, X-Ray Diffraction, 1108 Medical Microbiology, Cell Wall, Medicine and Health Sciences, Macromolecular Structure Analysis, Transferase, Chemical Precipitation, Staphylococcus Aureus, Biology (General), Enzyme Chemistry, Materials, degradation, chemistry.chemical_classification, 0303 health sciences, Crystallography, Phosphoglucosamine mutase, Physics, 030302 biochemistry & molecular biology, Chemical Reactions, Pseudomonas Aeruginosa, Staphylococcal Infections, Condensed Matter Physics, 3. Good health, Bacterial Pathogens, Enzymes, mobility mass-spectrometry, Chemistry, 1107 Immunology, Medical Microbiology, Physical Sciences, Crystal Structure, integrity, Pathogens, Phosphorus-Oxygen Lyases, Crystallization, Life Sciences & Biomedicine, Dinucleoside Phosphates, 0605 Microbiology, Research Article, Adenylyl Cyclases, Methicillin-Resistant Staphylococcus aureus, Protein Structure, QH301-705.5, functional-analysis, growth, Immunology, Protein domain, Materials Science, bcs, Cyclase, Microbiology, Enzyme Regulation, resistance, 03 medical and health sciences, Bacterial Proteins, Protein Domains, Virology, Pseudomonas, Scattering, Small Angle, Genetics, medicine, Solid State Physics, Dimers, Escherichia coli, Microbial Pathogens, Molecular Biology, 030304 developmental biology, Science & Technology, Bacteria, Mutagenesis, Organisms, Biology and Life Sciences, Proteins, RC581-607, Polymer Chemistry, enzyme, Enzyme, chemistry, Phosphoglucomutase, Oligomers, Adenylyl Cyclase Inhibitors, Enzymology, Parasitology, Immunologic diseases. Allergy, scanning protein-a
Abstract: c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell., Author summary c-di-AMP has recently emerged as an essential molecule in Gram-positive bacteria, controlling osmotic stress resistance and virulence. The molecule has also been linked to antibiotic resistance/sensitivity, as alterations in its levels have been shown to modulate the effect of cell wall-targeting antibiotics. c-di-AMP is produced and degraded by dedicated enzymes and the activities of these enzymes are tightly regulated in the cell for optimal bacterial growth. In the human pathogen Staphylococcus aureus, c-di-AMP is produced by the di-adenylate cyclase enzyme DacA, which is thought to form a complex and be regulated by YbbR and GlmM, two other conserved proteins. There are ongoing efforts to devise strategies to inhibit di-adenylate cyclase enzymes, as inhibition of c-di-AMP production will negatively impact bacterial growth and also re-sensitize methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. Here, we investigated the activity and regulation of the S. aureus c-di-AMP cyclase DacA. We determined the atomic structures of the S. aureus DacA and GlmM enzymes and show that GlmM readily binds to DacA in vitro and blocks its activity by masking its active site. With this, our work provides important insight into possible ways to ablate c-di-AMP production in bacterial cells.
Published: 2019

22. 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

23. HupA, the main undecaprenyl pyrophosphate and phosphatidylglycerol phosphate phosphatase in Helicobacter pylori is essential for colonization of the stomach

Author: Martin V. Douglass, Samia Hicham, Dominique Mengin-Lecreulx, Chantal Ecobichon, Sophie Roure, Thierry Touzé, M. Stephen Trent, Ivo G. Boneca, Xudong Tian, Elise Gasiorowski, Rodolphe Auger, Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 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), Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia [USA], NIH grants AI129940 and AI38576 to M. Stephen Trent, DIM-Malinf grant n°140053 to Dominique Mengin-Lecreulx and Ivo G. Boneca, FRM, fellowship FDT20170436808 support to Elise Gasiorowski, 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), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris] (IP), GRENET, OLIVIER, BLANC - Elucidation du métabolisme de l'undécaprényl phosphate, un lipide essentiel pour la biosynthèse des polymères de la paroi bactérienne - - BACTOPRENYL2011 - ANR-11-BSV3-0002 - BLANC - VALID, and Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID
Subjects: QH301-705.5, [SDV]Life Sciences [q-bio], Immunology, Phosphatase, Phosphatidate Phosphatase, Mice, Inbred Strains, Microbial Sensitivity Tests, Microbiology, Dephosphorylation, Lipid A, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Mice, Biosynthesis, Polyisoprenyl Phosphates, Cell Wall, Virology, Genetics, Escherichia coli, Animals, Amino Acid Sequence, Biology (General), Pyrophosphatases, Molecular Biology, 030304 developmental biology, Polymyxin B, 0303 health sciences, Helicobacter pylori, Chemistry, Escherichia coli Proteins, 030302 biochemistry & molecular biology, Cell Membrane, Stomach, Phosphatidic acid, Phosphatidate phosphatase, RC581-607, Phosphoric Monoester Hydrolases, 3. Good health, [SDV] Life Sciences [q-bio], DNA-Binding Proteins, Biochemistry, Parasitology, Female, Cell envelope, Immunologic diseases. Allergy, Carrier Proteins, Research Article, Bacterial Outer Membrane Proteins
Abstract: The biogenesis of bacterial cell-envelope polysaccharides requires the translocation, across the plasma membrane, of sugar sub-units that are produced inside the cytoplasm. To this end, the hydrophilic sugars are anchored to a lipid phosphate carrier (undecaprenyl phosphate (C55-P)), yielding membrane intermediates which are translocated to the outer face of the membrane. Finally, the glycan moiety is transferred to a nascent acceptor polymer, releasing the carrier in the “inactive” undecaprenyl pyrophosphate (C55-PP) form. Thus, C55-P is generated through the dephosphorylation of C55-PP, itself arising from either de novo synthesis or recycling. Two types of integral membrane C55-PP phosphatases were described: BacA enzymes and a sub-group of PAP2 enzymes (type 2 phosphatidic acid phosphatases). The human pathogen Helicobacter pylori does not contain BacA homologue but has four membrane PAP2 proteins: LpxE, LpxF, HP0350 and HP0851. Here, we report the physiological role of HP0851, renamed HupA, via multiple and complementary approaches ranging from a detailed biochemical characterization to the assessment of its effect on cell envelope metabolism and microbe-host interactions. HupA displays a dual function as being the main C55-PP pyrophosphatase (UppP) and phosphatidylglycerol phosphate phosphatase (PGPase). Although not essential in vitro, HupA was essential in vivo for stomach colonization. In vitro, the remaining UppP activity was carried out by LpxE in addition to its lipid A 1-phosphate phosphatase activity. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies., Author summary Helicobacter pylori colonizes the human’s gastric mucosa and infects around 50% of the world’s population. This pathogen is responsible for chronic gastritis, peptic ulcers and in worst cases leads to gastric cancer. It has been classified as a class I carcinogen by the World Health Organization in 1994. Here, we show that HP0851, renamed HupA, is the major undecaprenyl pyrophosphate (C55-PP) phosphatase (UppP) and the major phosphatidylglycerol phosphate phosphatase (PGPase). This enzyme is also involved in cationic antimicrobial peptide (CAMP) resistance to which H. pylori hupA mutant shows an increased sensitivity (4 fold). This mutant was unable to colonize the stomach in mouse model of infection showing that even if hupA was not essential in vitro, this gene was essential in vivo. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies.
Published: 2019

24. 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

25. 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

26. Kinetic mechanism of Enterococcus faeciumd-aspartate ligase

Author: Peter Molek, Veronika Škedelj, Jure Stojan, Anamarija Zega, Didier Blanot, Jean-Luc Mainardi, Urša Pečar Fonović, Stanislav Gobec, Sophie Magnet, 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), 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), 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), Faculty of Pharmacy, University of Ljubljana, Institute of Biochemistry, Faculty of Medicine, Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), 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, and 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)
Subjects: 0301 basic medicine, Progress curve analysis, Stereochemistry, bacterial cell-walls, precursor, [SDV]Life Sciences [q-bio], Enterococcus faecium, peptidoglycan, Biochemistry, Pentapeptide repeat, 03 medical and health sciences, chemistry.chemical_compound, pyruvate-kinase, Biosynthesis, Lactate dehydrogenase, Penicillin-Binding Proteins, Nucleotide, Computer Simulation, time, chemistry.chemical_classification, DNA ligase, 030102 biochemistry & molecular biology, Chemistry, Kinetic mechanism, Substrate (chemistry), General Medicine, enzyme, Kinetics, 030104 developmental biology, Models, Chemical, D-aspartate ligase (Asl(fm)), acid, Peptidoglycan, biosynthesis, Pyruvate kinase
Abstract: International audience; Enterococcus faecium D-aspartate ligase (Aslfm) is a peptide bond-forming enzyme that is involved in the peptidoglycan assembly pathway. It catalyzes the ATP-dependent ligation of the β-carboxylate of D-Asp to the ε-amino group of L-Lys in the nucleotide precursor UDP- MurNAc-pentapeptide. The enzyme is of interest as a target of new, potential, narrow-spectrum antibiotics directed against multiresistant E. faecium. The kinetic mechanism of Aslfm has not been fully characterized. To determine it, a progress curve analysis of Aslfm catalytic process using pyruvate kinase/lactate dehydrogenase ATPase detection assay was performed. With an inspection of the shape of measured progress curves and the results of specific qualitative experiments, the Aslfm reaction mechanism was singled out. The proposed Aslfm kinetics reaction scheme was evaluated by fitting the parameters of the corresponding differential equations to progress curves using the computer program ENZO. The complete kinetic analysis result is consistent with the substrate binding order 1) ATP, 2) D-Asp, and 3) UDP-MurNAc-pentapeptide. The analysis suggests that slowly establishing non-productive equilibria between the free and ATP-bound enzyme with the participating pentapeptide are responsible for initial reaction burst followed by a steady-state period before the complete depletion of the reactant added in the lowest concentration.
Published: 2018

27. A road map for prioritizing warheads for cysteine targeting covalent inhibitors

Author: Urša Pečar Fonović, Janko Kos, Ana Mitrović, Péter Ábrányi-Balogh, György M. Keserű, László Petri, Martina Hrast, David I. Roper, Stanislav Gobec, Krisztina Németh, Tímea Imre, Peter A. Szijj, György G. Ferenczy, Hélène Barreteau, Janez Ilaš, Kata Horváti, Andrea Scarpino, Faculty of Pharmacy, University of Ljubljana, 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), 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)-Institut de Biologie Intégrative de la Cellule (I2BC)
Subjects: 0301 basic medicine, electrophiles, [SDV]Life Sciences [q-bio], design, Ligands, 01 natural sciences, Cathepsin B, Covalent inhibitors, drug discovery, 03 medical and health sciences, Structure-Activity Relationship, Nucleophile, MurA, cathepsin-b, fragments, Nucleophilic substitution, Humans, kinase inhibitors, Cysteine, Enzyme Inhibitors, Electrophilic warheads, GSH reactivity assay, Pharmacology, Nucleophilic addition, Alkyl and Aryl Transferases, Dose-Response Relationship, Drug, Molecular Structure, 010405 organic chemistry, Ligand, Chemistry, Organic Chemistry, selectivity, General Medicine, Combinatorial chemistry, 0104 chemical sciences, Oligopeptide specificity assay, reactivity, Cathepsin X, 030104 developmental biology, Covalent bond, Drug Design, Proteome, Michael reaction, proteases
Abstract: WOS:000450383500008; International audience; Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.
Published: 2018

28. 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

29. 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

30. In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB

Author: Delphine Patin, Julie A. Tod, David I. Roper, Martina Hrast, Christopher G. Dowson, Hélène Barreteau, Marko Jukič, Stanislav Gobec, Faculty of Pharmacy, University of Ljubljana, 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), 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)-Institut de Biologie Intégrative de la Cellule (I2BC)
Subjects: 0301 basic medicine, RM, Staphylococcus aureus, MICROBIO, In silico, [SDV]Life Sciences [q-bio], ENVBAC, Tetrazoles, Context (language use), 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Structure-Activity Relationship, Bacterial Proteins, Catalytic Domain, Drug Discovery, Escherichia coli, Tetrazole, Enzyme Inhibitors, Pharmacology, chemistry.chemical_classification, Virtual screening, Binding Sites, Organic Chemistry, Antimicrobial, Chemical space, QR, 3. Good health, 0104 chemical sciences, Anti-Bacterial Agents, Molecular Docking Simulation, 010404 medicinal & biomolecular chemistry, Kinetics, 030104 developmental biology, Enzyme, chemistry, Molecular Medicine, Organic synthesis, Carbohydrate Dehydrogenases
Abstract: In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N- acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacopohore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. In order to explore chemical space around hit compounds, further similarity search and organic synthesis was employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µM, respectively. None of the reported compounds possessed in vitro antimicrobial activity against S. aureus and E. coli.
Published: 2017

31. Discovery of new MurA inhibitors using induced-fit simulation and docking

Author: Janez Konc, Kaja Rožman, Hélène Barreteau, Delphine Patin, Matej Sova, Stanislav Gobec, Martina Hrast, Samo Lešnik, Dušanka Janežič, Boris Brus, 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, Stereochemistry, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Pharmaceutical Science, Peptidoglycan, 01 natural sciences, Biochemistry, Molecular Docking Simulation, 03 medical and health sciences, Mura, MurA inhibitors, Drug Discovery, Binding site, Enzyme Inhibitors, Molecular Biology, chemistry.chemical_classification, Alkyl and Aryl Transferases, 010405 organic chemistry, Drug discovery, Organic Chemistry, computer.file_format, Protein Data Bank, Small molecule, Combinatorial chemistry, 0104 chemical sciences, Small-molecule inhibitors, ProBiS-CHARMMing web server, 030104 developmental biology, Enzyme, Antibacterial agents, chemistry, Carbohydrate Sequence, Docking (molecular), Molecular Medicine, computer, induced fit
Abstract: We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1μM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.
Published: 2016

32. 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

33. 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

34. 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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34 results on '"Enveloppes Bactériennes et Antibiotiques ( ENVBAC )"'

1. 5′-Methylene-triazole-substituted-aminoribosyl uridines as MraY inhibitors: synthesis, biological evaluation and molecular modeling

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

3. Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

4. Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION

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

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

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

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

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

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

11. The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux

12. Anthranilic acid inhibitors of undecaprenyl pyrophosphate synthase (UppS), an essential enzyme for bacterial cell wall biosynthesis

13. Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum

14. The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b

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

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

17. Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

18. Synthesis and structure–activity relationship study of novel quinazolinone-based inhibitors of MurA

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

20. H Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases

21. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM

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

23. HupA, the main undecaprenyl pyrophosphate and phosphatidylglycerol phosphate phosphatase in Helicobacter pylori is essential for colonization of the stomach

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

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

26. Kinetic mechanism of Enterococcus faeciumd-aspartate ligase

27. A road map for prioritizing warheads for cysteine targeting covalent inhibitors

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

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

30. In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB

31. Discovery of new MurA inhibitors using induced-fit simulation and docking

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

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

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

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

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