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Accueil > Départements > Microbiologie > Thierry TOUZÉ : Enveloppes Bactériennes et Antibiotiques



  • H. Barreteau, M. Vandervennet, L. Guedon, V. Point, S. Canaan, S. Rebuffat, J. Peduzzi, et A. Carre-Mlouka, « Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment », International Journal of Systematic and Evolutionary Microbiology, vol. 69, nᵒ 3, p. 732-738, mars 2019.
    Résumé : A halophilic organism, SWO25(T) , was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25(T) - grew optimally at 35-45 degrees C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25(T) to the genus Haloarcula. The DNA G+C content was 61.1 mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25(T) is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarcula sebkhae sp. nov. is proposed. The type strain is SWO25(T)(=CIP 110583(T) =JCM 19018(T)).
    Mots-clés : 16s ribosomal-rna, dna hybridization, ENVBAC, gen. nov., genera, haloarchaea, Haloarcula, halophilic archaeon, heterogeneity, hypersaline environments, MICROBIO, mukohataei, organic-solvent tolerance, rapid method, salt lake, sebkha, sequence.

  • E. Gasiorowski, R. Auger, X. Tian, S. Hicham, C. Ecobichon, S. Roure, M. V. Douglass, M. S. Trent, D. Mengin-Lecreulx, T. Touzé, et I. G. Boneca, « HupA, the main undecaprenyl pyrophosphate and phosphatidylglycerol phosphate phosphatase in Helicobacter pylori is essential for colonization of the stomach », PLoS pathogens, vol. 15, nᵒ 9, p. e1007972, sept. 2019.
    Résumé : 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.
    Mots-clés : ENVBAC, MICROBIO.

  • M. Hrasta, K. Rozman, I. Ogris, V. Skedelj, D. Patin, M. Sova, H. Barreteau, S. Gobec, S. G. Grdadolnik, et A. Zega, « H Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases », Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 34, nᵒ 1, p. 1010-1017, janv. 2019.
    Résumé : 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.
    Mots-clés : antibacterial agents, Bacterial Mur (MurC-MurF) ligases, Bacterial Mur (MurC–MurF) ligases, crystal-structure, cytoplasmic steps, d-glutamate ligase, design, discovery, drugs, efficiency, ENVBAC, enzymes, l-alanine, MICROBIO, NMR studies, published kinase inhibitor set, purification, steady-state kinetics measurements.

  • F. Laddomada, M. M. Miyachiro, M. Jessop, D. Patin, V. Job, D. Mengin-Lecreulx, A. Le Roy, C. Ebel, C. Breyton, I. Gutsche, et A. Dessen, « The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis », Scientific Reports, vol. 9, nᵒ 1, p. 4656, mars 2019.
    Résumé : Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.
    Mots-clés : complexes, crystal-structure, ENVBAC, escherichia-coli murg, macromolecules, MICROBIO, precursors, proteins, purification, reveals, small-angle scattering, ultracentrifugation.

  • L. Latino, D. Patin, D. Cherier, T. Touze, C. Pourcel, H. Barreteau, et D. Mengin-Lecreulx, « Impact of FiuA Outer Membrane Receptor Polymorphism on the Resistance of Pseudomonas aeruginosa toward Peptidoglycan Lipid II-Targeting PaeM Pyocins », Journal of Bacteriology, vol. 201, nᵒ 13, p. e00164, juill. 2019.
    Résumé : 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.
    Mots-clés : acquisition, bacteriocins, cell wall, colicin M, colicin-m, crystal-structure, DBG, emergence, ENVBAC, escherichia-coli, FiuA, lipid II, locus variable-number, mechanism, MICROBIO, PaeM pyocins, peptidoglycan, protein, Pseudomonas aeruginosa, receptor structure-function, sequence, SSFA, TonB, tonb gene.

  • J. Maire, C. Vincent-Monegat, S. Balmand, A. Vallier, M. Herve, F. Masson, N. Parisot, A. Vigneron, C. Anselme, J. Perrin, J. Orlans, I. Rahioui, P. Da Silva, M. - O. Fauvarque, D. Mengin-Lecreulx, A. Zaidman-Remy, et A. Heddi, « Weevil pgrp-lb prevents endosymbiont TCT dissemination and chronic host systemic immune activation », Proceedings of the National Academy of Sciences of the United States of America, vol. 116, nᵒ 12, p. 5623-5632, mars 2019.
    Résumé : Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil Sitophilus zeamais's association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC-independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. pgrp-lb silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas pgrp-lb was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.
    Mots-clés : bacteria, coevolution, drosophila, endosymbiosis, ENVBAC, infection, innate immunity, insect, MICROBIO, mitochondrial, mutualist wigglesworthia, neisseria-gonorrhoeae, nod1, peptidoglycan recognition proteins, PGRP.

  • A. Massarweh, M. Bosco, I. Chantret, T. Léger, L. Jamal, D. I. Roper, C. G. Dowson, P. Busca, A. Bouhss, C. Gravier-Pelletier, et S. E. H. Moore, « Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities », Molecules (Basel, Switzerland), vol. 24, nᵒ 11, p. 2135, juin 2019.
    Résumé : Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.
    Mots-clés : alanyl-d-alanine, congenital disorders of glycosylation, congenital disorders of glycosylation, endoplasmic reticulum, ENVBAC, glycosylation, lipid-linked oligosaccharide, MICROBIO, peptidoglycan biosynthesis, peptidoglycan biosynthesis, precursors, protein N-glycosylation.

  • V. Škedelj, U. P. Fonović, P. Molek, S. Magnet, J. - L. Mainardi, D. Blanot, S. Gobec, J. Stojan, et A. Zega, « Kinetic mechanism of Enterococcus faecium D-aspartate ligase », Biochimie, vol. 158, p. 217-223, janv. 2019.
    Résumé : 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.
    Mots-clés : acid, bacterial cell-walls, biosynthesis, D-aspartate ligase (Asl(fm)), Enterococcus faecium, ENVBAC, enzyme, Kinetic mechanism, MICROBIO, Progress curve analysis.

  • T. Tosi, F. Hoshiga, C. Millership, R. Singh, C. Eldrid, D. Patin, D. Mengin-Lecreulx, K. Thalassinos, P. Freemont, et A. Gründling, « Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM », PLoS pathogens, vol. 15, nᵒ 1, p. e1007537, janv. 2019.
    Résumé : 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.
    Mots-clés : cyclic dinucleotide, degradation, domain, ENVBAC, enzyme, functional-analysis, growth, integrity, MICROBIO, mobility mass-spectrometry, resistance, scanning protein-a.


  • P. Abranyi-Balogh, L. Petri, T. Imre, P. Szijj, A. Scarpino, M. Hrast, A. Mitrovic, U. P. Fonovic, K. Nemeth, H. Barreteau, D. I. Roper, K. Horvati, G. G. Ferenczy, J. Kos, J. Ilas, S. Gobec, et G. M. Keseru, « A road map for prioritizing warheads for cysteine targeting covalent inhibitors », European Journal of Medicinal Chemistry, vol. 160, p. 94-107, déc. 2018.
    Résumé : 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.
    Mots-clés : Cathepsin B, Cathepsin X, cathepsin-b, Covalent inhibitors, design, drug discovery, electrophiles, Electrophilic warheads, ENVBAC, fragments, GSH reactivity assay, kinase inhibitors, MICROBIO, MurA, Oligopeptide specificity assay.

  • M. El Ghachi, N. Howe, C. - Y. Huang, V. Olieric, R. Warshamanage, T. Touzé, D. Weichert, P. J. Stansfeld, M. Wang, F. Kerff, et M. Caffrey, « Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis », Nature Communications, vol. 9, nᵒ 1, p. 1078, 2018.
    Résumé : As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.
    Mots-clés : Amino Acid Sequence, Crystallography, X-Ray, ENVBAC, Escherichia coli Proteins, MICROBIO, Models, Biological, Molecular Sequence Data, Peptidoglycan, Phosphoric Monoester Hydrolases, Polyisoprenyl Phosphates, Protein Structure, Secondary, Pyrophosphatases.

  • M. J. Fer, L. Le Corre, N. Pietrancosta, N. Evrard-Todeschi, S. Olatunji, A. Bouhss, S. Calvet-Vitale, et C. Gravier-Pelletier, « Bacterial Transferase MraY, a Source of Inspiration towards New Antibiotics », Current Medicinal Chemistry, vol. 25, nᵒ 42, p. 6013-6029, 2018.
    Résumé : 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 structureactivity 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.
    Mots-clés : 1st membrane step, acetylmuramyl-pentapeptide-translocase, antimicrobial resistance, biological evaluation, biological evaluations, cell-wall, docking, ENVBAC, escherichia-coli, lysis protein e, MICROBIO, molecular modelling, MraY, mutagenesis, nucleoside antibiotics, peptidoglycan synthesis, ribofuranosyl nucleoside moiety, stereocontrolled synthesis, triazoles.

  • M. Fonvielle, A. Bouhss, C. Hoareau, D. Patin, D. Mengin-Lecreulx, L. Iannazzo, N. Sakkas, A. El Sagheer, T. Brown, M. Etheve-Quelquejeu, et M. Arthur, « Synthesis of Lipid-Carbohydrate-Peptidyl-RNA Conjugates to Explore the Limits Imposed by the Substrate Specificity of Cell Wall Enzymes on the Acquisition of Drug Resistance », Chemistry (Weinheim an Der Bergstrasse, Germany), vol. 24, nᵒ 56, p. 14911-14915, oct. 2018.
    Résumé : Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNA(Gly) and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 = 56 nm). The synthetic route was exploited to generate substrates and inhibitors containing D-lactate residue (D-Lac) instead of D-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.
    Mots-clés : Bacterial Proteins, Carbohydrates, Cell Wall, complex, Drug Discovery, Drug Resistance, Bacterial, ENVBAC, Enzyme Inhibitors, Fem, Humans, Lipids, mechanism, MICROBIO, mrsa, oligonucleotide, Peptidoglycan, proteins, RNA, RNA conjugates, small interfering rna, Solid-Phase Synthesis Techniques, Staphylococcal Infections, Staphylococcus aureus, staphylococcus-aureus, Substrate Specificity, targeted delivery, VanA, vancomycin.
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  • M. Hrast, M. Jukič, D. Patin, J. Tod, C. G. Dowson, D. I. Roper, H. Barreteau, et S. Gobec, « In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB », Chemical Biology & Drug Design, vol. 91, nᵒ 6, p. 1101-1112, 2018.

  • M. Jukič, K. Rožman, M. Sova, H. Barreteau, et S. Gobec, « Anthranilic Acid Inhibitors of Undecaprenyl Pyrophosphate Synthase (UppS), an Essential Enzyme for Bacterial Cell Wall Biosynthesis », Frontiers in Microbiology, vol. 9, p. 3322, 2018.
    Résumé : 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).
    Mots-clés : antibacterial agents, cell-wall, diphosphate synthase, ENVBAC, inhibitors, ligands, mechanism, MICROBIO, pharmacophore model, states, undecaprenyl pyrophosphate synthase, UppS.

  • X. Liu, N. Y. Meiresonne, A. Bouhss, et T. den Blaauwen, « FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli », Molecular Microbiology, vol. 109, nᵒ 6, p. 855-884, sept. 2018.
    Résumé : Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in E. coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of MurJ recruitment during cell division. This article is protected by copyright. All rights reserved.
    Mots-clés : 1st membrane step, bacillus-subtilis, beta-lactam antibiotics, divisome, E. coli, ENVBAC, flippase, flippase activity, lipid II, MICROBIO, penicillin-binding proteins, peptidoglycan, peptidoglycan synthesis, terminal domain, udp-n-acetylglucosamine, wall synthesis.

  • F. Ngadjeua, E. Braud, S. Saidjalolov, L. Iannazzo, D. Schnappinger, S. Ehrt, J. - E. Hugonnet, D. Mengin-Lecreulx, D. P. Patin, M. Ethève-Quelquejeu, M. Fonvielle, et M. Arthur, « Critical impact of peptidoglycan precursor amidation on the activity of L,D-transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis », Chemistry (Weinheim an Der Bergstrasse, Germany), vol. 24, p. 5743-5747, févr. 2018.
    Résumé : The bacterial cell wall peptidoglycan contains unusual L and D amino acids assembled in branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by L,D-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the L,D-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essentiality of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.
    Mots-clés : Amidation, Amidotransferase, ENVBAC, MICROBIO, Mycobacterium tuberculosis, Peptidoglycan, Transpeptidase.

  • F. Pompeo, D. Byrne, D. Mengin-Lecreulx, et A. Galinier, « Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth », Scientific Reports, vol. 8, nᵒ 1, p. 1660, 2018.


  • M. Bosco, A. Massarweh, S. Iatmanen-Harbi, A. Bouhss, I. Chantret, P. Busca, S. E. H. Moore, et C. Gravier-Pelletier, « Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP) », European Journal of Medicinal Chemistry, vol. 125, p. 952-964, janv. 2017.
    Résumé : Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [(3)H]OSP from [(3)H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.
    Mots-clés : Animals, Biological evaluation, CDG, Diphosphatase, Disubstituted diphosphates, Dolichol, Dolichol Phosphates, ENVBAC, Glycochemistry, Humans, liver, MICROBIO, Monoterpenes, Oligosaccharides, Phosphoric Diester Hydrolases, Phosphoric Monoester Hydrolases, Phosphosugars, Polyisoprenyl Phosphate Sugars, Polyisoprenyl Phosphates, Substrate Specificity.

  • S. Di Gregorio, S. Fernandez, A. Cuirolo, O. Verlaine, A. Amoroso, D. Mengin-Lecreulx, A. Famiglietti, B. Joris, et M. Mollerach, « Different Vancomycin-Intermediate Staphylococcus aureus Phenotypes Selected from the Same ST100-hVISA Parental Strain », Microbial Drug Resistance (Larchmont, N.Y.), vol. 23, nᵒ 1, p. 44-50, janv. 2017.
    Résumé : The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.
    Mots-clés : ENVBAC, hVISA, MICROBIO, MRSA, ST100, Staphylococcus aureus, vancomycin, VISA.

  • M. E. Ghachi, N. Howe, R. Auger, A. Lambion, A. Guiseppi, F. Delbrassine, G. Manat, S. Roure, S. Peslier, E. Sauvage, L. Vogeley, J. - C. Rengifo-Gonzalez, P. Charlier, D. Mengin-Lecreulx, M. Foglino, T. Touzé, M. Caffrey, et F. Kerff, « Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis », Cellular and molecular life sciences: CMLS, févr. 2017.
    Résumé : Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.
    Mots-clés : Bacterial lipids metabolism, ENVBAC, Membrane protein structure, MICROBIO, Peptidoglycan-related lipid, Undecaprenyl phosphate.

  • M. Hrast, K. Rožman, M. Jukič, D. Patin, S. Gobec, et M. Sova, « Synthesis and structure-activity relationship study of novel quinazolinone-based inhibitors of MurA », Bioorganic & Medicinal Chemistry Letters, mai 2017.
    Résumé : 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.
    Mots-clés : Antibacterial agents, ENVBAC, MICROBIO, MurA enzyme, Peptidoglycan, Quinazolinones.

  • K. Rožman, S. Lešnik, B. Brus, M. Hrast, M. Sova, D. Patin, H. Barreteau, J. Konc, D. Janežič, et S. Gobec, « Discovery of new MurA inhibitors using induced-fit simulation and docking », Bioorganic & Medicinal Chemistry Letters, vol. 27, nᵒ 4, p. 944-949, févr. 2017.
    Résumé : 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.
    Mots-clés : Antibacterial agents, ENVBAC, induced fit, MICROBIO, MurA inhibitors, ProBiS-CHARMMing web server, Small-molecule inhibitors.

  • M. - Y. Su, N. Som, C. - Y. Wu, S. - C. Su, Y. - T. Kuo, L. - C. Ke, M. - R. Ho, S. - R. Tzeng, C. - H. Teng, D. Mengin-Lecreulx, M. Reddy, et C. - I. Chang, « Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc », Nature Communications, vol. 8, nᵒ 1, p. 1516, nov. 2017.
    Résumé : 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.
    Mots-clés : ENVBAC, MICROBIO.


  • B. Al-Dabbagh, S. Olatunji, M. Crouvoisier, M. El Ghachi, D. Blanot, D. Mengin-Lecreulx, et A. Bouhss, « Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily », Biochimie, vol. 127, p. 249-257, août 2016.
    Résumé : The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the β-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed.
    Mots-clés : Amines, Bacillus subtilis, Biocatalysis, ENVBAC, Lipid Metabolism, MICROBIO, MraY, Peptidoglycan, Sequence Homology, Amino Acid, Substrate Specificity, Thermotoga maritima, Transferases, WecA.

  • D. Chérier, S. Giacomucci, D. Patin, A. Bouhss, T. Touzé, D. Blanot, D. Mengin-Lecreulx, et H. Barreteau, « Pectocin M1 (PcaM1) Inhibits Escherichia coli Cell Growth and Peptidoglycan Biosynthesis through Periplasmic Expression », Antibiotics (Basel, Switzerland), vol. 5, nᵒ 4, oct. 2016.
    Résumé : Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum, pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli, this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics.
    Mots-clés : bacteriocin, colicin, ENVBAC, MICROBIO, pectocin M1, Peptidoglycan, periplasmic expression.

  • M. Fonvielle, N. Sakkas, L. Iannazzo, C. Le Fournis, D. Patin, D. Mengin-Lecreulx, A. El-Sagheer, E. Braud, S. Cardon, T. Brown, M. Arthur, et M. Etheve-Quelquejeu, « Electrophilic RNA for Peptidyl-RNA Synthesis and Site-Specific Cross-Linking with tRNA-Binding Enzymes », Angewandte Chemie (International Ed. in English), vol. 55, nᵒ 43, p. 13553-13557, oct. 2016.
    Résumé : RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3' end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv . The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity.
    Mots-clés : crosslinking, ENVBAC, Fem transferases, MICROBIO, post-functionalization, RNA modifications, squarates.

  • J. - E. Hugonnet, D. Mengin-Lecreulx, A. Monton, T. den Blaauwen, E. Carbonnelle, C. Veckerlé, Y. V. Brun, M. van Nieuwenhze, C. Bouchier, K. Tu, L. B. Rice, et M. Arthur, « Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli », eLife, vol. 5, oct. 2016.
    Résumé : The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB.
    Mots-clés : (p)ppGpp, E. coli, ENVBAC, infectious disease, L,D-transpeptidase, mecillinam, MICROBIO, Microbiology, PBP1b, PBP5, β-lactam.

  • I. Iatsenko, S. Kondo, D. Mengin-Lecreulx, et B. Lemaitre, « PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway », Immunity, vol. 45, nᵒ 5, p. 1013-1023, nov. 2016.
    Résumé : Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.
    Mots-clés : ENVBAC, MICROBIO.

  • K. F. Naqvi, D. Patin, M. S. Wheatley, M. A. Savka, R. C. J. Dobson, H. M. Gan, H. Barreteau, D. Blanot, D. Mengin-Lecreulx, et A. O. Hudson, « Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136(T) », Frontiers in Microbiology, vol. 7, p. 362, 2016.
    Résumé : The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.
    Mots-clés : bacterial cell wall, ENVBAC, fusion enzyme, MICROBIO, MurB, MurC, Peptidoglycan, UDP-N-acetylenolpyruvoylglucosamine reductase, UDP-N-acetylmuramate:l-alanine ligase, Verrucomicrobium spinosum.

  • D. Patin, S. Turk, H. Barreteau, J. - L. Mainardi, M. Arthur, S. Gobec, D. Mengin-Lecreulx, et D. Blanot, « Unusual substrate specificity of the peptidoglycan MurE ligase from Erysipelothrix rhusiopathiae », Biochimie, vol. 121, p. 209-218, févr. 2016.
    Résumé : Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.
    Mots-clés : ENVBAC, Erysipelothrix, Erysipelothrix rhusiopathiae, Escherichia coli, l-alanine-adding enzyme, meso-diaminopimelate-adding enzyme, MICROBIO, MurE, Peptide Synthases, Peptidoglycan, Substrate Specificity.

  • R. Šink, M. Kotnik, A. Zega, H. Barreteau, S. Gobec, D. Blanot, A. Dessen, et C. Contreras-Martel, « Crystallographic Study of Peptidoglycan Biosynthesis Enzyme MurD: Domain Movement Revisited », PloS One, vol. 11, nᵒ 3, p. e0152075, 2016.
    Résumé : The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.
    Mots-clés : Crystallography, X-Ray, ENVBAC, Escherichia coli, MICROBIO, Peptide Synthases, Peptidoglycan, Protein Structure, Tertiary.


  • N. Cumby, K. Reimer, D. Mengin-Lecreulx, A. R. Davidson, et K. L. Maxwell, « The phage tail tape measure protein, an inner membrane protein and a periplasmic chaperone play connected roles in the genome injection process of E. coli phage HK97 », Molecular Microbiology, vol. 96, nᵒ 3, p. 437-447, mai 2015.
    Résumé : Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15-mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.
    Mots-clés : Coliphages, DNA, Viral, ENVBAC, Escherichia coli, Escherichia coli Proteins, Genome, Viral, Membrane Proteins, MICROBIO, Molecular Chaperones, Peptidylprolyl Isomerase, Periplasmic Proteins, Phosphoenolpyruvate Sugar Phosphotransferase System, Viral Tail Proteins, Virus Internalization.

  • M. J. Fer, A. Bouhss, M. Patrão, L. Le Corre, N. Pietrancosta, A. Amoroso, B. Joris, D. Mengin-Lecreulx, S. Calvet-Vitale, et C. Gravier-Pelletier, « 5'-Methylene-triazole-substituted-aminoribosyl uridines as MraY inhibitors: synthesis, biological evaluation and molecular modeling », Organic & Biomolecular Chemistry, vol. 13, nᵒ 26, p. 7193-7222, juill. 2015.
    Résumé : 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).
    Mots-clés : Anti-Bacterial Agents, Bacillus subtilis, Bacterial Proteins, Catalytic Domain, Chemistry Techniques, Synthetic, ENVBAC, Enzyme Inhibitors, Microbial Sensitivity Tests, MICROBIO, Models, Molecular, Transferases, Triazoles, Uridine.

  • M. Levefaudes, D. Patin, C. de Sousa-d'Auria, M. Chami, D. Blanot, M. Hervé, M. Arthur, C. Houssin, et D. Mengin-Lecreulx, « Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION », The Journal of Biological Chemistry, vol. 290, nᵒ 21, p. 13079-13094, mai 2015.
    Résumé : 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.
    Mots-clés : Amides, Amino Acid Sequence, Anti-Bacterial Agents, Antibiotics, bacterial metabolism, Bacterial Proteins, Blotting, Western, Cell Wall, Cells, Cultured, CORYNE, Corynebacteriales, Corynebacterium, DAP amidation, Diaminopimelic Acid, ENVBAC, enzyme, gene knockout, glutaminase, Immunoenzyme Techniques, lysozyme, MICROBIO, Microscopy, Electron, Transmission, Molecular Sequence Data, Muramidase, Mutation, Peptidoglycan, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transaminases.

  • A. Lipski, M. Hervé, V. Lombard, D. Nurizzo, D. Mengin-Lecreulx, Y. Bourne, et F. Vincent, « Structural and biochemical characterization of the β-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family », Glycobiology, vol. 25, nᵒ 3, p. 319-330, mars 2015.
    Résumé : 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.
    Mots-clés : Acetylglucosaminidase, Amino Acid Sequence, Bacterial Proteins, Catalytic Domain, catalytic mechanism, ENVBAC, MICROBIO, Molecular Sequence Data, Peptidoglycan, Phylogeny, Thermotoga maritima, X-ray structure, β-N-acetylglucosaminidase.

  • G. Manat, M. El Ghachi, R. Auger, K. Baouche, S. Olatunji, F. Kerff, T. Touzé, D. Mengin-Lecreulx, et A. Bouhss, « Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase », PloS One, vol. 10, nᵒ 11, p. e0142870, 2015.
    Résumé : Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane.
    Mots-clés : Amino Acid Motifs, Amino Acid Sequence, Arginine, Catalysis, Cell Membrane, ENVBAC, Escherichia coli, Escherichia coli Proteins, Genetic Complementation Test, Glutamine, Lipids, Membrane Proteins, MICROBIO, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphatidate Phosphatase, Phosphoric Monoester Hydrolases, Phosphorylation, Phosphotransferases (Phosphate Group Acceptor), Protein Structure, Tertiary, Sequence Homology, Amino Acid, Serine, Substrate Specificity.

  • D. Patin, M. Bayliss, D. Mengin-Lecreulx, P. Oyston, et D. Blanot, « Purification and biochemical characterisation of GlmU from Yersinia pestis », Archives of Microbiology, vol. 197, nᵒ 3, p. 371-378, avr. 2015.
    Résumé : Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents.
    Mots-clés : Acetyltransferases, Amino Acid Sequence, ENVBAC, Enzyme Activation, Escherichia coli, Hydrogen-Ion Concentration, Kinetics, Magnesium, Mercaptoethanol, MICROBIO, Molecular Sequence Data, Nucleotidyltransferases, Oxidants, Oxidation-Reduction, Sequence Alignment, Yersinia pestis.

  • A. Perdih, M. Hrast, K. Pureber, H. Barreteau, S. G. Grdadolnik, D. Kocjan, S. Gobec, T. Solmajer, et G. Wolber, « Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization », Journal of Computer-Aided Molecular Design, vol. 29, nᵒ 6, p. 541-560, juin 2015.
    Résumé : 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.
    Mots-clés : Adenosine Triphosphate, Anti-Bacterial Agents, Bacterial Proteins, Binding Sites, Carboxylic Acids, Drug Design, Drug Evaluation, Preclinical, ENVBAC, Enzyme Inhibitors, Furans, ligases, MICROBIO, Molecular Dynamics Simulation, Structure-Activity Relationship.

  • C. Soudais, F. Samassa, M. Sarkis, L. Le Bourhis, S. Bessoles, D. Blanot, M. Hervé, F. Schmidt, D. Mengin-Lecreulx, et O. Lantz, « In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells », Journal of Immunology (Baltimore, Md.: 1950), vol. 194, nᵒ 10, p. 4641-4649, mai 2015.
    Résumé : 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.
    Mots-clés : Animals, Disease Models, Animal, ENVBAC, Escherichia coli, Escherichia coli Infections, Flow Cytometry, Histocompatibility Antigens Class I, In Vitro Techniques, Ligands, Lymphocyte Activation, Mice, Mice, Inbred C57BL, Mice, Transgenic, MICROBIO, Minor Histocompatibility Antigens, Mucous Membrane, Natural Killer T-Cells, Riboflavin.

  • Q. Xu, D. Mengin-Lecreulx, X. W. Liu, D. Patin, C. L. Farr, J. C. Grant, H. - J. Chiu, L. Jaroszewski, M. W. Knuth, A. Godzik, S. A. Lesley, M. - A. Elsliger, A. M. Deacon, et I. A. Wilson, « Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains », mBio, vol. 6, nᵒ 5, p. e02327-02314, sept. 2015.
    Résumé : Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCE: Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.
    Mots-clés : Aminopeptidases, Bacterial Proteins, Catalytic Domain, Crystallography, X-Ray, DNA Mutational Analysis, ENVBAC, MICROBIO, Models, Molecular, Mutant Proteins, Protein Conformation, src Homology Domains, Substrate Specificity.
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Liste de publications (2008-2014)


Xu Q., Mengin-Lecreulx D., Patin D., Grant J.C., Chiu H.-J., Jaroszewski L., Knuth M.W., Godzik A., Lesley S.A., Elsliger M.A., Deacon A.M., and Wilson I.A. 2014. Structure-guided functional characterization of DUF1460 reveals a highly specific NlpC/P60 amidase family. Structure 22, 1799-1809.

Denoël T., Zervosen A., Lemaire C., Joris B., Hervé M., Blanot D., Zaragoza G., and Luxen A. 2014. Enantioselective synthesis of α-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan. Org. Biomol. Chem. 12, 9853-9863.

Denoël T., Zervosen A., Gerards T., Lemaire C., Joris B., Blanot D., and Luxen A. 2014. Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli. Bioorg. Med. Chem. 22, 4621-4628.

Perdih A., Hrast M., Barreteau H., Gobec S., Wolber G., and Solmajer T. 2014. Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF). Bioorg. Med. Chem. 22, 4124-4134.

Manat G., Roure S., Auger R., Bouhss A., Barreteau H., Mengin-Lecreulx D., and Touzé T. 2014. Deciphering the metabolism of undecaprenyl-phosphate : the bacterial cell-wall unit carrier at the membrane frontier. Microb. Drug Resist. 20, 199-214.

Perdih A., Hrast M., Barreteau H., Gobec S., Wolber G., and Solmajer T. 2014. Inhibitor design strategy based on an enzyme structural flexibility : A case of bacterial MurD ligase. J. Chem. Inf. Model. 54, 1451-1466.

Hrast M., Anderluh M., Knez D., Randall C.P., Barreteau H., O’Neill A.J., Blanot D., and Gobec S. 2014. Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold. Eur. J. Med. Chem. 73, 83-96.


Šink R., Barreteau H., Patin D., Mengin-Lecreulx D., Gobec S., and Blanot D. 2013. MurD enzymes : some recent developments. BioMol. Concepts 4, 539-556.

Das D., Hervé M., Elsliger M.-A., Kadam R.U., Grant J., Chiu H.-J., Knuth M.W., Klock H.E., Miller M.D., Godzik A., Lesley S.A., Deacon A.M., Mengin-Lecreulx D., and Wilson I.A. 2013. Structure and function of a novel LD-carboxypeptidase A involved in peptidoglycan recycling. J. Bacteriol. 195, 5555-5566.

Ruane K.M., Lloyd A.J., Fülöp V., Dowson C.G., Barreteau H., Boniface A., Dementin S., Blanot D., Mengin-Lecreulx D., Gobec S., Dessen A., and Roper D.I. 2013. Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE ternary complex. J. Biol. Chem. 288, 33439-33448.

Fer M.J., Olatunji S., Bouhss A., Calvet-Vitale S., and Gravier-Pelletier C. 2013. Toward analogs of MraY natural inhibitors : Synthesis of 5’-triazole-substituted-aminoribosyl uridines through a Cu-catalyzed azide-alkyne cycloaddition. J. Org. Chem. 78, 10088-10105.

Škedelj V., Perdih A., Brvar M., Kroflič A., Dubbée V., Savage V., O’Neill A.J., Solmajer T., Bešter-Rogač M., Blanot D., Hugonnet J.-E., Magnet S., Arthur M., Mainardi J.-L., Stojan J., and Zega A. 2013. Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm). Eur. J. Med. Chem. 67, 208-220.

Bazot S., Barthes L., Blanot D. and Fresneau C. 2013. Distribution of non-structural nitrogen and carbohydrate reserves in mature oak trees in a temperate forest at four key phenological stages. Trees-Structure and Function 27, 1023-1034.

Ammam F., Meziane-cherif D., Mengin-Lecreulx D., Blanot D., Patin D., Boneca I.G., Courvalin P., Lambert T., and Candela T. 2013. The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance. Mol. Microbiol. 89, 612-625.

Turk S., Hrast M., Sosič I, Barreteau H., Mengin-Lecreulx D., Blanot D., and Gobec S. 2013. Biochemical characterization of MurF from Streptococcus pneumoniae and the identification of a new MurF inhibitor through ligand-based virtual screening. Acta Chim. Slov. 60, 294-299.

Hrast M., Turk S., Sosič I., Knez D., Randall C.P., Barreteau H., Contreras-Martel C., Dessen A., O’Neill A.J., Mengin-Lecreulx D., Blanot D., and Gobec S. 2013. Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF. Eur. J. Med. Chem. 66, 32-45.

Fonvielle M., Li de La Sierra-Gallay I., El-Sagheer A.H., Lecerf M., Patin D., Mellal D., Mayer C., Blanot D., Gale N., Brown T., van Tilbeurgh H., Ethève-Quelquejeu M., and Arthur M. 2013. The structure of FemXWv in complex with a peptidyl-RNA conjugate : Mechanism of aminoacyl transfer from Ala-tRNAAla to peptidoglycan precursors. Angew. Chem. Int. Ed. 52, 7278-7281.

Rubino S.J., Magalhaes J.G., Philpott D., Bahr G.M., Blanot D., Girardin S.E. 2013. Identification of a synthetic muramyl peptide derivative with enhanced Nod2 stimulatory capacity. Innate Immun. 19, 493-503.

McGroty S.E., Pattaniyil D.T., Patin D., Blanot D., Ravichandran A.C., Suzuki H., Dobson R.C.J., Savka M.A., and Hudson A.O. 2013. Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate : meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T. PLoS One 8, e66458.

Fonvielle M., Mellal D., Patin D., Lecerf M., Blanot D., Bouhss A., Santarem M., Mengin-Lecreulx D., Sollogoub M., Arthur M., and Ethève-Quelquejeu M. 2013. Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemXWv aminoacyl-transferase. Chemistry 19, 1357-1363.

Hervé M., Kovač A., Cardoso C., Patin D., Brus B., Barreteau H., Mengin-Lecreulx D., Gobec S., and Blanot D. 2013. Synthetic tripeptides as alternate substrates of murein peptide ligase (Mpl). Biochimie 95, 1120-1126.


Simčič M., Sosič I., Hodošček M., Barreteau H., Blanot D., Gobec S., and Golič Grdadolnik S. 2012. The binding mode of second-generation sulfonamide inhibitors of MurD : clues for the rational design of potent MurD inhibitors. PLoS One 7, e52817.

Touzé T., Barreteau H., El Ghachi M., Bouhss A., Barnéoud-Arnoulet A., Patin D., Sacco E., Blanot D., Arthur M., Duché D., Lloubès R., and Mengin-Lecreulx D. 2012. Colicin M, a peptidoglycan lipid II-degrading enzyme : potential use for antibacterial means ? Biochem. Soc. Trans. 40, 1522-1527.

Maqbool A., Hervé M., Mengin-Lecreulx D., Wilkinson A.J., and Thomas G.H. 2012. MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan derived peptides in γ-proteobacteria. Biochem. J. 448, 329-341.

Barreteau H., Tiouajni M., Graille M., Josseaume N., Bouhss A., Patin D., Blanot D., Fourgeaud M., Mainardi J.-L., Arthur M., van Tilbeurgh H., Mengin-Lecreulx D., and Touzé T. 2012. Functional and structural characterization of PaeM, a colicin M-like bacteriocin from Pseudomonas aeruginosa. J. Biol. Chem. 287, 37395-37405.

Tomašić, T., Sink, R., Zidar, N., Fic, A., Contreras-Martel, C., Dessen, A., Patin, D., Blanot, D., Müller-Premru, M., Gobec, S., Zega, A., Kikelj, D., and Peterlin Mašič, L. 2012. Dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus. ACS Med. Chem. Lett. 3, 626-630.

Barreteau H., Sosič I., Turk S., Humljan J., Tomašić T., Zidar N., Hervé M., Boniface A., Peterlin-Mašič L., Kikelj D., Mengin-Lecreulx D., Gobec S., and Blanot D. 2012. MurD enzymes from different bacteria : evaluation of inhibitors. Biochem. Pharmacol. 84, 625-632.

Barreteau H., El Ghachi M., Barnéoud-Arnoulet A., Sacco E., Touzé T., Duché D., Gérard F., Brooks M., Patin D., Bouhss A., Blanot D., van Tilbeurgh H., Arthur M., Lloubès R., and Mengin-Lecreulx D. 2012. Characterization of colicin M and its orthologues targeting bacterial cell wall peptidoglycan biosynthesis. Microb. Drug Resist. 18, 222-229.

Amoroso A., Boudet J., Berzigotti S., Duval V., Teller N., Mengin-Lecreulx D., Luxen A., Simorre J.-P., and B. Joris. 2012. A peptidoglycan fragment triggers β-lactam resistance in Bacillus licheniformis. PLoS Pathogens 8, e1002571.

Patin, D., Bostock J., Chopra I., Mengin-Lecreulx D., and Blanot D. 2012. Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem. Arch. Microbiol.194, 505-512.

Patin, D., Barreteau H., Auger G., Magnet S., Crouvoisier M., Bouhss A., Touzé T., Arthur M., Mengin-Lecreulx D., and Blanot D. 2012. Colicin M hydrolyses branched lipids II from Gram-positive bacteria. Biochimie 94, 985-990.

Tomašić T., Kovač A., Klebe G., Blanot D., Gobec S., Kikelj D., and Peterlin Mašič L. 2012. Virtual screening for potential inhibitors of bacterial MurC and MurD ligases. J. Mol. Model. 18, 1063-1072.

Marchand C.H., Salmeron C., Bou Raad R., Méniche X., Chami M., Masi M., Blanot D., Daffé M., Tropis M., Huc E., Le Maréchal P., Decottignies P., and Bayan N. 2012. Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum. J Bacteriol. 194, 587-597.

Blanot D., Lee J., and Girardin SE. 2012. Synthesis and biological evaluation of biotinyl hydrazone derivatives of muramyl peptides. Chem. Biol. Drug Des. 79, 2-8.


Auberger N., Frlan R., Al-Dabbagh B., Bouhss A., Crouvoisier M., Gravier-Pelletier C., and Le Merrer Y. 2011. Synthesis and biological evaluation of potential new inhibitors of the bacterial transferase MraY with a β-ketophosphonate structure. Org. Biomol. Chem. 9, 8301-8312.

Tanino T., Al-Dabbagh B., Mengin-Lecreulx D., Bouhss A., Oyama H., Ichikawa S., and Matsuda A. 2011. Mechanistic analysis of muraymycin analogues : A guide to the design of MraY inhibitors. J. Med. Chem. 54, 8421-8439.

Zidar N., Tomašić T., Sink R., Kovač A., Patin D., Blanot D., Contreras-Martel C., Dessen A., Premru M.M., Zega A., Gobec S., Mašič L.P., and Kikelj D. 2011. New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase : Design, synthesis, crystal structures, and biological evaluation. Eur. J. Med. Chem. 46, 5512-5523.

Ma Y., Muench D., Schneider T., Sahl H.G., Bouhss A., Ghoshdastider U., Wang J., Doetsch V., Wang X., and Bernhard F. 2011. Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes. J. Biol. Chem. 286, 38844-38853.

Maqbool A., Levdikov V.M., Blagova E.V., Hervé M., Horler R.S., Wilkinson A.J., and Thomas G.H. 2011. Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein. J. Biol. Chem. 286, 31512-31521.

Tomašić T., Kovač A., Simčič M., Blanot D., Grdadolnik S.G., Gobec S., Kikelj D., and Peterlin Mašič L. 2011. Novel 2-thioxothiazolidin-4-one inhibitors of bacterial MurD ligase targeting D-Glu- and diphosphate-binding sites. Eur. J. Med. Chem. 46, 3964-3975.

Tomasic T., Zidar N., Šink R., Kovac A., Blanot D., Contreras-Martel C., Dessen A., Müller-Premru M., Zega A., Gobec S., Kikelj D., and Peterlin Mašič L. 2011. Structure-based design of a new series of D-glutamic acid-based inhibitors of bacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). J. Med. Chem. 54, 4600-4610.

Sosič I., Barreteau H., Simčič M., Sink R., Cesar J., Zega A., Grdadolnik S.G., Contreras-Martel C., Dessen A., Amoroso A., Joris B., Blanot D., and Gobec S. 2011. Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme : Activity optimisation with conformationally rigid analogues of D-glutamic acid. Eur. J. Med. Chem. 46, 2880-2894.

Frlan R., Kovač A., Blanot D., Gobec S., Pečar S., and Obreza A. 2011. Design, synthesis and in vitro biochemical activity of novel amino acid sulfonohydrazide inhibitors of MurC. Acta Chim. Sloven. 58, 295-310.

Živec M., Turk S., Blanot D., and Gobec, S. 2011. Design and synthesis of new peptidomimetics as potential inhibitors of MurE. Acta Chim. Slov. 58, 95-109.

Olrichs N. K., Aarsman M. E. G., Verheul J., Arnusch C. J., Martin N. I., Hervé M., Vollmer W., de Kruijff B., Breukink E. and den Blaauwen T. 2011. A novel in vivo cell-wall labeling approach sheds new light on peptidoglycan synthesis in Escherichia coli. ChemBioChem 12, 1124-1133.

Mohammadi T., van Dam V., Sijbrandi R., Vernet T., Zapun A., Bouhss A., Diepeveen-de Bruin M., Nguyen-Distèche M., de Kruijff B., and Breukink E. 2011. Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane. EMBO J. 30, 1425-1432.

Das D., Hervé M., Feuerhelm J., Farr C. L., Chiu H.-J., Elsliger M.-A., Knuth M.W., Klock H.E., Miller M.D., Godzik A., Lesley S.A., Deacon A.M., Mengin-Lecreulx D., and Wilson I.A. 2011. Structure and function of the first full-length murein peptide ligase (Mpl) cell wall recycling protein. PLoS One 6, e17624.

Zaidman-Rémy A., Poidevin M., Hervé M., Welchman D.P., Paredes J.C., Fahlander C., Steiner H., Mengin-Lecreulx D., and Lemaitre B. 2011. Drosophila immunity : analysis of PGRP-SB1 expression, enzymatic activity and function. PLoS One 6, e17231.

Mravljak J., Monasson O., Al-Dabbagh B., Crouvoisier M., Bouhss A., Gravier-Pelletier C., and Le Merrer Y. 2011. Synthesis and biological evaluation of a diazepanone-based library of liposidomycins analogs as MraY inhibitors. Eur. J. Med. Chem. 46, 1582-1592.

Gérard F., Brooks M.A., Barreteau H., Touzé T., Graille M., Bouhss A., Blanot D., van Tilbeurgh H., and Mengin-Lecreulx D. 2011. X-ray structure and site-directed mutagenesis analysis of the Escherichia coli colicin M immunity protein. J. Bacteriol. 193, 205-214.


Paracuellos P., Ballandras A., Robert X., Kahn R., Hervé M., Mengin-Lecreulx D., Cozzone A.J., Duclos B., and Gouet P. 2010. The extended conformation of the 2.9 Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus. J. Mol. Biol. 404, 847-858.

Zidar N., Tomašić T., Šink R., Rupnik V., Kovač A., Turk S., Patin D., Blanot D., Contreras Martel C., Dessen A., Müller Premru M., Zega A., Gobec S., Peterlin Mašič L., and Kikelj D. 2010. Discovery of novel 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD ligase. J. Med. Chem. 53, 6584-6594.

Tanino T., Ichikawa S., Al-Dabbagh B., Bouhss A., Oyama H., and Matsuda A. 2010. Synthesis and biological evaluation of muraymycin analogues against anti-drug-resistant bacteria. ACS Med. Chem. Lett. 1, 758-762.

Barnéoud-Arnoulet A., Barreteau H., Touzé T., Mengin-Lecreulx D., Lloubès R., and Duché D. 2010. Toxicity of the colicin M catalytic domain exported to the periplasm is FkpA independent. J. Bacteriol. 192, 5212-5219.

Patin D., Boniface A., Kovač A., Hervé M., Dementin S., Barreteau H., Mengin-Lecreulx D., and Blanot D. 2010. Purification and biochemical characterization of Mur ligases from Staphylococcus aureus. Biochimie 92, 1793-1800.

Fonvielle M., Chemama M., Lecerf M., Villet R., Busca P., Bouhss A., Ethève-Quelquejeu M., and Arthur M. 2010. Decoding the logic of the tRNA regiospecificity of nonribosomal FemXWv aminoacyl transferase. Angew. Chem. Int. Ed. 49, 5115-5119.

Lecerclé D., Clouet A., Al-Dabbagh B., Crouvoisier M., Bouhss A., Gravier-Pelletier C., and Le Merrer Y. 2010. Bacterial transferase MraY inhibitors : synthesis and biological evaluation. Bioorg. Med. Chem. 18, 4560-4569.

Ii K., Ichikawa S., Al-Dabbagh B., Bouhss A., and Matsuda A. 2010. Function-oriented synthesis of simplified caprazamycins : discovery of oxazolidine-containing uridine derivatives as antibacterial agents against drug-resistant bacteria. J. Med. Chem. 53, 3793-3813.

Barreteau H., Bouhss A., Gérard F., Duché D., Boussaid B., Blanot D., Lloubès R., Mengin-Lecreulx D., and Touzé T. 2010. Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II degrading enzyme. J. Biol. Chem. 285, 12378-12389.

Sosič I., Štefane B., Kovač A., Turk A., Blanot D., and Gobec S. 2010. The synthesis of novel 2,4,6-trisubstituted 1,3,5-triazines : a search for potential MurF enzyme inhibitors. Heterocycles 81, 91-115.

Sacco E., Hugonnet J.E., Josseaume N., Cremniter J., Dubost L., Marie A., Patin D., Blanot D., Rice L.B., Mainardi J.-L., and Arthur M. 2010. Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium. Mol. Microbiol. 75, 874-875.

Tomašić T., Zidar N., Kovač A., Turk S., Simčič M., Blanot D., Müller-Premru M., Filipič M., Golič Grdadolnik S., Zega A., Anderluh M., Gobec S., Kikelj D., and Peterlin Mašič L. 2010. 5-Benzylidenethiazolidin-4-ones as multitarget inhibitors of bacterial Mur ligases. ChemMedChem 5, 286-295.


Gendrin M., Welchman D.P., Poidevin M., Hervé M., and Lemaitre B. 2009. Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila. PLoS Pathogens 5, e1000694.

Patin D., Bostock J., Blanot D., Mengin-Lecreulx D., and Chopra I. 2009. Functional and biochemical analysis of Chlamydia trachomatis MurE ligase. J. Bacteriol. 191, 7430-7435.

Sova M., Kovač A., Turk S., Hrast M., Blanot D., and Gobec S. 2009. Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF. Bioorg Chem. 37, 217-222.

Hynninen A., Touzé T., Pitkänen L., Mengin-Lecreulx D., and Virta M. 2009. An efflux transporter PbrA and a phosphatase PbrB cooperate in a lead-resistance mechanism in bacteria. Mol. Microbiol. 74, 384-394.

Bouhss A., Al-Dabbagh B., Vincent M., Odaert B., Aumont-Nicaise M., Bressolier P., Desmadril M., Mengin-Lecreulx D., Urdaci M.C., and Gallay J. 2009. Specific interactions of clausin, a new lantibiotic, with lipid precursors of the bacterial cell wall. Biophysical J. 97, 1390-1397.

Boniface A., Parquet C., Arthur M., Mengin-Lecreulx D., and Blanot D. 2009. The elucidation of the structure of Thermotoga maritima peptidoglycan reveals two novel types of cross-link. J. Biol. Chem. 284, 21856-21862.

Al-Dabbagh B., Blanot D., Mengin-Lecreulx D., and Bouhss A. 2009. Preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-N-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers. Anal. Biochem. 391, 163-165.

Perdih A., Kovač A., Wolber G., Blanot D., Gobec S., and Solmajer T. 2009. Discovery of novel benzene 1,3-dicarboxylic acid inhibitors of bacterial MurD and MurE ligases by structure-based virtual screening approach. Bioorg. Med. Chem. Lett. 19, 2668-2673.

Barreteau H., Bouhss A., Fourgeaud M., Mainardi J.-L., Touzé T., Gérard F., Blanot D., Arthur M., and Mengin-Lecreulx D. 2009. Human and plant pathogenic Pseudomonas species produce bacteriocins exhibiting colicin M-like hydrolase activity towards peptidoglycan precursors. J. Bacteriol. 191, 3657-3664.

Blake K.L., O’Neill A.J., Mengin-Lecreulx D., Henderson P.J., Bostock J.M., Dunsmore C.J., Simmons K.J., Fishwick C.W., Leeds J.A., and Chopra I. 2009. The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors. Mol. Microbiol. 72, 335-343.

Turk S., Kovač A., Boniface A., Bostock J.M., Chopra I., Blanot D., and Gobec S. 2009. Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening. Bioorg. Med. Chem. 17, 1884-1889.

Pennartz A., Généreux C., Parquet C., Mengin-Lecreulx D., and Joris B. 2009. Substrate-induced inactivation of the Escherichia coli AmiD N-acetylmuramoyl-L-alanine amidase highlights a new strategy to inhibit this class of enzyme. Antimicrob. Agents Chemother. 53, 2991-2997.

Fonvielle M., Chemama M., Villet R., Lecerf M., Bouhss A., Valéry J.M., Ethève-Quelquejeu M., and Arthur M. 2009. Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis. Nucleic Acids Res. 37, 1589-1601.

Barreteau H., Magnet S., El Ghachi M., Touzé T., Arthur M., Mengin-Lecreulx D. and Blanot D. 2009. Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes. J. Chromatogr. B 877, 213-220.

Tomašić T., Zidar N., Rupnik V., Kovač A., Blanot D., Gobec S., Kikelj D., Peterlin Mašič L. 2009. Synthesis and biological evaluation of new glutamic acid-based inhibitors of MurD ligase. Bioorg. Med. Chem. Lett. 19, 153-157.

Bui N.K., Gray J., Schwarz H., Schumann P., Blanot D., and Vollmer W. 2009. The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development. J. Bacteriol. 191, 494-505.


Humljan J., Kotnik M., Contreras-Martel C., Blanot D., Urleb U., Dessen A., Šolmajer T., and Gobec S. 2008. Novel naphthalene-N-sulfonyl-D-glutamic acid derivatives as inhibitors of MurD, a key peptidoglycan biosynthesis enzyme. J. Med. Chem. 51, 7486-7494.

Fiuza M., Canova M.J., Patin D., Letek M., Zanella-Cléon I., Becchi M., Mateos L.M., Mengin-Lecreulx D., Molle V., and Gil J.A. 2008. The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum. J. Biol. Chem. 283, 36553-36563.

Al-Dabbagh B., Mengin-Lecreulx D., and Bouhss A. 2008. Purification and characterization of the bacterial UDP-GlcNAc : undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA. J. Bacteriol. 190, 7141-7146. 18723618

Terrak M., Sauvage E., Derouaux A., Dehareng D., Bouhss A., Breukink E., Jeanjean S., and Nguyen-Distèche M. 2008. Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli. J. Biol. Chem. 283, 28464-28470.

Sink R., Kovac A., Tomašic T., Rupnik V., Boniface A., Bostock J., Chopra I., Blanot D., Mašic L.P., Gobec S., and Zega A. 2008. Synthesis and biological evaluation of N-acylhydrazones as inhibitors of MurC and MurD ligases. ChemMedChem. 3, 1362-1370.

Al-Dabbagh B., Henry X., El Ghachi M., Auger G., Blanot D., Parquet C., Mengin-Lecreulx D., and Bouhss A. 2008. Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis. Biochemistry 47, 8919-8928.

Marr N., Tirsoaga A., Blanot D., Fernandez R., and Caroff M. 2008. Glucosamine found as a substituent of both phosphate groups in Bordetellae lipid A backbones : role of a BvgAS-activated ArnT ortholog. J. Bacteriol. 190, 4281-4290.

Lavollay M., Arthur M., Fourgeaud M., Dubost L., Marie A., Veziris N., Blanot D., Gutmann L., and Mainardi J.L. 2008. The peptidoglycan of stationary phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J. Bacteriol. 190, 4360-4366.

Touzé T., Blanot D., and Mengin-Lecreulx D. 2008. Substrate specificity and membrane topology of Escherichia coli PGPB, an undecaprenyl pyrophosphate phosphatase. J. Biol. Chem. 283, 16573-16583.

Absalon C., Hamze K., Blanot D., Fréhel C., Carballido-Lopez R., Holland I.B., van Heijenoort J., and Séror S.J. 2008. The GTPase, CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis. J. Bacteriol. 190, 3786-3790.

Clouet, A., Gravier-Pelletier C., Al-Dabbagh B., Bouhss A., and Le Merrer Y. 2008. Efficient synthesis of a bacterial translocase MraY inhibitor. Tetrahedron : Asymmetry 19, 397-400.

Barreteau H., Kovač A., Boniface A., Sova M., Gobec S., and Blanot D. 2008. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 168-207.

Frlan R., Kovač A., Blanot D., Gobec S., Pečar S., and Obreza A. 2008. Design and synthesis of novel N-benzylidenesulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents. Molecules 13, 11-30.

Nuevo M., Auger G., Blanot D., and d’Hendecourt L. 2008. A detailed analysis of the amino acids produced after the vacuum UV irradiation of interstellar ice analogs. Origins Life Evol. Biosph. 38, 37-56.

Vollmer W., Blanot D., and de Pedro M.A. 2008. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149-167.

Bouhss A., Trunkfield A.E., Bugg T.D,, and Mengin-Lecreulx D. 2008. The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol. Rev. 32, 208-233.

Touzé, T., Tran A.X., Hankins J.V., Mengin-Lecreulx D., and Trent M.S. 2008. Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate. Mol. Microbiol. 67, 264-277.

Touzé T., and Mengin-Lecreulx D. 2008. Undecaprenyl phosphate synthesis. EcoSal Plus.

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