76 results on '"Anne Marie Di Guilmi"'
Search Results
2. Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori
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Prashant P. Damke, Anne Marie Di Guilmi, Paloma Fernández Varela, Christophe Velours, Stéphanie Marsin, Xavier Veaute, Mérick Machouri, Gaurav V. Gunjal, Desirazu N. Rao, Jean-Baptiste Charbonnier, and J. Pablo Radicella
- Subjects
Science - Abstract
Some bacteria can take up DNA molecules from the environment. Here, Damke et al. identify a DNA-binding protein in Helicobacter pylori that is required for DNA import into the periplasm and that interacts with an inner-membrane channel that translocates the DNA into the cytoplasm.
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- 2019
- Full Text
- View/download PDF
3. Streptococcus pneumoniae GAPDH Is Released by Cell Lysis and Interacts with Peptidoglycan.
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Rémi Terrasse, Ana Amoroso, Thierry Vernet, and Anne Marie Di Guilmi
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Medicine ,Science - Abstract
Release of conserved cytoplasmic proteins is widely spread among Gram-positive and Gram-negative bacteria. Because these proteins display additional functions when located at the bacterial surface, they have been qualified as moonlighting proteins. The GAPDH is a glycolytic enzyme which plays an important role in the virulence processes of pathogenic microorganisms like bacterial invasion and host immune system modulation. However, GAPDH, like other moonlighting proteins, cannot be secreted through active secretion systems since they do not contain an N-terminal predicted signal peptide. In this work, we investigated the mechanism of GAPDH export and surface retention in Streptococcus pneumoniae, a major human pathogen. We addressed the role of the major autolysin LytA in the delivery process of GAPDH to the cell surface. Pneumococcal lysis is abolished in the ΔlytA mutant strain or when 1% choline chloride is added in the culture media. We showed that these conditions induce a marked reduction in the amount of surface-associated GAPDH. These data suggest that the presence of GAPDH at the surface of pneumococcal cells depends on the LytA-mediated lysis of a fraction of the cell population. Moreover, we demonstrated that pneumococcal GAPDH binds to the bacterial cell wall independently of the presence of the teichoic acids component, supporting peptidoglycan as a ligand to surface GAPDH. Finally, we showed that peptidoglycan-associated GAPDH recruits C1q from human serum but does not activate the complement pathway.
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- 2015
- Full Text
- View/download PDF
4. A multidimensional strategy to detect polypharmacological targets in the absence of structural and sequence homology.
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Jacob D Durrant, Rommie E Amaro, Lei Xie, Michael D Urbaniak, Michael A J Ferguson, Antti Haapalainen, Zhijun Chen, Anne Marie Di Guilmi, Frank Wunder, Philip E Bourne, and J Andrew McCammon
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Biology (General) ,QH301-705.5 - Abstract
Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.
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- 2010
- Full Text
- View/download PDF
5. Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA sampling and recruitment to oxidized bases in living cells
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Ostiane D’Augustin, Virginie Gaudon, Capucine Siberchicot, Rebecca Smith, Catherine Chapuis, Jordane Depagne, Xavier Veaute, Didier Busso, Anne-Marie Di Guilmi, Bertrand Castaing, J Pablo Radicella, Anna Campalans, Sébastien Huet, Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Stabilité génétique, cellules souches et radiations (SGCSR (U_1274 / UMR_E_008)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université Paris Cité (UPCité), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), Ligue contre le Cancer du Grand-Ouest [committee 22], Ligue contre le Cancer du Grand-Ouest [committee 29], Ligue contre le Cancer du Grand-Ouest [committee 35], Ligue contre le Cancer du Grand-Ouest [committee 37], Ligue contre le Cancer du Grand-Ouest [committee 45], Institut Universitaire de France, Region Centre-Val de Loire [2013-00082978, 2017-00117252], Canceropole Grand-Ouest [project CONCERTO] [2018-001240994], Fondation ARC pour la recherche sur le cancer [PDF20181208405, PJA20181207762], Commissariat a l'Energie Atomique (CEA) Radiobiology program, Agence Nationale de la Recherche [PRCI-2018 TG-TOX], Region Bretagne, CEA, Ligue Nationale Contre le Cancer, and Commissariat al'Energie Atomique
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[SDV.GEN]Life Sciences [q-bio]/Genetics ,Genetics - Abstract
The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly samples the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This sampling process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the sampling of the DNA and recruitment to oxidative lesions.
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- 2023
6. OGG1 competitive inhibitors show important off-target effects by directly inhibiting efflux pumps and disturbing mitotic progression
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Xhaferr Tanushi, Guillaume Pinna, Marie Vandamme, Capucine Siberchicot, Ostiane D’Augustin, Anne-Marie Di Guilmi, J. Pablo Radicella, Bertrand Castaing, Rebecca Smith, Sebastien Huet, François Leteurtre, Anna Campalans, Stabilité génétique, cellules souches et radiations (SGCSR (U_1274 / UMR_E_008)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université Paris Cité (UPCité), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), Campalans lab received funding from the Commissariat à l'Energie Atomique (CEA) Radiobiology program, Electricité de France and from the Agence Nationale de la Recherche (ANR PRCI-2018 TG-TOX). Castaing lab received funding from Région Centre Val de Loire (MoOGly-2017-00117252)., and Jonchère, Laurent
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SU0268 ,OGG1 inhibitor ,TH5487 ,Cell Biology ,8-oxoG ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Base Excision Repair (BER) ,OGG1 ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,Developmental Biology - Abstract
One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity—by interfering with metaphase completion—resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.
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- 2023
7. OGG1 at the Crossroads Between Repair and Transcriptional Regulation
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Anne-Marie Di Guilmi, Nuria Fonknechten, and Anna Campalans
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- 2023
8. Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells
- Author
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Ostiane D’Augustin, Virginie Gaudon, Capucine Siberchicot, Rebecca Smith, Catherine Chapuis, Jordane Depagne, Xavier Veaute, Didier Busso, Anne-Marie Di Guilmi, Bertrand Castaing, J. Pablo Radicella, Anna Campalans, and Sébastien Huet
- Abstract
The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.
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- 2022
9. ComF is a key mediator in single-stranded DNA transport and handling during natural transformation
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Anne Marie Di Guilmi, Pierre Legrand, Stéphanie Marsin, Sumedha M. Kondekar, Sophie Quevillon-Cheruel, Julien Vercruyssen, Hélène Walbott, Jordane Depagne, Louisa Celma, Raphael Guerois, Prashant P. Damke, Xavier Veaute, J. Pablo Radicella, Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut 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), Synchrotron SOLEIL (SSOLEIL), and Centre National de la Recherche Scientifique (CNRS)
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0303 health sciences ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,030306 microbiology ,Chemistry ,DNA transport ,Virulence ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Genome ,Cell biology ,Cell membrane ,03 medical and health sciences ,Transformation (genetics) ,chemistry.chemical_compound ,medicine.anatomical_structure ,Mediator ,Cytoplasm ,medicine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,DNA ,030304 developmental biology - Abstract
Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram-positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.
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- 2021
10. Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae
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Jennyfer, Trouve, André, Zapun, Christopher, Arthaud, Claire, Durmort, Anne Marie, Di Guilmi, Bill, Söderström, Anais, Pelletier, Christophe, Grangeasse, Dominique, Bourgeois, Yung-Sing, Wong, Cecile, Morlot, Jennyfer, Trouve, André, Zapun, Christopher, Arthaud, Claire, Durmort, Anne Marie, Di Guilmi, Bill, Söderström, Anais, Pelletier, Christophe, Grangeasse, Dominique, Bourgeois, Yung-Sing, Wong, and Cecile, Morlot
- Abstract
Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis., source:https://www.sciencedirect.com/science/article/pii/S0960982221005765?via%3Dihub
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- 2021
11. Nanoscale Dynamics of Peptidoglycan Assembly During the Cell Cycle of Streptococcus pneumoniae
- Author
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Bill Soederstroem, Christopher Arthaud, Dominique Bourgeois, Jennyfer Trouve, Anne Marie Di Guilmi, Christophe Grangeasse, Yung-Sing Wong, Cécile Morlot, André Zapun, and Claire Durmort
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Cell wall ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Cell division ,Cell morphogenesis ,Cell ,Morphogenesis ,medicine ,Peptidoglycan ,Cell cycle ,Cleavage (embryo) ,Cell biology - Abstract
Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatio-temporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions, and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct phases of peripheral and septal synthesis.
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- 2020
12. One-Pot Two-Step Metabolic Labeling of Teichoic Acids and Direct Labeling of Peptidoglycan Reveals Tight Coordination of Both Polymers Inserted into Pneumococcus Cell Wall
- Author
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Anne Marie Di Guilmi, Yung-Sing Wong, Thierry Vernet, J. Bonnet, Claire Durmort, André Zapun, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département de pharmacochimie moléculaire (DPM ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'Ingénierie des Macromolécules (LIM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
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0301 basic medicine ,Azides ,Fluorophore ,Alkyne ,Peptidoglycan ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Choline ,Cell wall ,Cyclooctanes ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Wall ,Fluorescent Dyes ,chemistry.chemical_classification ,Teichoic acid ,Cycloaddition Reaction ,General Medicine ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Cycloaddition ,0104 chemical sciences ,Teichoic Acids ,carbohydrates (lipids) ,Streptococcus pneumoniae ,030104 developmental biology ,chemistry ,Alkynes ,Molecular Probes ,Click chemistry ,Molecular Medicine ,Click Chemistry ,Azide - Abstract
International audience; A method for labeling teichoic acids in the human pathogen Streptococcus pneumoniae has been developed using a one-pot two-step metabolic labeling approach. The essential nutriment choline modified with an azido-group was incorporated and exposed at the cell surface more rapidly than it reacted with the strain promoted azide alkyne cycloaddition (SPAAC) partner also present in the medium. Once at the cell surface on teichoic acids, coupling of the azido group could then occur within 5 min by the bio-orthogonal click reaction with a DIBO-linked fluorophore. This fast and easy method allowed pulse-chase experiments and was combined with another fluorescent labeling approach to compare the insertion of teichoic acids with peptidoglycan synthesis with unprecedented temporal resolution. It has revealed that teichoic acid and peptidoglycan processes are largely concomitant, but teichoic acid insertion persists later at the division site.
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- 2018
13. Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae
- Author
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Dominique Bourgeois, Bill Söderström, Cécile Morlot, Yung-Sing Wong, Anne Marie Di Guilmi, Christophe Grangeasse, Anais Pelletier, Christopher Arthaud, Jennyfer Trouve, Claire Durmort, André Zapun, Groupe Pneumocoque (IBS-PG), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), iThree Institute, University of Technology Sydney (UTS), Okinawa Institute of Science and Technology Graduate University (OIST), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Groupe Imagerie intégrée de la réponse au stress / Integrated Imaging of Stress Response Group (IBS-I2SR), Département de pharmacochimie moléculaire (DPM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), ANR-16-CE11-0016,DIVinHD,Imagerie super-résolue de la division bactérienne(2016), and ANR-19-CE15-0011,LookingForPegase,A la recherche de protéines régulant des enzymes responsables de la synthèse du peptidoglycane(2019)
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0301 basic medicine ,Cell division ,Cell ,Morphogenesis ,Peptidoglycan ,Biology ,Cleavage (embryo) ,General Biochemistry, Genetics and Molecular Biology ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacterial Proteins ,Cell Wall ,medicine ,Bacteria ,Cell morphogenesis ,Cell Cycle ,Cell cycle ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Cell biology ,Streptococcus pneumoniae ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,06 Biological Sciences, 11 Medical and Health Sciences, 17 Psychology and Cognitive Sciences ,General Agricultural and Biological Sciences ,Cell Division ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
International audience; Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization micro- scopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis.
- Published
- 2021
14. Peptidoglycan O-acetylation is functionally related to cell wall biosynthesis and cell division inStreptococcus pneumoniae
- Author
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Cécile Morlot, Yves V. Brun, J. Bonnet, Nathalie Campo, Thierry Vernet, Maxime Jacq, Michael S. VanNieuwenhze, Claire Durmort, Isabelle Mortier-Barrière, Christine Moriscot, Anne Marie Di Guilmi, Christopher Arthaud, and Benoit Gallet
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0301 basic medicine ,Glycan ,Cell division ,biology ,Autolysin ,Pseudopeptidoglycan ,Microbiology ,carbohydrates (lipids) ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Biochemistry ,Acetylation ,biology.protein ,Peptidoglycan ,Lysozyme ,Cell envelope ,Molecular Biology - Abstract
Summary The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. It is formed by linear glycan chains composed of N-acetylmuramic acid-(β-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodeled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus. This article is protected by copyright. All rights reserved.
- Published
- 2017
15. Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides
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Anne-Marie Di Guilmi, Hélène Rogniaux, Mathieu Fanuel, Cedric Montanier, Antoine Bouchoux, David Ropartz, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), FRISBI (ANR-10-INSB-05-02), GRAL within the Grenoble Partnership for Structural Biology (PSB) ( ANR-10-LABX-49-01), French National Research Agency (INRA-CEPIA), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)
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0301 basic medicine ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Immobilized enzyme ,lcsh:Medicine ,Biotechnologies ,Polysaccharide ,Article ,Substrate Specificity ,Fungal Proteins ,03 medical and health sciences ,Hydrolysis ,0302 clinical medicine ,Cell Wall ,Polysaccharides ,lcsh:Science ,chemistry.chemical_classification ,Multidisciplinary ,Endo-1,4-beta Xylanases ,Chemistry ,lcsh:R ,Substrate (chemistry) ,Enzymes, Immobilized ,Xylan ,Wood ,030104 developmental biology ,Enzyme ,Biocatalysis ,Biophysics ,Xylanase ,lcsh:Q ,Neocallimastix ,030217 neurology & neurosurgery - Abstract
Enzymes are involved in various types of biological processes. In many cases, they are part of multi-component machineries where enzymes are localized in close proximity to each-other. In such situations, it is still not clear whether inter-enzyme spacing actually plays a role or if the colocalization of complementary activities is sufficient to explain the efficiency of the system. Here, we focus on the effect of spatial proximity when identical enzymes are immobilized onto a surface. By using an innovative grafting procedure based on the use of two engineered protein fragments, Jo and In, we produce model systems in which enzymes are immobilized at surface densities that can be controlled precisely. The enzyme used is a xylanase that participates to the hydrolysis of plant cell wall polymers. By using a small chromogenic substrate, we first show that the intrinsic activity of the enzymes is fully preserved upon immobilization and does not depend on surface density. However, when using beechwood xylan, a naturally occurring polysaccharide, as substrate, we find that the enzymatic efficiency decreases by 10–60% with the density of grafting. This unexpected result is probably explained through steric hindrance effects at the nanoscale that hinder proper interaction between the enzymes and the polymer. A second effect of enzyme immobilization at high densities is the clear tendency for the system to release preferentially shorter oligosaccharides from beechwood xylan as compared to enzymes in solution.
- Published
- 2019
16. The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae
- Author
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Maxime Jacq, Christopher Arthaud, Sylvie Manuse, Chryslène Mercy, Laure Bellard, Katharina Peters, Benoit Gallet, Jennifer Galindo, Thierry Doan, Waldemar Vollmer, Yves V. Brun, Michael S. VanNieuwenhze, Anne Marie Di Guilmi, Thierry Vernet, Christophe Grangeasse, Cecile Morlot, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institute for Cell and Molecular Biosciences, Newcastle University [Newcastle], Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Indiana University [Bloomington], Indiana University System, ANR-12-BSV3-0008,PiBaKi,Rôle cellulaire et mode d'action de deux protéine-kinases centrales chez les bactéries(2012), ANR-14-CE14-0003,ORBiMP,Déjouer la résistance aux beta-lactamines des méningocoques et pneumocoques(2014), ANR-16-CE11-0016,DIVinHD,Imagerie super-résolue de la division bactérienne(2016), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-12-BS07-0017,GRAL,Une nouvelle Génération de dRogues pour la maladie d'ALzheimer basées sur des études in vitro et in silico des oligomères de la protéine beta-amyloide et des tests in vivo(2012), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
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Models, Molecular ,lcsh:R ,Amino Acid Motifs ,lcsh:Medicine ,Protein Structure, Secondary ,Article ,Cytoskeletal Proteins ,Protein Transport ,Streptococcus pneumoniae ,Bacterial Proteins ,Microscopy, Fluorescence ,Cell Wall ,Sequence Homology, Nucleic Acid ,[CHIM]Chemical Sciences ,lcsh:Q ,Muramidase ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,lcsh:Science ,Cell Division ,Gene Deletion - Abstract
International audience; Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.
- Published
- 2018
17. Single molecule force spectroscopy reveals two-domain binding mode of pilus-1 tip protein RrgA of $Streptococcus\ pneumoniae$ to fibronectin
- Author
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Hauke Clausen-Schaumann, Raimund Gürster, Arndt F. Schilling, Tanja D Becke, Markus Hilleringmann, Stefanie Sudhop, Anne-Marie di Guilmi, Stefan Ness, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), University of Applied Sciences [Munich], Ludwig-Maximilians-Universität München (LMU), University Medical Center Göttingen (UMG), Laboratoire de Recherche sur l'Instabilité Génétique (LRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Service d'Instabilité Génétique Réparation Recombinaison (SIGRR)
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0301 basic medicine ,Virulence Factors ,General Physics and Astronomy ,Microscopy, Atomic Force ,Pneumococcal Infections ,Pilus ,Extracellular matrix ,03 medical and health sciences ,Protein Domains ,fibronectin ,Humans ,General Materials Science ,RrgA ,Binding site ,single molecule force spectroscopy ,Binding Sites ,biology ,Strain (chemistry) ,Chemistry ,General Engineering ,Force spectroscopy ,Adhesion ,Gram-positive bacteria ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Fibronectins ,3. Good health ,Cell biology ,Fibronectin ,Bacterial adhesin ,030104 developmental biology ,Streptococcus pneumoniae ,Fimbriae, Bacterial ,biology.protein ,pili ,Fimbriae Proteins ,AFM ,Protein Binding - Abstract
International audience; For host cell adhesion and invasion, surface piliation procures benefits for bacteria. A detailed investigation of how pili adhere to host cells is therefore a key aspect inunderstanding their role during infection. Streptococcus pneumoniae TIGR 4, a clinical relevant serotype 4 strain, is capable of expressing pilus-1 with terminal RrgA, an adhesin interacting with host extracellular matrix (ECM) proteins. We used single molecule force spectroscopy to investigate the binding of full-length RrgA and single RrgA domains to fibronectin. Our results show that full-length RrgA and its terminal domains D3 and D4 bind to fibronectin with forces of 51.6 (full length), 52.8 (D3), and 46.2 pN (D4) at force-loading rates of around 1500 pN/s. Selective saturation of D3 and D4 binding sites on fibronectin showed that both domains can interact simultaneously with fibronectin, revealing a two-domain binding mechanism for the pilus-1 tip protein. The high off rates and the corresponding short lifetime of the RrgA Fn bond (τ = 0.26 s) may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces and thus to efficiently scan the surface for specific receptors promoting host cell adhesion and invasion. These molecular properties could be essential for S. pneumoniae pili to mediate initial contact to the host cells andshared with other piliated Gram-positive bacteria_favor host invasion
- Published
- 2018
18. Peptidoglycan O-acetylation is functionally related to cell wall biosynthesis and cell division in Streptococcus pneumoniae
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Julie, Bonnet, Claire, Durmort, Maxime, Jacq, Isabelle, Mortier-Barrière, Nathalie, Campo, Michael S, VanNieuwenhze, Yves V, Brun, Christopher, Arthaud, Benoit, Gallet, Christine, Moriscot, Cécile, Morlot, Thierry, Vernet, and Anne Marie, Di Guilmi
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carbohydrates (lipids) ,Streptococcus pneumoniae ,Cell Wall ,Muramic Acids ,Gram-Negative Bacteria ,Acetylation ,N-Acetylmuramoyl-L-alanine Amidase ,Peptidoglycan ,Cell Division ,Article ,Acetylglucosamine - Abstract
The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. It is formed by linear glycan chains composed of N-acetylmuramic acid-(β-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodelled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus.
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- 2017
19. Deciphering key residues involved in the virulence-promoting interactions between Streptococcus pneumoniae and human plasminogen
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Anne Marie Di Guilmi, Christine Gaboriaud, Christophe Moreau, Nicole M. Thielens, Thierry Vernet, Rémi Terrasse, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
0301 basic medicine ,crystal structure ,bacterial pathogenesis ,Protein Conformation ,Host–pathogen interaction ,030106 microbiology ,Virulence ,cell surface receptor ,host-pathogen interaction ,medicine.disease_cause ,Biochemistry ,Protein–protein interaction ,protein-protein interaction ,03 medical and health sciences ,Immune system ,Cell surface receptor ,Streptococcus pneumoniae ,medicine ,Humans ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Glyceraldehyde 3-phosphate dehydrogenase ,peptide array ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Streptococcus ,Cell Biology ,Surface Plasmon Resonance ,Kinetics ,030104 developmental biology ,gram-positive bacteria ,Protein Structure and Folding ,biology.protein ,plasminogen ,Thermodynamics ,Protein Binding - Abstract
International audience; Bacterial pathogens recruit circulating proteins to their own surfaces, coopting the host protein functions as a mechanism of virulence. Particular attention has focused on the binding of plasminogen (Plg) to bacterial surfaces, as it has been shown that this interaction contributes to bacterial adhesion to host cells, invasion of host tissues and evasion of the immune system. Several bacterial proteins are known to serve as receptors for Plg including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytoplasmic enzyme that appears on the cell surface in this moonlighting role. Although Plg typically binds to these receptors via several lysine-binding domains, the specific interactions that occur have not been documented in all cases. However, identification of the relevant residues could help define strategies for mitigating the virulence of important human pathogens, like Streptococcus pneumoniae (Sp). To shed light on this question, we have described a combination of peptide-spot array screening, competition and SPR assays, high-resolution crystallography and mutational analyses to characterize the interaction between SpGAPDH and Plg. We identified three SpGAPDH lysire residues that were instrumental in defining the kinetic and thermodynamic parameters of the interaction. Altogether, the integration of the data presented in this work allows us to propose a structural model for the molecular interaction of the SpGAPDH-Plg complex.
- Published
- 2017
20. Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae
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Amandine Maccagni, Munan Shaik, Andréa Dessen, Anne Marie Di Guilmi, Guillaume Tourcier, Thomas, Frank, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Molecular Sequence Data ,MESH: Amino Acid Sequence ,Plasma protein binding ,medicine.disease_cause ,Biochemistry ,Pilus ,Bacterial cell structure ,MESH: Fimbriae, Bacterial ,Microbiology ,Fimbriae Proteins ,MESH: Protein Structure, Tertiary ,chemistry.chemical_compound ,Bacterial Proteins ,MESH: Aminoacyltransferases ,Streptococcus pneumoniae ,medicine ,MESH: Protein Binding ,Amino Acid Sequence ,MESH: Bacterial Proteins ,Molecular Biology ,MESH: Molecular Sequence Data ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,biology ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Aminoacyltransferases ,MESH: Fimbriae Proteins ,Protein Structure, Tertiary ,3. Good health ,Bacterial adhesin ,Cysteine Endopeptidases ,chemistry ,Fimbriae, Bacterial ,Pilin ,Protein Structure and Folding ,biology.protein ,bacteria ,Peptidoglycan ,MESH: Streptococcus pneumoniae ,Protein Binding ,MESH: Cysteine Endopeptidases - Abstract
International audience; Pili are surface-attached, fibrous virulence factors that play key roles in the pathogenesis process of a number of bacterial agents. Streptococcus pneumoniae is a causative agent of pneumonia and meningitis, and the appearance of drug-resistance organisms has made its treatment challenging, especially in developing countries. Pneumococcus-expressed pili are composed of three structural proteins: RrgB, which forms the polymerized backbone, RrgA, the tip-associated adhesin, and RrgC, which presumably associates the pilus with the bacterial cell wall. Despite the fact that the structures of both RrgA and RrgB were known previously, structural information for RrgC was still lacking, impeding the analysis of a complete model of pilus architecture. Here, we report the structure of RrgC to 1.85 Å and reveal that it is a three-domain molecule stabilized by two intradomain isopeptide bonds. RrgC does not depend on pilus-specific sortases to become attached to the cell wall; instead, it binds the preformed pilus to the peptidoglycan by employing the catalytic activity of SrtA. A comprehensive model of the type 1 pilus from S. pneumoniae is also presented.
- Published
- 2014
21. PatA and PatB Form a Functional Heterodimeric ABC Multidrug Efflux Transporter Responsible for the Resistance of Streptococcus pneumoniae to Fluoroquinolones
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Jean-Michel Jault, Thierry Vernet, Benoît Bernay, Anne Marie Di Guilmi, Christine Ebel, Jacques Croize, Claire Durmort, and Emilie Boncoeur
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Mutant ,ATP-binding cassette transporter ,medicine.disease_cause ,Biochemistry ,Pneumococcal Infections ,Microbiology ,chemistry.chemical_compound ,Drug Resistance, Bacterial ,medicine ,Humans ,Escherichia coli ,Mutation ,Walker motifs ,Transporter ,Anti-Bacterial Agents ,Streptococcus pneumoniae ,chemistry ,ATP-Binding Cassette Transporters ,Efflux ,Protein Multimerization ,Vanadates ,Ethidium bromide ,Gene Deletion ,Fluoroquinolones - Abstract
All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.
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- 2012
22. Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae
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Anne-Marie Di Guilmi, Frédéric Galisson, Christophe Grangeasse, Céline Freton, Sébastien Guiral, Aurore Fleurie, Isabelle Zanella-Cléon, and Caroline Cluzel
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0303 health sciences ,Cell division ,030306 microbiology ,Kinase ,Biology ,Microbiology ,Phenotype ,Serine ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Cytoplasm ,Phosphorylation ,Peptidoglycan ,Kinase activity ,Molecular Biology ,030304 developmental biology - Abstract
Eukaryotic-like serine/threonine-kinases are involved in the regulation of a variety of physiological processes in bacteria. In Streptococcus pneumoniae, deletion of the single serine/threonine-kinase gene stkP results in an aberrant cell morphology suggesting that StkP participates in pneumococcus cell division. To understand the function of StkP, we have engineered various pneumococcus strains expressing truncated or kinase-dead forms of StkP. We show that StkP kinase activity, but also its extracellular and cytoplasmic domains per se, are required for pneumococcus cell division. Indeed, we observe that mutant cells show round or elongated shapes with non-functional septa and a chain phenotype, delocalized sites of peptidoglycan synthesis and diffused membrane StkP localization. To gain understanding of the underlying StkP-mediated regulatory mechanism, we show that StkP specifically phosphorylates in vivo the cell division protein DivIVA on threonine 201. Pneumococcus cells expressing non-phosphorylatable DivIVA-T201A possess an elongated shape with a polar bulge and aberrant spatial organization of nascent peptidoglycan. This brings the first evidence of the importance of StkP in relationship to the phosphorylation of one of its substrates in cell division. It is concluded that StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division.
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- 2012
23. Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae
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Anne Marie Di Guilmi, Lamya El Mortaji, Andréa Dessen, Thierry Vernet, and Rémi Terrasse
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Proteolysis ,Molecular Sequence Data ,medicine.disease_cause ,Biochemistry ,Pilus ,Bacterial Proteins ,Sortase ,medicine ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,Sequence Deletion ,chemistry.chemical_classification ,Isopeptide bond ,Binding Sites ,Sequence Homology, Amino Acid ,medicine.diagnostic_test ,biology ,Protein Stability ,Cell Biology ,Aminoacyltransferases ,Recombinant Proteins ,Cysteine Endopeptidases ,Protein Subunits ,Streptococcus pneumoniae ,Amino Acid Substitution ,chemistry ,Covalent bond ,Multiprotein Complexes ,Pilin ,Protein Structure and Folding ,Mutagenesis, Site-Directed ,biology.protein ,Fimbriae Proteins ,Protein stabilization - Abstract
Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.
- Published
- 2010
24. Structural Basis of Host Cell Recognition by the Pilus Adhesin from Streptococcus pneumoniae
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Thierry Vernet, Clothilde Manzano, Rémy Terrasse, Thierry Izoré, Lamya El Mortaji, Anne Marie Di Guilmi, Carlos Contreras-Martel, and Andréa Dessen
- Subjects
Models, Molecular ,Protein Folding ,MICROBIO ,PROTEINS ,Molecular Sequence Data ,Fimbria ,Integrin ,Virulence ,Sequence alignment ,Crystallography, X-Ray ,Pilus ,Microbiology ,Conserved sequence ,Structural Biology ,Amino Acid Sequence ,Adhesins, Bacterial ,Molecular Biology ,Peptide sequence ,Conserved Sequence ,biology ,Extracellular Matrix ,Protein Structure, Tertiary ,Bacterial adhesin ,Streptococcus pneumoniae ,Fimbriae, Bacterial ,biology.protein ,Sequence Alignment ,Protein Binding - Abstract
SummaryPili are fibrous virulence factors associated directly to the bacterial surface that play critical roles in adhesion and recognition of host cell receptors. The human pathogen Streptococcus pneumoniae carries a single pilus-related adhesin (RrgA) that is key for infection establishment and provides protection from bacterial challenge in animal infection models, but details of these roles remain unclear. Here we report the high-resolution crystal structure of RrgA, a 893-residue elongated macromolecule whose fold contains four domains presenting both eukaryotic and prokaryotic origins. RrgA harbors an integrin I collagen-recognition domain decorated with two inserted “arms” that fold into a positively charged cradle, as well as three “stalk-forming” domains. We show by site-specific mutagenesis, mass spectrometry, and thermal shift assays that intradomain isopeptide bonds play key roles in stabilizing RrgA's stalk. The high sequence similarity between RrgA and its homologs in other Gram-positive microorganisms suggests common strategies for ECM recognition and immune evasion.
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- 2010
25. Sortase Activity Is Controlled by a Flexible Lid in the Pilus Biogenesis Mechanism of Gram-Positive Pathogens
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Thierry Izoré, Anne Marie Di Guilmi, Clothilde Manzano, Andréa Dessen, and Viviana Job
- Subjects
Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Virulence ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Pilus ,Microbiology ,Bacterial Proteins ,Tandem Mass Spectrometry ,Sortase ,Streptococcus pneumoniae ,Catalytic triad ,medicine ,Transferase ,Amino Acid Sequence ,Gene ,Sequence Homology, Amino Acid ,biology ,Active site ,Aminoacyltransferases ,Recombinant Proteins ,Cysteine Endopeptidases ,Fimbriae, Bacterial ,Biocatalysis ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Chromatography, Liquid - Abstract
Pili are surface-linked virulence factors that play key roles in infection establishment in a variety of pathogenic species. In Gram-positive pathogens, pilus formation requires the action of sortases, dedicated transpeptidases that covalently associate pilus building blocks. In Streptococcus pneumoniae, a major human pathogen, all genes required for pilus formation are harbored in a single pathogenicity islet which encodes three structural proteins (RrgA, RrgB, RrgC) and three sortases (SrtC-1, SrtC-2, SrtC-3). RrgB forms the backbone of the streptococcal pilus, to which minor pilins RrgA and RrgC are covalently associated. SrtC-1 is the main sortase involved in polymerization of the RrgB fiber and displays a lid which encapsulates the active site, a feature present in all pilus-related sortases. In this work, we show that catalysis by SrtC-1 proceeds through a catalytic triad constituted of His, Arg, and Cys and that lid instability affects protein fold and catalysis. In addition, we show by thermal shift analysis that lid flexibility can be stabilized by the addition of substrate-like peptides, a feature shared by other periplasmic transpeptidases. We also report the characterization of a trapped acyl-enzyme intermediate formed between SrtC-1 and RrgB. The presence of lid-encapsulated sortases in the pilus biogenesis systems in many Gram-positive pathogens points to a common mechanism of substrate recognition and catalysis that should be taken into consideration in the development of sortase inhibitors.
- Published
- 2009
26. Sortase-Mediated Pilus Fiber Biogenesis in Streptococcus pneumoniae
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Lamya El Mortaji, Anne Marie Di Guilmi, Guy Schoehn, Andréa Dessen, Thierry Izoré, Thierry Vernet, Clothilde Manzano, Daphna Fenel, and Carlos Contreras-Martel
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Models, Molecular ,PROTEINS ,Molecular Sequence Data ,medicine.disease_cause ,Pilus ,Microbiology ,Bacterial Proteins ,Sortase ,Structural Biology ,Streptococcus pneumoniae ,medicine ,Transferase ,Pilus biogenesis ,Amino Acid Sequence ,Pathogen ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,biochemical phenomena, metabolism, and nutrition ,SIGNALING ,Fimbriae, Bacterial ,Pilin ,Mutation ,biology.protein ,bacteria ,Fimbriae Proteins ,Biogenesis ,Protein Binding - Abstract
Summary Streptococcus pneumoniae is a piliated pathogen whose ability to circumvent vaccination and antibiotic treatment strategies is a cause of mortality worldwide. Pili play important roles in pneumococcal infection, but little is known about their biogenesis mechanism or the relationship between components of the pilus-forming machinery, which includes the fiber pilin (RrgB), two minor pilins (RrgA, RrgC), and three sortases (SrtC-1, SrtC-2, SrtC-3). Here we show that SrtC-1 is the main pilus-polymerizing transpeptidase, and electron microscopy analyses of RrgB fibers reconstituted in vitro reveal that they structurally mimic the pneumococcal pilus backbone. Crystal structures of both SrtC-1 and SrtC-3 reveal active sites whose access is controlled by flexible lids, unlike in non-pilus sortases, and suggest that substrate specificity is dictated by surface recognition coupled to lid opening. The distinct structural features of pilus-forming sortases suggest a common pilus biogenesis mechanism that could be exploited for the development of broad-spectrum antibacterials.
- Published
- 2008
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27. The Interaction of Streptococcus pneumoniae with Plasmin Mediates Transmigration across Endothelial and Epithelial Monolayers by Intercellular Junction Cleavage
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Cécile Attali, Thierry Vernet, Anne Marie Di Guilmi, and Claire Durmort
- Subjects
Plasmin ,Immunology ,Cell ,Fluorescent Antibody Technique ,Biology ,Microbiology ,Cell junction ,Bacterial Adhesion ,Pneumococcal Infections ,Cell Line ,Adherens junction ,medicine ,Humans ,Fibrinolysin ,Cellular Microbiology: Pathogen-Host Cell Molecular Interactions ,Cadherin ,Endothelial Cells ,Epithelial Cells ,Plasminogen ,Cell migration ,Cadherins ,Cell biology ,Intercellular Junctions ,Streptococcus pneumoniae ,Infectious Diseases ,medicine.anatomical_structure ,Cell culture ,Parasitology ,medicine.drug - Abstract
The precise mechanisms by which Streptococcus pneumoniae overcomes epithelial and endothelial barriers to access underlying human tissues remain to be determined. The plasminogen system is highly important for the tissue barrier degradation which allows cell migration. Plasminogen is known to interact with pneumococci via enolase, glyceraldehyde-3-phosphate dehydrogenase, and choline-binding protein E. These observations prompted us to evaluate the role of this proteolytic system in the pneumococcal invasion process. We observed that coating of S. pneumoniae R6 strain with plasminogen or inactivated plasmin increased adherence to pulmonary epithelial A549 and vascular endothelial EaHy cells in vitro. This indicates that plasminogen-mediated adherence is independent of the protease activity and involves plasminogen binding to receptors on eukaryotic cell surfaces. Conversely, decreased adherence of bacterial cells coated with active plasmin was observed, indicating that the protease activity limits bacterial attachment on the cell surface. We were then interested in investigating the role of the proteolytic plasmin activity in the traversal of tissue barriers. We observed that adherence of plasmin-coated D39 (encapsulated) or R6 (unencapsulated) pneumococci induced sporadic disruptions of EaHy and A549 monolayer cell junctions. This was not observed when plasmin was inhibited by aprotinin. Endothelial junction disorganization may proceed by proteolysis of the cell junction components. This is supported by our observation of the in vitro cleavage by plasmin bound to pneumococci of recombinant vascular endothelial cadherin, the main component of endothelial adherens junctions. Finally, junction damage induced by plasmin may be related to tissue barrier traversal, as we measured an increase of S. pneumoniae transmigration across epithelial A549 and endothelial EaHy layers when active plasmin was present on the bacterial surface. Our results highlight a novel function for the plasminogen recruitment at the bacterial surface in facilitating adherence of pneumococci to endothelial and epithelial cells, while active plasmin degrades intercellular junctions. This process promotes migration of pneumococci through cell barriers by a pericellular route, a prerequisite for dissemination of S. pneumoniae in the host organism.
- Published
- 2008
28. Streptococcus pneumoniaeCholine-Binding Protein E Interaction with Plasminogen/Plasmin Stimulates Migration across the Extracellular Matrix
- Author
-
Cecile Frolet, Claire Durmort, Julien Offant, Cécile Attali, Thierry Vernet, and Anne Marie Di Guilmi
- Subjects
Proteases ,Protein family ,Plasmin ,Immunology ,Receptors, Cell Surface ,Plasma protein binding ,Biology ,Microbiology ,Kringle domain ,Protein structure ,Protein Interaction Mapping ,medicine ,Humans ,Phosphorylcholine ,Binding protein ,Plasminogen ,Molecular Pathogenesis ,Extracellular Matrix ,Protein Structure, Tertiary ,Streptococcus pneumoniae ,Infectious Diseases ,Biochemistry ,Mutagenesis, Site-Directed ,Parasitology ,Gene Deletion ,Protein Binding ,medicine.drug - Abstract
The virulence mechanisms leadingStreptococcus pneumoniaeto convert from nasopharyngeal colonization to a tissue-invasive phenotype are still largely unknown. Proliferation of infection requires penetration of the extracellular matrix, which occurs by recruitment of host proteases to the bacterial cell surface. We present evidence supporting the role of choline-binding protein E (CBPE) (a member of the surface-exposed choline-binding protein family) as an important receptor for human plasminogen, the precursor of plasmin. The results of ligand overlay blot analyses, solid-phase binding assays, and surface plasmon resonance experiments support the idea of an interaction between CBPE and plasminogen. We have shown that the phosphorylcholine esterase (Pce) domain of CBPE interacts with the plasminogen kringle domains. Analysis of the crystal structure of the Pce domain, followed by site-directed mutagenesis, allowed the identification of the plasminogen-binding region composed in part by lysine residues, some of which map in a linear fashion on the surface of the Pce domain. The biological relevance of the CBPE-plasminogen interaction is supported by the fact that, compared to the wild-type strain, a mutant of pneumococcus with thecbpEgene deleted (i) displays a reduced level of plasminogen binding and plasmin activation and (ii) shows reduced ability to cross the extracellular matrix in an in vitro model. These results support the idea of a physiological role for the CBPE-plasminogen interaction in pneumococcal dissemination into human tissue.
- Published
- 2008
29. Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316
- Author
-
Thibault Chaze, Eric Morello, Patrick Trieu-Cuot, Adeline Mallet, Liliana Oliveira, Shaynoor Dramsi, Michel-Yves Mistou, Anne-Marie Di Guilmi, Yoan Konto-Ghiorghi, Giulia Oliva, Biologie des Bactéries pathogènes à Gram-positif, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Microscopie Ultrastructurale (Plate-forme), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Spectrométrie de Masse structurale et protéomique, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), French National Research Agency ANR-10-BLANC-1314 Glyco-Path, French Government's Investissement d'Avenir program, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' ANR-10-LABX-62-IBEID, ANR-10-BLAN-1314,Glyco-path,Glycosylation des protéines chez une bactérie pathogène à Gram-positif(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Dramsi, Shaynoor
- Subjects
Models, Molecular ,Ribosome Inactivating Proteins ,lcsh:Medicine ,CELL-SHAPE ,medicine.disease_cause ,Bacterial Adhesion ,Pilus ,DISEASE ,chemistry.chemical_compound ,lcsh:Science ,Multidisciplinary ,biology ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Biochemistry ,Glucosyltransferases ,Fimbriae Proteins ,Asparagine ,Plant Lectins ,Autre (Sciences du Vivant) ,GROUP-B STREPTOCOCCUS ,LINKED PROTEIN GLYCOSYLATION ,BIOSYNTHESIS ,PATHOGENS ,SRR1 ,Research Article ,Protein Binding ,Glycosylation ,Streptococcus agalactiae ,Microbiology ,Bacterial Proteins ,medicine ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Adhesins, Bacterial ,Organisms, Genetically Modified ,lcsh:R ,Lectin ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,N-Acetylneuraminic Acid ,Protein Structure, Tertiary ,Sialic acid ,Bacterial adhesin ,Protein Subunits ,chemistry ,Fimbriae, Bacterial ,Pilin ,biology.protein ,lcsh:Q ,Protein Processing, Post-Translational ,N-Acetylneuraminic acid - Abstract
International audience; Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.
- Published
- 2015
30. A Structural Snapshot of Type II Pilus Formation in Streptococcus pneumoniae
- Author
-
Munan Shaik, Andréa Dessen, Guy Schoehn, Daniel M. Trindade, Daphna Fenel, Charlotte Lombardi, Anne Marie Di Guilmi, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Preuves, Programmes et Systèmes (PPS), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Thomas, Frank
- Subjects
Molecular model ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Virulence factor ,Pilus ,Microbiology ,Structure-Activity Relationship ,Bacterial Proteins ,Sortase ,Hydrolase ,Streptococcus pneumoniae ,medicine ,Humans ,Cell adhesion ,Protein Structure, Quaternary ,Molecular Biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Protein Structure, Tertiary ,Sortase A ,Fimbriae, Bacterial ,Protein Structure and Folding ,bacteria - Abstract
International audience; Pili are fibrous appendages expressed on the surface of a vast number of bacterial species, and their role in surface adhesion is important for processes such as infection, colonization, andbiofilm formation. The human pathogen Streptococcus pneumoniae expresses two different types of pili, PI-1 and PI-2, both of which require the concerted action of structural proteins and sortases for their polymerization. The type PI-1 streptococcal pilus is a complex, well studied structure, but the PI-2 type, present in a number of invasive pneumococcal serotypes, has to date remained less well understood. The PI-2 pilus consists of repeated units of a single protein, PitB, whose covalent association is catalyzed by cognate sortase SrtG-1 and partner protein SipA. Here we report the high resolution crystal structures of PitB and SrtG1 and use molecular modeling to visualize a "trapped" 1:1 complex between the two molecules. X-ray crystallography and electron microscopy reveal that the pneumococcal PI-2 backbone fiber is formed by PitB monomers associated in head-to-tail fashion and that short, flexible fibers can be formed even in the absence of coadjuvant proteins. These observations, obtained with a simple pilus biosynthetic system, are likely to be applicable to other fiber formation processes in a variety of Gram-positive organisms.
- Published
- 2015
31. Remodeling of the Z-Ring Nanostructure during the Streptococcus pneumoniae Cell Cycle Revealed by Photoactivated Localization Microscopy
- Author
-
Dominique Bourgeois, Thierry Vernet, Virgile Adam, Cécile Morlot, Maxime Jacq, Anne-Marie Di Guilmi, and Christine Moriscot
- Subjects
Programmed cell death ,biology ,Cell division ,Cell Cycle ,Cell cycle ,medicine.disease_cause ,Microbiology ,QR1-502 ,Nanostructures ,Cell biology ,Cell wall ,Cytoskeletal Proteins ,Streptococcus pneumoniae ,Bacterial Proteins ,Microscopy, Fluorescence ,Virology ,biology.protein ,medicine ,Photoactivated localization microscopy ,FtsZ ,Cytoskeleton ,Cell Division ,Fluorescent Dyes ,Research Article - Abstract
Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring’s morphology during the division cycle at the nanoscale level. We show that changes in the ring’s axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci., IMPORTANCE The Gram-positive human pathogen S. pneumoniae is responsible for 1.6 million deaths per year worldwide and is increasingly resistant to various antibiotics. FtsZ is a cytoskeletal protein polymerizing at midcell into a ring-like structure called the Z-ring. FtsZ is a promising new antimicrobial target, as its inhibition leads to cell death. A precise view of the Z-ring architecture in vivo is essential to understand the mode of action of inhibitory drugs (see T. den Blaauwen, J. M. Andreu, and O. Monasterio, Bioorg Chem 55:27–38, 2014, doi:10.1016/j.bioorg.2014.03.007, for a review on FtsZ inhibitors). This is notably true in ovococcoid bacteria like S. pneumoniae, in which FtsZ is the only known cytoskeletal protein. We have used superresolution microscopy to obtain molecular details of the pneumococcus Z-ring that have so far been inaccessible with conventional microscopy. This study provides a nanoscale description of the Z-ring architecture and remodeling during the division of ovococci.
- Published
- 2015
32. Remodeling of the Z-Ring Nanostructure during the Streptococcus pneumoniae Cell Cycle Revealed by Photoactivated Localization Microscopy
- Author
-
Maxime Jacq, Virgile ADAM, Dominique Bourgeois, Christine Moriscot, Anne-Marie Di Guilmi, Thierry Vernet, Cécile Morlot, European Synchrotron Radiation Facility (ESRF), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Thomas, Frank, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,[SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] - Abstract
International audience; Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring's morphology during the division cycle at the nanoscale level. We show that changes in the ring's axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci. The Gram-positive human pathogen S. pneumoniae is responsible for 1.6 million deaths per year worldwide and is increasingly resistant to various antibiotics. FtsZ is a cytoskeletal protein polymerizing at midcell into a ring-like structure called the Z-ring. FtsZ is a promising new antimicrobial target, as its inhibition leads to cell death. A precise view of the Z-ring architecture in vivo is essential to understand the mode of action of inhibitory drugs (see T. den Blaauwen, J. M. Andreu, and O. Monasterio, Bioorg Chem 55:27-38, 2014, doi:10.1016/j.bioorg.2014.03.007, for a review on FtsZ inhibitors). This is notably true in ovococcoid bacteria like S. pneumoniae, in which FtsZ is the only known cytoskeletal protein. We have used superresolution microscopy to obtain molecular details of the pneumococcus Z-ring that have so far been inaccessible with conventional microscopy. This study provides a nanoscale description of the Z-ring architecture and remodeling during the division of ovococci.
- Published
- 2015
33. Bifunctional Penicillin-Binding Proteins as an Antibacterial Target: Update on Enzymatic Properties and Cellular Functions
- Author
-
Anne Marie Di Guilmi
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Penicillin binding proteins ,Biochemistry ,Chemistry ,Drug Discovery ,Cellular functions ,Molecular Medicine ,Bifunctional - Published
- 2005
34. Crystal Structure of Phosphorylcholine Esterase Domain of the Virulence Factor Choline-binding Protein E from Streptococcus pneumoniae
- Author
-
Otto Dideberg, David Lemaire, Gianpiero Garau, Anne Marie Di Guilmi, and Thierry Vernet
- Subjects
chemistry.chemical_classification ,Teichoic acid ,biology ,Phosphorylcholine ,Active site ,Cell Biology ,medicine.disease_cause ,Biochemistry ,Esterase ,Microbiology ,chemistry.chemical_compound ,Enzyme ,chemistry ,Streptococcus pneumoniae ,Hydrolase ,medicine ,biology.protein ,Molecular Biology ,Choline binding - Abstract
Streptococcus pneumoniae is the worldwide leading cause of deaths from invasive infections such as pneumoniae, sepsis, and meningitidis in children and the elderly. Nasopharyngeal colonization, which plays a key role in the development of pneumococcal disease, is highly dependent on a family of surface-exposed proteins, the choline-binding proteins (CBPs). Here we report the crystal structure of phosphorylcholine esterase (Pce), the catalytic domain of choline-binding protein E (CBPE), which has been shown to be crucial for host/pathogen interaction processes. The unexpected features of the Pce active site reveal that this enzyme is unique among the large family of hydrolases harboring the metallo-β-lactamase fold. The orientation and calcium stabilization features of an elongated loop, which lies on top of the active site, suggest that the cleft may be rearranged. Furthermore, the structure of Pce complexed with phosphorylcholine, together with the characterization of the enzymatic role played by two iron ions located in the active site allow us to propose a reaction mechanism reminiscent of that of purple acid phosphatase. This mechanism is supported by site-directed mutagenesis experiments. Finally, the interactions of the choline binding domain and the Pce region of CBPE with chains of teichoic acids have been modeled. The ensemble of our biochemical and structural results provide an initial understanding of the function of CBPE.
- Published
- 2005
35. Structure-Based Phylogeny of the Metallo-β-Lactamases
- Author
-
Anne Marie Di Guilmi, Gianpiero Garau, and Barry G. Hall
- Subjects
Models, Molecular ,Molecular Sequence Data ,Sequence alignment ,Biology ,beta-Lactamases ,Homology (biology) ,Evolution, Molecular ,Protein structure ,Mechanisms of Resistance ,Phylogenetics ,polycyclic compounds ,Pharmacology (medical) ,Amino Acid Sequence ,Gene ,Peptide sequence ,Phylogeny ,Pharmacology ,Genetics ,Bacteria ,Phylogenetic tree ,biochemical phenomena, metabolism, and nutrition ,Protein superfamily ,bacterial infections and mycoses ,enzymes and coenzymes (carbohydrates) ,Infectious Diseases ,bacteria ,Sequence Alignment ,Software - Abstract
The metallo-β-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-β-lactamases and five structurally homologous proteins from the metallo-β-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-β-lactamases. The presence of genes from Eubacteria , Archaebacteria , and Eukaryota on that tree is consistent with a very ancient origin of the metallo-β-lactamase family.
- Published
- 2005
36. Active site restructuring regulates ligand recognition in class A penicillin-binding proteins
- Author
-
Thierry Vernet, Otto Dideberg, Andréa Dessen, Anne Marie Di Guilmi, Pauline Macheboeuf, and Viviana Job
- Subjects
Penicillin binding proteins ,Cell division ,Mutation, Missense ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Ligands ,beta-Lactams ,Peptidoglycan biosynthetic process ,beta-Lactam Resistance ,chemistry.chemical_compound ,Protein structure ,polycyclic compounds ,Penicillin-Binding Proteins ,Amino Acid Sequence ,Binding site ,Peptide sequence ,Binding Sites ,Multidisciplinary ,Molecular Structure ,Biological Sciences ,Protein Structure, Tertiary ,Streptococcus pneumoniae ,chemistry ,Biochemistry ,Peptidoglycan ,Sequence Alignment ,Protein Binding - Abstract
Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for β-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b * ) has been solved to 1.9 Å, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b * is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b * complexed with β-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b * suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.
- Published
- 2005
37. Biochemical Characterization of Streptococcus pneumoniae Penicillin-Binding Protein 2b and Its Implication in β-Lactam Resistance
- Author
-
Thierry Vernet, Laurent Chesnel, Julie Hopkins, Jacques Croize, Estelle Pagliero, Anne Marie Di Guilmi, and Otto Dideberg
- Subjects
DNA, Bacterial ,Penicillin binding proteins ,Molecular Sequence Data ,Penicillins ,Muramoylpentapeptide Carboxypeptidase ,Biology ,medicine.disease_cause ,Pneumococcal Infections ,beta-Lactam Resistance ,law.invention ,Microbiology ,chemistry.chemical_compound ,Bacterial Proteins ,Mechanisms of Resistance ,law ,Streptococcus pneumoniae ,polycyclic compounds ,medicine ,Humans ,Penicillin-Binding Proteins ,Pharmacology (medical) ,Amino Acid Sequence ,Cloning, Molecular ,Serotyping ,Peptide sequence ,Pharmacology ,Mutation ,Strain (chemistry) ,Penicillin G ,Aminoacyltransferases ,Penicillin ,Infectious Diseases ,Hexosyltransferases ,chemistry ,Peptidyl Transferases ,Recombinant DNA ,Peptidoglycan ,Carrier Proteins ,medicine.drug - Abstract
Extensive use of β-lactam antibiotics has led to the selection of pathogenic streptococci resistant to β-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 β-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S 443 SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.
- Published
- 2004
38. The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains
- Author
-
Otto Dideberg, Anne Marie Di Guilmi, Thierry Vernet, and Andréa Dessen
- Subjects
Glycan ,Penicillin binding proteins ,Peptidyl transferase ,Polymers ,Molecular Sequence Data ,Peptide ,Peptidoglycan ,Muramoylpentapeptide Carboxypeptidase ,Microbiology ,Catalysis ,chemistry.chemical_compound ,Bacterial Proteins ,Polysaccharides ,Humans ,Penicillin-Binding Proteins ,Amino Acid Sequence ,Peptide Synthases ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,biology ,Lipid II ,Glycosyltransferases ,Enzymes and Proteins ,Uridine Diphosphate N-Acetylmuramic Acid ,Transmembrane protein ,Protein Structure, Tertiary ,Kinetics ,Streptococcus pneumoniae ,Hexosyltransferases ,chemistry ,Biochemistry ,Peptidyl Transferases ,biology.protein ,Carrier Proteins - Abstract
The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N -acetylglucosaminyl- N -acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat / K m were measured at 40.6 μM (± 15.5) and 1 × 10 −3 M −1 s −1 , respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S . pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.
- Published
- 2003
39. New approaches towards the identification of antibiotic and vaccine targets in Streptococcus pneumoniae
- Author
-
Andréa Dessen and Anne Marie Di Guilmi
- Subjects
medicine.drug_class ,Antibiotics ,Drug Resistance ,Reviews ,Virulence ,Drug resistance ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Pneumococcal Infections ,Microbiology ,Species Specificity ,Cell Wall ,Streptococcus pneumoniae ,Genetics ,medicine ,Humans ,Molecular Biology ,Infectivity ,Vaccines ,medicine.disease ,Virology ,Anti-Bacterial Agents ,Pneumococcal infections ,Mutagenesis ,Drug Design ,Bacteremia ,Pneumonia (non-human) ,Genome, Bacterial - Abstract
Streptococcus pneumoniae causes more than one million deaths every year, mostly of young children in developing countries, due to pneumonia, bacteremia and meningitis. The emergence and dissemination of drug-resistant pneumococcal strains, coupled to changing patterns of virulence and the inadequacy of available vaccines, calls for an aggressive search for novel targets for antibiotic and vaccine development. Microbial genomics techniques allow genetic and biochemical tools to be employed to tackle discovery, design and development of new anti-infective agents based on the identification of hundreds of new targets. In this review, novel approaches employed to identify potential antibiotic and vaccine targets in S. pneumoniae are highlighted. Recently identified virulence factors, as well as molecules essential for bacterial viability, cell wall integrity and infectivity, are discussed.
- Published
- 2002
40. Human L-ficolin recognizes phosphocholine moieties of pneumococcal teichoic acid
- Author
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Nicole M. Thielens, Anne-Marie Di Guilmi, Monique Lacroix, Richard R. Schmidt, Christian Pedersen, Emilie Vassal-Stermann, Lydie Martin, Ulrich Zähringer, Ana Maria Amoroso, Evelyne Gout, Emmanuelle Laffly, Christine Gaboriaud, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Proteases ,[SDV]Life Sciences [q-bio] ,Phosphorylcholine ,Immunology ,Plasma protein binding ,Biology ,Pneumococcal Infections ,chemistry.chemical_compound ,Cell Wall ,Lectins ,Complement C4b ,Immunology and Allergy ,Humans ,Binding site ,Complement Activation ,Phosphocholine ,chemistry.chemical_classification ,Teichoic acid ,Fibrinogen ,Acetylation ,Surface Plasmon Resonance ,Immunity, Innate ,Complement system ,Amino acid ,Protein Structure, Tertiary ,Teichoic Acids ,Streptococcus pneumoniae ,chemistry ,Biochemistry ,Complement C3b ,Host-Pathogen Interactions ,[SDV.IMM]Life Sciences [q-bio]/Immunology ,Lipoteichoic acid ,Protein Binding - Abstract
Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.
- Published
- 2014
41. O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry
- Author
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Michel-Yves Mistou, Thibault Chaze, Julia Chamot-Rooke, Shaynoor Dramsi, Benoît Valot, Patrick Trieu-Cuot, Anne-Marie Di Guilmi, Alain Guillot, Olivier Langella, Spectrométrie de Masse structurale et protéomique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Génétique Quantitative et Evolution - Le Moulon (Génétique Végétale) (GQE-Le Moulon), Institut National de la Recherche Agronomique (INRA)-Université Paris-Sud - Paris 11 (UP11)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie des Bactéries Pathogènes à Gram-positif, We thank the French National Research Agency (ANR) for supporting the 'GlycoPath' project. The DIM Malinf from the regionIle-de-France is also acknowledged for funding of the LTQ-Orbitrap Velos mass spectrometer., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-AgroParisTech-Université Paris-Sud - Paris 11 (UP11)-Institut National de la Recherche Agronomique (INRA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
MESH: Adhesins, Bacterial ,Glycan ,Glycosylation ,Collision-induced dissociation ,[SDV]Life Sciences [q-bio] ,Tandem mass spectrometry ,Biochemistry ,Analytical Chemistry ,Streptococcus agalactiae ,Serine ,chemistry.chemical_compound ,MESH: Software ,Fragmentation (mass spectrometry) ,Tandem Mass Spectrometry ,MESH: Monosaccharides ,MESH: Serine ,Threonine ,Adhesins, Bacterial ,Molecular Biology ,MESH: Glycopeptides ,biology ,Chemistry ,Research ,Monosaccharides ,Glycopeptides ,MESH: Tandem Mass Spectrometry ,MESH: Glycosylation ,MESH: Streptococcus agalactiae ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Electron-transfer dissociation ,biology.protein ,MESH: Chromatography, Liquid ,Software ,Chromatography, Liquid - Abstract
International audience; Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.
- Published
- 2014
42. Mutations in the Active Site of Penicillin-binding Protein PBP2x from Streptococcus pneumoniae
- Author
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Nicolas Mouz, Regine Hakenbeck, Anne Marie Di Guilmi, Thierry Vernet, Otto Dideberg, and E.J. Gordon
- Subjects
Mutation ,Penicillin binding proteins ,Cefotaxime ,medicine.drug_class ,Mutant ,Antibiotics ,Cell Biology ,Biology ,medicine.disease_cause ,Biochemistry ,Microbiology ,Penicillin ,Streptococcus pneumoniae ,polycyclic compounds ,medicine ,Molecular Biology ,Escherichia coli ,medicine.drug - Abstract
Penicillin-binding protein 2x (PBP2x) isolated from clinical β-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for β-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from β-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 → Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 → Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand β3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand β3 conflicts with the negative charge of the β-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. Thein vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.
- Published
- 1999
43. Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Is Membrane Associated
- Author
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JoAnn Hoskins, Anne Marie Di Guilmi, Otto Dideberg, Lydie Martin, S. Richard Jaskunas, Nicolas Mouz, and Thierry Vernet
- Subjects
Penicillin binding proteins ,Protein Conformation ,Molecular Sequence Data ,Cefotaxime ,Muramoylpentapeptide Carboxypeptidase ,medicine.disease_cause ,Microbiology ,Protein structure ,Bacterial Proteins ,Glycosyltransferase ,polycyclic compounds ,medicine ,Penicillin-Binding Proteins ,Trypsin ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Sequence Homology, Amino Acid ,biology ,Cell Membrane ,Cell Polarity ,Glycosyltransferases ,Membrane Proteins ,Drug Resistance, Microbial ,biochemical phenomena, metabolism, and nutrition ,Lipids ,Enzymes and Proteins ,Fusion protein ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Transmembrane protein ,Streptococcus pneumoniae ,Hexosyltransferases ,Biochemistry ,Membrane protein ,Peptidyl Transferases ,biology.protein ,bacteria ,Carrier Proteins - Abstract
Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to β-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S -transferase fusion protein. The acylation kinetic parameters of PBP 2a* for β-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other β-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.
- Published
- 1999
44. Identification, Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1a
- Author
-
Nicolas Mouz, Anne Marie Di Guilmi, Jean-Pierre Andrieu, S. Richard Jaskunas, Jean Gagnon, JoAnn Hoskins, Otto Dideberg, and Thierry Vernet
- Subjects
Enzyme complex ,Recombinant Fusion Proteins ,Ear infection ,Muramoylpentapeptide Carboxypeptidase ,medicine.disease_cause ,Peptide Mapping ,Microbiology ,Bacterial Proteins ,Multienzyme Complexes ,Endopeptidases ,Streptococcus pneumoniae ,polycyclic compounds ,medicine ,Penicillin-Binding Proteins ,Trypsin ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Glutathione Transferase ,biology ,Proteolytic enzymes ,Glycosyltransferases ,Periplasmic space ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Enzymes and Proteins ,Peptide Fragments ,Hexosyltransferases ,Solubility ,Biochemistry ,Peptidyl Transferases ,bacteria ,Carrier Proteins ,Bacteria - Abstract
The synthesis of the bacterial cell wall requires cytoplasmic and periplasmic enzymes. The final steps of peptidoglycan biosynthesis occur outside the cytoplasmic membrane, and they are catalyzed by membrane-bound penicillin-binding proteins (PBPs). PBPs play essential roles in cell division and morphology (6, 20, 31). Based upon their molecular sizes and amino acid sequence similarities, PBPs can be classified into two groups (6): low-molecular-weight (low-Mr) PBPs, which act as d,d-carboxypeptidases, and high-molecular-weight (high-Mr) PBPs, which carry transpeptidase (TP) and glycosyltransferase (GT) activities. The high-Mr group can be further divided into bifunctional enzymes with TP and GT activities (class A) and monofunctional TP enzymes (class B). β-Lactam antibiotics bind with high affinity specifically to d,d-carboxypeptidase and TP domains because of their structural similarity to the natural substrates, the stem peptides. This binding results in the formation of a covalent acyl-PBP enzyme complex, leading to the inactivation of PBPs. High-Mr PBPs are multidomain proteins (6). The three-dimensional structure of Streptococcus pneumoniae PBP 2x (class B high-Mr PBP) illustrates this domain organization (25). The only non-penicillin-binding domain of known function is the GT domain, corresponding to the N-terminal region of class A PBPs. This GT activity, clearly identified in Escherichia coli PBP 1b, is difficult to measure (23, 29, 31–35). It is insensitive to penicillin but sensitive to moenomycin, an antibiotic which is not used for human therapy (23, 29, 32, 33). S. pneumoniae is one of the major human pathogens of the upper respiratory tract, causing pneumonia, meningitis, and ear infections. Six PBPs have been identified in S. pneumoniae: high-Mr PBPs 1a, 1b, 2a, 2x, and 2b and low-Mr PBP 3 (8). PBPs 1a, 1b, and 2a belong to class A, while PBPs 2x and 2b are monofunctional class B proteins. Deletion of pbp2x and pbp2b in S. pneumoniae is lethal for the bacteria, while the deletion of pbp1a is tolerated (11), probably due to compensation by PBP 1b. This has been observed for E. coli class A PBP 1a, whose deletion can be compensated for by PBP 1b (36). In clinical isolates of resistant pneumococci, pbp1a, pbp2x, and pbp2b genes were shown to present a mosaic organization, encoding PBPs with reduced affinity for β-lactam antibiotics (2, 5, 15, 18). The specific resistance to ceftriaxone and cefotaxime of S. pneumoniae from the hospital environment is mediated by modification of PBP 2x and PBP 1a (22). Furthermore, gene transfer of pbp1a, pbp2x, and pbp2b from resistant strains conferred penicillin resistance on sensitive S. pneumoniae strains under laboratory conditions (2–4, 14, 15, 27, 30). The effort to overcome resistance to antibiotics in S. pneumoniae might therefore benefit from a detailed understanding of the molecular basis of TP and GT activities. The GT domain represents a new potential target for novel nonpenicillin antibiotics. Here, we delineate the GT and TP domains of S. pneumoniae PBP 1a* (a water-soluble form of PBP 1a) by limited proteolytic digestion and expression of recombinant domains. The TP activity of PBP 1a* and that of the isolated TP domain were compared. We also present evidence for an interaction between the isolated GT domain and moenomycin.
- Published
- 1998
45. Human adenovirus serotype 3 (Ad3) and the AD fiber protein bind to a 130-kDa membrane protein on HeLa cells
- Author
-
Anne Marie Di Guilmi, Evelyne Gout, Jadwiga Chroboczek, Paul Kitts, and Annie Barge
- Subjects
Cancer Research ,Vesicle-associated membrane protein 8 ,viruses ,Plasma protein binding ,Biology ,biology.organism_classification ,Cell biology ,Retinoblastoma-like protein 1 ,Cell membrane ,HeLa ,Infectious Diseases ,medicine.anatomical_structure ,Membrane protein ,Virology ,Protein A/G ,medicine ,biology.protein ,Protein G - Abstract
The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.
- Published
- 1995
46. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein
- Author
-
Anne Marie Di Guilmi, Christine Moriscot, Philippe Frachet, Rémi Terrasse, Thierry Vernet, Guy Schoehn, Nicole M. Thielens, Pascale Tacnet-Delorme, and Julien Pérard
- Subjects
Immunology ,chemical and pharmacologic phenomena ,Apoptosis ,Plasma protein binding ,Biology ,Ligands ,Biochemistry ,Cell Membrane Structures ,HeLa ,Cell membrane ,Classical complement pathway ,stomatognathic system ,immune system diseases ,medicine ,Humans ,Molecular Biology ,Complement Activation ,Glyceraldehyde 3-phosphate dehydrogenase ,Innate immune system ,Complement C1q ,Cell Membrane ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Plasminogen ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Surface Plasmon Resonance ,biology.organism_classification ,Molecular biology ,In vitro ,Complement system ,Solutions ,Kinetics ,Protein Transport ,medicine.anatomical_structure ,Immobilized Proteins ,Streptococcus pneumoniae ,Solubility ,Mutation ,biology.protein ,HeLa Cells ,Protein Binding - Abstract
C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.
- Published
- 2012
47. The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization
- Author
-
Anne Marie Di Guilmi, Ilaria Ferlenghi, Monica Moschioni, Andréa Dessen, Carlos Contreras-Martel, Thierry Vernet, Clothilde Manzano, and Lamya El Mortaji
- Subjects
Models, Molecular ,Protein subunit ,Amino Acid Motifs ,Molecular Conformation ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Models, Biological ,Pilus ,Streptococcus pneumoniae ,medicine ,Molecular Biology ,Escherichia coli ,chemistry.chemical_classification ,Mineral Fibers ,Isopeptide bond ,biology ,Chemistry ,Structural protein ,Hydrogen Bonding ,Cell Biology ,Crystallography ,Polymerization ,Pilin ,Fimbriae, Bacterial ,biology.protein ,Mutagenesis, Site-Directed ,Fimbriae Proteins ,Protein Multimerization ,Crystallization - Abstract
RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1–D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus. Abbreviations: EM, electron microscopy; ESI–MS, electrospray ionization MS; TOF, time-of-flight
- Published
- 2011
48. Structural Basis for the Substrate Specificity of a Novel β-N-Acetylhexosaminidase StrH Protein from Streptococcus pneumoniae R6*
- Author
-
Jun-Wei Zhang, Anne-Marie Di Guilmi, Yuxing Chen, Yong-Liang Jiang, Cecile Frolet, Thierry Vernet, Wei-Li Yu, and Cong-Zhao Zhou
- Subjects
Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,chemistry.chemical_compound ,Structure-Activity Relationship ,Bacterial Proteins ,TIM barrel ,Hydrolase ,Glycoside hydrolase ,Enzyme kinetics ,Molecular Biology ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,biology ,Active site ,Cell Biology ,beta-N-Acetylhexosaminidases ,Sialic acid ,Enzyme ,Streptococcus pneumoniae ,chemistry ,Protein Structure and Folding ,biology.protein ,Neuraminidase - Abstract
The β-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the β-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of β(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and β-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 Å crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (β/α)(8) TIM barrel with the active site residing at the center of the β-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-β-d-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k(cat)/K(m)) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAGβ(1,2)Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the β(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the β(1,2)-linked β-N-acetylglucosides.
- Published
- 2011
49. Structural and enzymatic characterization of the streptococcal ATP/diadenosine polyphosphate and phosphodiester hydrolase Spr1479/SapH
- Author
-
Jun-Wei Zhang, Cecile Frolet, Anne-Marie Di Guilmi, Wang Cheng, Yuxing Chen, Thierry Vernet, Cong-Zhao Zhou, Wei-Li Yu, Yong-Liang Jiang, and Chen-Chen Zhang
- Subjects
Protein Folding ,Stereochemistry ,Hypothetical protein ,Hydrolase activity ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Inorganic phosphate ,Apoenzymes ,Phosphodiester hydrolase ,Bacterial Proteins ,Molecular Biology ,chemistry.chemical_classification ,Adenosine Triphosphatases ,Binding Sites ,biology ,Polyphosphate ,Active site ,Cell Biology ,Phosphate ,Acid Anhydride Hydrolases ,Enzyme ,Streptococcus pneumoniae ,chemistry ,Protein Structure and Folding ,biology.protein - Abstract
Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.
- Published
- 2011
50. Biochemical characterization of the histidine triad protein PhtD as a cell surface zinc-binding protein of Pneumococcus
- Author
-
Elodie Loisel, Anne Imberty, Claire Durmort, Suneeta Chimalapati, Anne Marie Di Guilmi, Thierry Vernet, Catherine Bougault, Jeremy S. Brown, Benoit Gallet, Carret, Michèle, inconnu, Inconnu, Centre de Recherches sur les Macromolécules Végétales (CERMAV), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)
- Subjects
Operon ,Hydrolases ,Lipoproteins ,Amino Acid Motifs ,Virulence ,chemistry.chemical_element ,ATP-binding cassette transporter ,Zinc ,Biology ,Biochemistry ,In vitro ,Recombinant Proteins ,Streptococcus pneumoniae ,chemistry ,Bacterial Proteins ,Carrier Proteins ,Pathogen ,Gene ,Cation Transport Proteins ,Function (biology) ,ComputingMilieux_MISCELLANEOUS - Abstract
Zinc homeostasis is critical for pathogen host colonization. Indeed, during invasion, Streptococcus pneumoniae has to finely regulate zinc transport to cope with a wide range of Zn(2+) concentrations within the various host niches. AdcAII was identified as a pneumococcal Zn(2+)-binding protein; its gene is present in an operon together with the phtD gene. PhtD belongs to the histidine triad protein family, but to date, its function has not been clarified. Using several complementary biochemical methods, we provide evidence that like AdcAII, PhtD is a metal-binding protein specific for zinc. When Zn(2+) binds (K(d) = 131 ± 10 nM), the protein displays substantial thermal stabilization. We also present the first direct evidence of a joint function of AdcAII and PhtD by demonstrating that their expression is corepressed by Zn(2+), that they interact directly in vitro, and that they are colocalized at the bacterial surface. These results suggest the common involvement of the AdcAII-PhtD system in pneumococcal zinc homeostasis.
- Published
- 2011
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