21 results on '"Meriem El Ghachi"'
Search Results
2. Molecular architecture of the PBP2–MreC core bacterial cell wall synthesis complex
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Carlos Contreras-Martel, Alexandre Martins, Chantal Ecobichon, Daniel Maragno Trindade, Pierre-Jean Matteï, Samia Hicham, Pierre Hardouin, Meriem El Ghachi, Ivo G. Boneca, and Andréa Dessen
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Science - Abstract
Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.
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- 2017
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3. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.
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Guillaume Manat, Meriem El Ghachi, Rodolphe Auger, Karima Baouche, Samir Olatunji, Frédéric Kerff, Thierry Touzé, Dominique Mengin-Lecreulx, and Ahmed Bouhss
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Medicine ,Science - Abstract
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.
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- 2015
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4. Crosstalk between Helicobacter pylori and gastric epithelial cells is impaired by docosahexaenoic acid.
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Marta Correia, Valérie Michel, Hugo Osório, Meriem El Ghachi, Mathilde Bonis, Ivo G Boneca, Hilde De Reuse, António A Matos, Pascal Lenormand, Raquel Seruca, Ceu Figueiredo, Jose Carlos Machado, and Eliette Touati
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Medicine ,Science - Abstract
H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.
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- 2013
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5. Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.
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Marcelo Hill, Ala-Eddine Deghmane, Mercedes Segovia, Maria Leticia Zarantonelli, Gaëlle Tilly, Philippe Blancou, Gaëlle Bériou, Régis Josien, Ignacio Anegon, Eva Hong, Corinne Ruckly, Aude Antignac, Meriem El Ghachi, Ivo Gomperts Boneca, Muhamed-Kheir Taha, and Maria Cristina Cuturi
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Medicine ,Science - Abstract
Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1), CD80 (LOGEC50 = 4.88 µg/ml ± 0.15) and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.
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- 2011
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6. Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis
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Martin Caffrey, Frédéric Kerff, Chia-Ying Huang, R. Warshamanage, Vincent Olieric, Nicole Howe, Meitian Wang, Meriem El Ghachi, Phillip J. Stansfeld, Dietmar Weichert, Thierry Touzé, Centre d'Ingénierie des Protéines, Université de Liège, Trinity College Dublin, The Swiss Light Source (SLS) (SLS-PSI), Paul Scherrer Institute (PSI), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry [Oxford], University of Oxford [Oxford], ANR-11-BSV3-0002,BACTOPRENYL,Elucidation du métabolisme de l'undécaprényl phosphate, un lipide essentiel pour la biosynthèse des polymères de la paroi bactérienne(2011), Centre d’Ingénierie des Protéines [Université de Liège] = Centre for Protein Engineering [University of Liège] (CIP), and University of Oxford
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0301 basic medicine ,Science ,Molecular Sequence Data ,General Physics and Astronomy ,Peptidoglycan ,Crystallography, X-Ray ,Models, Biological ,01 natural sciences ,Article ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,Bacterial cell structure ,03 medical and health sciences ,chemistry.chemical_compound ,Polyisoprenyl Phosphates ,0103 physical sciences ,QD ,Amino Acid Sequence ,Pyrophosphatases ,lcsh:Science ,Multidisciplinary ,010304 chemical physics ,Escherichia coli Proteins ,General Chemistry ,Flippase ,Periplasmic space ,QP ,Phosphoric Monoester Hydrolases ,Cell biology ,QR ,A-site ,030104 developmental biology ,chemistry ,Membrane protein ,Structural biology ,Cytoplasm ,[SDV.IB]Life Sciences [q-bio]/Bioengineering ,lcsh:Q - Abstract
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., Bacterial cell wall components are assembled in a transmembrane cycle that involves the membrane integral pyrophosphorylase, BacA. Here the authors solve the crystal structure of BacA which shows an interdigitated inverted topology repeat that hints towards a flippase function for BacA.
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- 2018
7. Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis
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Alexandre Lambion, L. Vogeley, Dominique Mengin-Lecreulx, Sophie Roure, Juan-Carlos Rengifo-Gonzalez, Eric Sauvage, Sabine Peslier, Thierry Touzé, Paulette Charlier, Meriem El Ghachi, Frédéric Kerff, Maryline Foglino, Nicole Howe, Annick Guiseppi, Martin Caffrey, Guillaume Manat, François Delbrassine, Rodolphe Auger, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Trinity College Dublin, Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Schneider Electric (Grenoble), Schneider Electric, Transporteurs membranaires, chimioresistance et drug-design (TMCD2), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Club Rhumatismes et Inflammation, Centre d'Ingénierie des Protéines, Université de Liège-Institut de Chimie B6, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Laboratoire de chimie bactérienne ( LCB ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Transporteurs membranaires, chimioresistance et drug-design ( TMCD2 ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Schneider Electric ( SE), Centre d’Ingénierie des Protéines [Université de Liège] = Centre for Protein Engineering [University of Liège] (CIP), and Université de Liège
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Models, Molecular ,0301 basic medicine ,030106 microbiology ,Phosphatase ,Phosphatidate Phosphatase ,Bacillus subtilis ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Glycerophospholipids ,Crystallography, X-Ray ,Substrate Specificity ,[ SDE ] Environmental Sciences ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Catalytic Domain ,Hydrolase ,Escherichia coli ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Molecular Biology ,[ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Pharmacology ,chemistry.chemical_classification ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,biology ,[ SDV.BC ] Life Sciences [q-bio]/Cellular Biology ,Cell Membrane ,Genetic Complementation Test ,Active site ,Phosphatidylglycerols ,Cell Biology ,Phosphatidic acid ,biology.organism_classification ,Transmembrane protein ,Enzyme ,Solubility ,Biochemistry ,chemistry ,Genes, Bacterial ,[SDE]Environmental Sciences ,Mutagenesis, Site-Directed ,biology.protein ,Molecular Medicine ,[ SDV.GEN ] Life Sciences [q-bio]/Genetics - Abstract
International audience; 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 (C-55-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 C-55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 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.
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- 2017
8. Penicillin Resistance Compromises Nod1-Dependent Proinflammatory Activity and Virulence Fitness of Neisseria meningitidis
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Francina Langa Vives, Aude Antignac, Muhamed-Kheir Taha, Ala-Eddine Deghmane, Anna Skoczynska, Faridabano Nato, Philippe J. Sansonetti, Stephen E. Girardin, Ivo G. Boneca, Lucie Peduto, Françoise Thouron, Martine Fanton d'Andon, Thierry Pedron, Meriem El Ghachi, Marek Szatanik, Dana J. Philpott, Catherine Werts, Lionel LeBourhis, Maria Leticia Zarantonelli, Céline Mulet, Gérard Eberl, Neisseria, Institut Pasteur [Paris], National Reference Centre for Bacterial Meningitis [Warsaw, Poland] (NRCBM), National Medicines Institute - Narodowy Instytut Leków [Warsaw] (NIL), Infections Bactériennes Invasives, Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Groupe Avenir, Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Développement des Tissus Lymphoïdes, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre d'Ingénierie génétique murine - Mouse Genetics Engineering Center (CIGM), Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Immunité Innée et Signalisation, Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), Our research is supported by Institut Pasteur grants (PTR153 and PTR187), an ACI 0321 (Action Concertée Incitative Intégrée Microbiology) Grant from the Ministère chargé de la Recherche (INSERM No. MIC 0321), and a European Research Council starting grant (202283-PGNfromSHAPEtoVIR). A.S. was supported by a Marie Curie Intra-European Fellowship (No. 23188) within the Sixth European Community Framework Programme and Marie Curie European Reintegration Grants (No. 234876) within the Seventh European Community Framework Programme. M.E.G. was supported by an Institut Pasteur Roux fellowship., European Project: 234876,EC:FP7:PEOPLE,FP7-PEOPLE-ERG-2008,NMEN-PBP(2009), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Collège de France - Chaire Microbiologie et Maladies infectieuses
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Cancer Research ,Penicillin Resistance ,Virulence ,MESH: Nod1 Signaling Adaptor Protein ,Biology ,Neisseria meningitidis ,medicine.disease_cause ,Microbiology ,MESH: Neisseria meningitidis ,Proinflammatory cytokine ,Sepsis ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,MESH: Mutant Proteins ,Antibiotic resistance ,MESH: Cell Wall ,Cell Wall ,Virology ,Nod1 Signaling Adaptor Protein ,Immunology and Microbiology(all) ,medicine ,Animals ,Humans ,Penicillin-Binding Proteins ,MESH: Animals ,Pathogen ,MESH: Mice ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,MESH: Penicillin-Binding Proteins ,MESH: Humans ,030306 microbiology ,medicine.disease ,MESH: Penicillin Resistance ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,3. Good health ,Penicillin ,chemistry ,Parasitology ,Mutant Proteins ,[SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie ,Peptidoglycan ,medicine.drug - Abstract
International audience; Neisseria meningitidis is a life-threatening human bacterial pathogen responsible for pneumonia, sepsis, and meningitis. Meningococcal strains with reduced susceptibility to penicillin G (Pen(I)) carry a mutated penicillin-binding protein (PBP2) resulting in a modified peptidoglycan structure. Despite their antibiotic resistance, Pen(I) strains have failed to expand clonally. We analyzed the biological consequences of PBP2 alteration among clinical meningococcal strains and found that peptidoglycan modifications of the Pen(I) strain resulted in diminished in vitro Nod1-dependent proinflammatory activity. In an influenza virus-meningococcal sequential mouse model mimicking human disease, wild-type meningococci induced a Nod1-dependent inflammatory response, colonizing the lungs and surviving in the blood. In contrast, isogenic Pen(I) strains were attenuated for such response and were out-competed by meningococci sensitive to penicillin G. Our results suggest that antibiotic resistance imposes a cost to the success of the pathogen and may potentially explain the lack of clonal expansion of Pen(I) strains.
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- 2013
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9. Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily
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Didier Blanot, Samir Olatunji, Meriem El Ghachi, Bayan Al-Dabbagh, Ahmed Bouhss, Muriel Crouvoisier, Dominique Mengin-Lecreulx, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, College of Science, United Arab Emirates University (UAEU), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), and United Arab Emirates University
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0301 basic medicine ,Stereochemistry ,Substrate specificity ,[SDV]Life Sciences [q-bio] ,030106 microbiology ,Bacillus subtilis ,Peptidoglycan ,Biochemistry ,WecA ,MraY ,03 medical and health sciences ,chemistry.chemical_compound ,Transferases ,Uridine monophosphate ,Transferase ,Nucleotide ,Thermotoga maritima ,Amines ,chemistry.chemical_classification ,Aquifex aeolicus ,biology ,[ SDV ] Life Sciences [q-bio] ,Sequence Homology, Amino Acid ,Catalytic mechanism ,General Medicine ,biology.organism_classification ,Lipid Metabolism ,carbohydrates (lipids) ,030104 developmental biology ,chemistry ,Biocatalysis ,Cell envelope - Abstract
International audience; 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.
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- 2016
10. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori
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Sylviane Hoos, Christine Ebel, Alexandre Martins, Christine Schmitt, Frank Gabel, Chantal Ecobichon, Patrick England, Andréa Dessen, Pierre-Jean Matteï, Ivo G. Boneca, Meriem El Ghachi, Marie-Christine Prévost, and Frédéric Colland
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chemistry.chemical_classification ,0303 health sciences ,030306 microbiology ,Cell ,Periplasmic space ,biochemical phenomena, metabolism, and nutrition ,Biology ,Cell morphology ,Microbiology ,Yeast ,Bacterial cell structure ,Amino acid ,law.invention ,03 medical and health sciences ,medicine.anatomical_structure ,chemistry ,Biochemistry ,law ,polycyclic compounds ,Recombinant DNA ,Biophysics ,medicine ,Surface plasmon resonance ,Molecular Biology ,030304 developmental biology - Abstract
Summary The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.
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- 2011
11. Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes
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Didier Blanot, Meriem El Ghachi, Dominique Mengin-Lecreulx, Thierry Touzé, Hélène Barreteau, Michel Arthur, and Sophie Magnet
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Staphylococcus aureus ,Clinical Biochemistry ,medicine.disease_cause ,Biochemistry ,High-performance liquid chromatography ,Analytical Chemistry ,Cell wall ,Membrane Lipids ,chemistry.chemical_compound ,Polyisoprenyl Phosphates ,Escherichia coli ,medicine ,Chromatography, High Pressure Liquid ,Chromatography ,Terpenes ,Chemistry ,Cell Membrane ,Cell Biology ,General Medicine ,Membrane ,Undecaprenyl phosphate ,Peptidoglycan ,Undecaprenyl pyrophosphate - Abstract
Undecaprenyl phosphate is the essential lipid involved in the transport of hydrophilic motifs across the bacterial membranes during the synthesis of cell wall polymers such as peptidoglycan. A HPLC procedure was developed for the quantification of undecaprenyl phosphate and its two derivatives, undecaprenyl pyrophosphate and undecaprenol. During the exponential growth phase, the pools of undecaprenyl phosphate and undecaprenyl pyrophosphate were ca. 75 and 270 nmol/g of cell dry weight, respectively, in Escherichia coli, and ca. 50 and 150 nmol/g, respectively, in Staphylococcus aureus. Undecaprenol was detected in S. aureus (70 nmol/g), but not in E. coli (
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- 2009
12. The bacA Gene of Escherichia coli Encodes an Undecaprenyl Pyrophosphate Phosphatase Activity
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Didier Blanot, Dominique Mengin-Lecreulx, Ahmed Bouhss, and Meriem El Ghachi
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Cytoplasm ,Time Factors ,Detergents ,Mutant ,Transferases (Other Substituted Phosphate Groups) ,Bacitracin ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Catalysis ,Gene product ,Bacterial Proteins ,Polyisoprenyl Phosphates ,Transferases ,Drug Resistance, Bacterial ,Escherichia coli ,medicine ,Molecular Biology ,Gene ,chemistry.chemical_classification ,Expression vector ,Escherichia coli Proteins ,Cell Membrane ,Membrane Transport Proteins ,Cell Biology ,Phosphoric Monoester Hydrolases ,Phenotype ,Enzyme ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mutation ,Phosphorylation ,Electrophoresis, Polyacrylamide Gel ,Chromatography, Thin Layer ,Bacillus subtilis ,Plasmids ,medicine.drug - Abstract
The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784-3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by approximately 75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.
- Published
- 2004
13. Penicillin Binding Proteins as Danger Signals: Meningococcal Penicillin Binding Protein 2 Activates Dendritic Cells through Toll-Like Receptor 4
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Gaelle Beriou, Eva Hong, Philippe Blancou, Aude Antignac, Ala-Eddine Deghmane, Meriem El Ghachi, Gaëlle Tilly, Ignacio Anegon, Muhamed-Kheir Taha, Corinne Ruckly, Ivo G. Boneca, Maria Leticia Zarantonelli, Régis Josien, Marcelo Hill, Maria-Cristina Cuturi, Mercedes Segovia, INSERM UMR 643, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Universidad de la República [Montevideo] (UDELAR), Infections Bactériennes Invasives, Institut Pasteur [Paris] (IP), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Centre hospitalier universitaire de Nantes (CHU Nantes), Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), This work is supported by the Institut Pasteur, Fondation Progreffe and IMBIO. Meriem El Ghachi was supported by a post-doctoral Roux Fellowship (Institut Pasteur). Work in the group of Ivo G. Boneca is supported by an ERC starting Grant (PGNfromSHAPEtoVIR nu 202283)., European Project: 202283,EC:FP7:ERC,ERC-2007-StG,PGNFROMSHAPETOVIR(2008), Universidad de la República [Montevideo] (UCUR), Institut Pasteur [Paris], Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), and Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes
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Bacterial Diseases ,Penicillin binding proteins ,Neisseria meningitidis ,MESH: Dose-Response Relationship, Drug ,Mice ,0302 clinical medicine ,penicillin binding protein ,polycyclic compounds ,MESH: Animals ,Immune Response ,Cells, Cultured ,0303 health sciences ,Toll-like receptor ,MESH: Penicillin-Binding Proteins ,Multidisciplinary ,MESH: Dendritic Cells ,MESH: Toll-Like Receptor 4 ,méningocoque ,3. Good health ,Infectious Diseases ,Host-Pathogen Interactions ,B7-1 Antigen ,Medicine ,cellule dendritique ,Antibody ,MESH: B7-2 Antigen ,Research Article ,MESH: Cells, Cultured ,Science ,Immune Cells ,Immunology ,Biology ,MESH: Neisseria meningitidis ,Microbiology ,03 medical and health sciences ,Immune system ,In vivo ,Animals ,Humans ,Penicillin-Binding Proteins ,MESH: Mice ,030304 developmental biology ,CD86 ,MESH: Humans ,Dose-Response Relationship, Drug ,Pathogen-associated molecular pattern ,MESH: Host-Pathogen Interactions ,Immunity ,Histocompatibility Antigens Class II ,Dendritic Cells ,biochemical phenomena, metabolism, and nutrition ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Toll-Like Receptor 4 ,Immune System ,biology.protein ,MESH: Histocompatibility Antigens Class II ,MESH: B7-1 Antigen ,B7-2 Antigen ,CD80 ,030215 immunology - Abstract
International audience; Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to b-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 mg/ml60.1), CD80 (LOGEC50 = 4.88 mg/ml60.15) and CD86 (LOGEC50 = 5.36 mg/ml60.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.765.1% cells versus 1262.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed that PBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.
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- 2011
14. Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?
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Delphine Patin, Dominique Mengin-Lecreulx, Thierry Touzé, Aurélie Barnéoud-Arnoulet, Roland Lloubès, Ahmed Bouhss, Didier Blanot, Michel Arthur, Hélène Barreteau, Denis Duché, Emmanuelle Sacco, Meriem El Ghachi, Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Enveloppes Bactériennes et Antibiotiques, Département Microbiologie (Dpt Microbio), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Département Microbiologie ( Dpt Microbio ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -IFR58-Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris Descartes - Paris 5 ( UPD5 ), Laboratoire d'ingénierie des systèmes macromoléculaires ( LISM ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), and Enveloppes Bactériennes et Antibiotiques ( ENVBAC )
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Models, Molecular ,Isomerase activity ,Protein Conformation ,Colicins ,Peptidoglycan ,Biology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Bacteriocin ,Bacteriocins ,Antibiosis ,medicine ,Escherichia coli ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,030304 developmental biology ,0303 health sciences ,Lipid II ,030306 microbiology ,Periplasmic space ,Uridine Diphosphate N-Acetylmuramic Acid ,Anti-Bacterial Agents ,chemistry ,Colicin - Abstract
International audience; Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis-trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA-TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolated catalytic domain. Genes encoding proteins with similarity to the catalytic domain of ColM were identified in pathogenic strains of Pseudomonas and other genera. ColM acts on several structures of lipid II representative of the diversity of peptidoglycan chemotypes. All together, these data open the way to the potential use of ColM-related bacteriocins as broad spectrum antibacterial agents.
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- 2012
15. Characterization of colicin M and its orthologs targeting bacterial cell wall peptidoglycan biosynthesis
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Delphine Patin, Meriem El Ghachi, Ahmed Bouhss, Michel Arthur, Mark A. Brooks, Thierry Touzé, Dominique Mengin-Lecreulx, Hélène Barreteau, Denis Duché, Emmanuelle Sacco, Fabien Gérard, Roland Lloubès, Aurélie Barnéoud-Arnoulet, Herman van Tilbeurgh, Didier Blanot, Enveloppes Bactériennes et Antibiotiques, Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Département Microbiologie ( Dpt Microbio ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -IFR58-Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'ingénierie des systèmes macromoléculaires ( LISM ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Université Paris Descartes - Paris 5 ( UPD5 ), and Enveloppes Bactériennes et Antibiotiques ( ENVBAC )
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Microbiology (medical) ,Models, Molecular ,Lysis ,Immunology ,Colicins ,Peptidoglycan ,Biology ,medicine.disease_cause ,Microbiology ,Bacterial cell structure ,Substrate Specificity ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Bacteriocin ,Bacteriocins ,Cell Wall ,Pseudomonas ,medicine ,Escherichia coli ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,030304 developmental biology ,Pharmacology ,chemistry.chemical_classification ,0303 health sciences ,030306 microbiology ,Uridine Diphosphate N-Acetylmuramic Acid ,Protein Structure, Tertiary ,Enzyme ,chemistry ,Biochemistry ,Colicin - Abstract
International audience; For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.
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- 2012
16. 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
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Xavier Henry, Dominique Mengin-Lecreulx, Ahmed Bouhss, Claudine Parquet, Bayan Al-Dabbagh, Meriem El Ghachi, Didier Blanot, and Geneviève Auger
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Mutant ,Molecular Sequence Data ,Magnesium Chloride ,Transferases (Other Substituted Phosphate Groups) ,Bacillus subtilis ,Peptidoglycan ,Biochemistry ,Models, Biological ,Transformation, Genetic ,Bacterial Proteins ,Protein-fragment complementation assay ,Transferases ,Transferase ,Amino Acid Sequence ,Binding site ,Peptide sequence ,Integral membrane protein ,Binding Sites ,biology ,Genetic Complementation Test ,Active site ,Hydrogen-Ion Concentration ,biology.organism_classification ,Enzyme Activation ,Mutation ,biology.protein ,Mutagenesis, Site-Directed - Abstract
The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.
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- 2008
17. Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors
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Dominique Mengin-Lecreulx, Ahmed Bouhss, Thierry Touzé, Geneviève Auger, Didier Blanot, Meriem El Ghachi, and Hélène Barreteau
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Lysis ,Molecular Sequence Data ,Colicins ,Peptidoglycan ,Biology ,Biochemistry ,Cell wall ,chemistry.chemical_compound ,Bacitracin ,Biosynthesis ,Polyisoprenyl Phosphates ,medicine ,Escherichia coli ,Nucleotide ,Molecular Biology ,chemistry.chemical_classification ,Lipid II ,Base Sequence ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Lipids ,carbohydrates (lipids) ,chemistry ,Mechanism of action ,Models, Chemical ,Colicin ,Mutation ,bacteria ,lipids (amino acids, peptides, and proteins) ,Chromatography, Thin Layer ,medicine.symptom ,Plasmids - Abstract
Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.
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- 2006
18. BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance
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Dominique Mengin-Lecreulx, François Denizot, Remi Bernard, Meriem El Ghachi, and Marc Chippaux
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Reserpine ,Protein family ,Phosphatase ,Molecular Sequence Data ,Drug Resistance ,ATP-binding cassette transporter ,Bacitracin ,Bacillus subtilis ,medicine.disease_cause ,Biochemistry ,law.invention ,Bacterial Proteins ,Polyisoprenyl Phosphates ,law ,medicine ,Escherichia coli ,Cloning, Molecular ,Pyrophosphatases ,Molecular Biology ,biology ,Adrenergic Uptake Inhibitors ,Base Sequence ,Dose-Response Relationship, Drug ,Computational Biology ,Cell Biology ,biology.organism_classification ,Molecular biology ,Phosphoric Monoester Hydrolases ,Anti-Bacterial Agents ,Mutation ,Recombinant DNA ,ATP-Binding Cassette Transporters ,Electrophoresis, Polyacrylamide Gel ,Bacteria ,Gene Deletion ,medicine.drug ,Plasmids ,Protein Binding - Abstract
Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.
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- 2005
19. Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli
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Ahmed Bouhss, Anne Derbise, Dominique Mengin-Lecreulx, and Meriem El Ghachi
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Hot Temperature ,Time Factors ,Genotype ,Phosphatase ,Amino Acid Motifs ,Molecular Sequence Data ,Oligonucleotides ,Peptidoglycan ,Biology ,medicine.disease_cause ,Biochemistry ,Models, Biological ,Catalysis ,Gene product ,chemistry.chemical_compound ,Bacitracin ,Biosynthesis ,Polyisoprenyl Phosphates ,Cell Wall ,medicine ,Escherichia coli ,Nucleotide ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,chemistry.chemical_classification ,Base Sequence ,Escherichia coli Proteins ,Cell Membrane ,Temperature ,Cell Biology ,Lipids ,Phosphoric Monoester Hydrolases ,Phenotype ,Membrane protein ,chemistry ,Mutation ,Gene Deletion ,Plasmids - Abstract
The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106-30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant delta bacA,delta ybjG,delta pgpB in which the expression of bacA is impaired at 42 degrees C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.
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- 2005
20. Crosstalk between Helicobacter pylori and Gastric Epithelial Cells Is Impaired by Docosahexaenoic Acid
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Pascal Lenormand, Ceu Figueiredo, A. Alves de Matos, Marta Correia, José Carlos Machado, Hugo Osório, Valérie Michel, Eliette Touati, Raquel Seruca, Meriem El Ghachi, Mathilde Bonis, Ivo G. Boneca, Hilde De Reuse, Universidade do Porto = University of Porto, Pathogenèse de Helicobacter, Institut Pasteur [Paris] (IP), Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Centro Hospitalar de Lisboa Central E.P.E, Protéomique (Plate-Forme), This study was supported by the ERA-NET Pathogenomics (ERA-PTG/0001/2010) and by the Portuguese Foundation for Science and Technology (FCT -PTDC/SAU-SAP/120024/2010). MC is supported by an FCT fellowship BD/36689/2007. The Acçoes integradas Luso-Francesas (CRUP-PAULIF) Portugal and Francealso provided financial support (AF-8/09). Mathilde Bonis was supported by a PhD fellowship (Ministère de l’Enseignement Supérieur et de la Recherche, France).This study was also supported by the ERC starting grant (PGNfromSHAPEtoVIR number 202283) to Ivo G. Bonec, European Project: 202283,EC:FP7:ERC,ERC-2007-StG,PGNFROMSHAPETOVIR(2008), European Project: 116470,FCT::,ERA-PTG/2010,ERA-PTG/0001/2010(2011), Universidade do Porto, and Institut Pasteur [Paris]
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Bacterial Diseases ,MESH: Inflammation ,Antibacterial factors ,Lipopolysaccharide ,Anti-Inflammatory Agents ,Biochemistry ,Bacterial Adhesion ,Bacterial cell structure ,MESH: Stomach ,chemistry.chemical_compound ,Cell Wall ,Gastrointestinal Infections ,Helicobacter Infections/microbiology ,0303 health sciences ,education.field_of_study ,Multidisciplinary ,biology ,Fatty Acids ,Stomach ,Lipids ,3. Good health ,MESH: Docosahexaenoic Acids ,Infectious Diseases ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,MESH: Epithelial Cells ,Docosahexaenoic acid ,Medicine ,medicine.symptom ,Bacterial outer membrane ,Research Article ,Bacterial Outer Membrane Proteins ,Drugs and Devices ,Docosahexaenoic Acids ,Science ,Population ,Inflammation ,Gastroenterology and Hepatology ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Microbiology ,03 medical and health sciences ,MESH: Cell Wall ,medicine ,MESH: Bacterial Adhesion ,education ,Biology ,030304 developmental biology ,Helicobacter pylori ,030306 microbiology ,MESH: Bacterial Outer Membrane Proteins ,Immunity ,Epithelial Cells ,biology.organism_classification ,Pharmacodynamics ,chemistry ,MESH: Anti-Inflammatory Agents ,MESH: Helicobacter pylori ,Docosahexanoic acid DHA ,Bacteria - Abstract
International audience; H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.
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- 2013
21. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori
- Author
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El Ghachi, Meriem, Matteï, Pierre-Jean, Ecobichon, Chantal, Martins, Alexandre, Hoos, Sylviane, Schmitt, Christine, Colland, Frédéric, Ebel, Christine, Prévost, Marie-Christine, Gabel, Frank, England, Patrick, Dessen, Andréa, Boneca, Ivo G., Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Institut Pasteur [Paris] (IP), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale (plate-forme), Hybrigenics [Paris], Hybrigenics, Meriem El Ghachi was supported by a Roux fellowship (Institut Pasteur), ERC starting Grant (PGNfromSHAPEtoVIR n°202283) and INSERM. Ivo G. Boneca was supported by a Roux fellowship (Institut Pasteur) and a post-doctoral Fellowship from the Fundação de Ciências e Tecnologia, Portugal. Work in the group of IGB was supported by ERC starting grant (PGNfromSHAPEtoVIR n°202283). Work in the Dessen laboratory was supported by the Fondation pour la Recherche Médicale (FRM DEQ20090515390) and the European Commission (LSHM-CT-2004–512138). Pierre-Jean Matteï received a PhD fellowship from the Rhône Alpes region., We would like to acknowledge the technical help of Christian Vanderbergh with the production of the rabbit polyclonal antibodies, Hybrigenics for the Yeast two-hybrid screen, as well as Adam Round (BioSAXS beamline ID14-3, ESRF Grenoble, Partnership for Structural Biology) for help with SAXS data collection and analysis. We thank Aline Le Roy (analytical ultracentrifugation platform, IBS, PSB) for access to and assistance with AUC measurements., and European Project: 202283,EC:FP7:ERC,ERC-2007-StG,PGNFROMSHAPETOVIR(2008)
- Subjects
MESH: Penicillin-Binding Proteins ,MESH: Molecular Sequence Data ,Microbial Viability ,Helicobacter pylori ,[SDV]Life Sciences [q-bio] ,Molecular Sequence Data ,MESH: Sequence Alignment ,MESH: Amino Acid Sequence ,biochemical phenomena, metabolism, and nutrition ,MESH: Two-Hybrid System Techniques ,Protein Structure, Tertiary ,MESH: Protein Structure, Tertiary ,Bacterial Proteins ,Two-Hybrid System Techniques ,polycyclic compounds ,MESH: Helicobacter pylori ,MESH: Protein Binding ,Penicillin-Binding Proteins ,Amino Acid Sequence ,MESH: Bacterial Proteins ,Sequence Alignment ,MESH: Microbial Viability ,Protein Binding - Abstract
International audience; The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.
- Published
- 2011
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