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13 results on '"Marchetti, Roberta"'

1. Solid State NMR Studies of Intact Lipopolysaccharide Endotoxin

Author

Cédric Laguri, Alessandra Polissi, Antonio Molinaro, Paul Schanda, Alba Silipo, Jean-Pierre Simorre, Alessandra M. Martorana, Roberta Marchetti, 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), Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, Department of Pharmacological Sciences and Department of Biomolecular Sciences and Biotechnology, University of Milan, ISBG, UMS 3518 CNRS-CEA-UJF-EMBL, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), European Project: 721484, Train2target (ETN), Laguri, Cedric, Silipo, Alba, Martorana, Alessandra M, Schanda, Paul, Marchetti, Roberta, Polissi, Alessandra, Molinaro, Antonio, Simorre, Jean-Pierre, 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), University of Naples Federico II = Università degli studi di Napoli Federico II, and Università degli Studi di Milano = University of Milan (UNIMI)

Subjects
Lipopolysaccharides, 0301 basic medicine, Magnetic Resonance Spectroscopy, Oligosaccharides, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, Biochemistry, Lipid A, 03 medical and health sciences, Glycolipid, Escherichia coli, medicine, Humans, Pseudomonas Infections, Escherichia coli Infections, chemistry.chemical_classification, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, Vesicle, O Antigens, General Medicine, Nuclear magnetic resonance spectroscopy, Oligosaccharide, Ligand (biochemistry), biology.organism_classification, 0104 chemical sciences, 030104 developmental biology, Pseudomonas aeruginosa, Biophysics, Molecular Medicine, lipids (amino acids, peptides, and proteins), Bacteria
Abstract

International audience; Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of Gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E. coli and pathogenic P. aeruginosa with long O-antigen polysaccharides assembled in solution as vesicles or elongated micelles. Solid-state NMR with magic-angle spinning permitted the identification of NMR signals arising from regions with different flexibilities in the LPS, from the lipid components to the O-antigen polysaccharides. Atomic scale data on the LPS enabled the study of the interaction of gentamicin antibiotic bound to P. aeruginosa LPS, for which we could confirm that a specific oligosaccharide is involved in the antibiotic binding. The possibility to study LPS alone and bound to a ligand when it is assembled in membrane-like structures opens great prospects for the investigation of proteins and antibiotics that specifically target such an important molecule at the surface of Gram-negative bacteria.

Published
2018

2. Enzymatic and acidic degradation of high molecular weight dextran into low molecular weight and its characterizations using novel Diffusion-ordered NMR spectroscopy

Author

Afsheen Aman, Alba Silipo, Roberta Marchetti, Shah Ali Ul Qader, Samina Iqbal, Antonio Molinaro, Iqbal, Samina, Marchetti, Roberta, Aman, Afsheen, Silipo, Alba, Qader, Shah Ali Ul, and Molinaro, Antonio

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Bacillus, 02 engineering and technology, Biochemistry, Enzymatic hydrolysi, Diffusion, 03 medical and health sciences, chemistry.chemical_compound, Hydrolysis, Exopolysaccharide, Structural Biology, Enzymatic hydrolysis, Organic chemistry, Low molecular weight dextran, Molecular Biology, chemistry.chemical_classification, Chromatography, biology, Dextrans, Acid hydrolysi, Glycosidic bond, Dextranase, General Medicine, Nuclear magnetic resonance spectroscopy, Hydrogen-Ion Concentration, 021001 nanoscience & nanotechnology, biology.organism_classification, Structural characterization, 030104 developmental biology, Dextran, chemistry, Leuconostoc mesenteroides, Acid hydrolysis, 0210 nano-technology, Two-dimensional nuclear magnetic resonance spectroscopy, Leuconostoc
Abstract

Low molecular weight fractions were derived from native high molecular weight dextran produced by Leuconostoc mesenteroides KIBGE-IB26. Structural characterization of native and low molecular weight fractions obtained after acidic and enzymatic hydrolysis was done using FTIR and NMR spectroscopy. The molecular weight was estimated using Diffusion Ordered NMR spectroscopy. Native dextran (892 kDa) is composed of α-(1 → 6) glycosidic linkage along with α-(1 → 3) branching. Major proportion of 528 kDa dextran was obtained after prolong enzymatic hydrolysis however, an effective acidic treatment at pH-1.4 up to 02 and 04 h of exposure resulted in the formation of 77 kDa and 57 kDa, respectively. The increment in pH from 1.4 to 1.8 lowered the hydrolysis efficiency and resulted in the formation of 270 kDa dextran fraction. The results suggest that derived low molecular weight water soluble fractions can be utilized as a drug delivery carrier along with multiple application relating pharmaceutical industries.

Published
2017

3. Rhodopseudomonas palustris Strain CGA009 Produces an O-Antigen Built up by a C-4-Branched Monosaccharide: Structural and Conformational Studies

Author

Eric Giraud, Emiliano Bedini, Roberta Marchetti, Djamel Gully, Antonio Molinaro, Alba Silipo, Rosa Lanzetta, Marchetti, Roberta, Bedini, Emiliano, Gully, Djamel, Lanzetta, Rosa, Giraud, Eric, Molinaro, Antonio, Silipo, Alba, Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant’Angelo, University of Naples Federico II = Università degli studi di Napoli Federico II, Laboratoire des symbioses tropicales et méditerranéennes (UMR LSTM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), European Project: 642157,H2020,H2020-MSCA-ITN-2014,TOLLerant(2015), Università degli studi di Napoli Federico II, and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)

Subjects
Magnetic Resonance Spectroscopy, Stereochemistry, Molecular Conformation, 01 natural sciences, Biochemistry, 03 medical and health sciences, Molecular dynamics, Monosaccharide, Physical and Theoretical Chemistry, Optical rotation, Spectroscopy, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Strain (chemistry), 010405 organic chemistry, Chemistry, Organic Chemistry, Monosaccharides, Absolute configuration, O Antigens, biology.organism_classification, 0104 chemical sciences, Rhodopseudomonas, Rhodopseudomonas palustris, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Two-dimensional nuclear magnetic resonance spectroscopy
Abstract

International audience; Here, the analysis of the peculiar homopolymeric O-chain, isolated from the lipopolysaccharide (LPS) of Rhodopseudomonas palustris strain CGA009, is reported. The O-chain is built up of a novel 4-C-branched sugar (12-deoxy-4-C-(D-altro-5,7,8,9-tetrahydroxyhexyl))-3-O-methyl-D-galactopyranose)) whose structure, absolute configuration, and conformational features were deduced by 2D NMR spectroscopy, optical rotation measurements, and molecular dynamics simulations.

Published
2018

4. Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis

Author

Paul J. Brett, Roberta Marchetti, Mark A. Hubbard, Mary N. Burtnick, David P. AuCoin, Michael J. Dillon, Charles Gauthier, Marielle Tamigney Kenfack, Alba Silipo, Antonio Molinaro, Rosa Lanzetta, Yves Blériot, Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP), Université de Poitiers-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Marchetti, Roberta, Dillon, Mj, Burtnick, Mn, Hubbard, Ma, Kenfack, Mt, Blériot, Y, Gauthier, C, Brett, Pj, Aucoin, Dp, Lanzetta, Rosa, Silipo, Alba, and Molinaro, Antonio

Subjects
Bacterial capsule, Burkholderia pseudomallei, Melioidosis, medicine.drug_class, Immunoblotting, Biological Warfare Agents, Monoclonal antibody, Biochemistry, Epitope, Microbiology, Antigen, medicine, Humans, Bacterial Capsules, biology, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Antibodies, Monoclonal, General Medicine, biology.organism_classification, medicine.disease, Virology, 3. Good health, Bacterial vaccine, Bacterial Vaccines, biology.protein, Molecular Medicine, Antibody, Protein Binding
Abstract

International audience; Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

Published
2015

5. Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope

Author

Takema Fukatsu, Ye Rang Huh, Roberta Marchetti, Dae Woo Son, Jae Hyun Cho, Jiyeun Kate Kim, Ha-Young Park, Hiroshi Nakayama, Alba Silipo, Luisa Sturiale, Chan-Hee Kim, Antonio Molinaro, Bok Luel Lee, Kim, Jk, Son, Dw, Kim, Ch, Cho, Jh, Marchetti, Roberta, Silipo, Alba, Sturiale, L, Park, Hy, Huh, Yr, Nakayama, H, Fukatsu, T, Molinaro, Antonio, and Lee, B. L.

Subjects
animal structures, Burkholderia, Host–pathogen interaction, Immunology, Antimicrobial peptides, Biochemistry, Bacterial cell structure, Microbiology, Heteroptera, Symbiosis, Cell Wall, Animals, skin and connective tissue diseases, Molecular Biology, Innate immune system, biology, Host (biology), Cell Membrane, fungi, O Antigens, food and beverages, Cell Biology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Gastrointestinal Tract, bacteria, sense organs, Cell envelope, Antimicrobial Cationic Peptides
Abstract

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.

Published
2015

6. Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens

Author

Charles Gauthier, Yves Blériot, Roberta Marchetti, Mary N. Burtnick, Narisara Chantratita, Teerapat Nualnoi, Abba Ngassimou, Antonio Molinaro, Kitisak Sintiprungrat, Jérôme Marrot, Paul J. Brett, Marcelina Mazur, Marielle Tamigney Kenfack, David P. AuCoin, Alba Silipo, Teresa L. Shaffer, Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP), Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Poitiers-Institut de Chimie du CNRS (INC), Wroclaw University of Environmental and Life Sciences, School of Medicine [Reno], University of Nevada [Reno], Prince of Songkla University (PSU), University of South Alabama, Institut Lavoisier de Versailles (ILV), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Dipartimento di Scienze Chimiche [Naples], Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Mahidol University [Bangkok], Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Institut Armand Frappier (INRS-IAF), Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS), This work was supported by a grant from the Agence Nationale de la Recherche Programme Jeunes Chercheuses Jeunes Chercheurs (ANR-JCJC-12-JS07-0003-01), the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2016-04950), and the Department of Energy-funded (DE-FG02-93ER-20097) Center for Plant and Microbial Complex Carbohydrates. N.C. is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (U01AI115520). M.M. thanks the Wroclaw Center of Biotechnology for PDoc funding within the framework of the Leading National Research Center (KNOW), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Tamigney Kenfack, Marielle, Mazur, Marcelina, Nualnoi, Teerapat, Shaffer, Teresa L., Ngassimou, Abba, Blériot, Yve, Marrot, Jérôme, Marchetti, Roberta, Sintiprungrat, Kitisak, Chantratita, Narisara, Silipo, Alba, Molinaro, Antonio, Aucoin, David P., Burtnick, Mary N., Brett, Paul J., and Gauthier, Charles

Subjects
0301 basic medicine, Lipopolysaccharides, Melioidosis, Burkholderia pseudomallei, medicine.drug_class, Science, [SDV]Life Sciences [q-bio], 030106 microbiology, General Physics and Astronomy, Monoclonal antibody, Burkholderia mallei, General Biochemistry, Genetics and Molecular Biology, Epitope, Article, Microbiology, 03 medical and health sciences, Epitopes, Mice, Antigen, Bacterial Proteins, medicine, Animals, Antigens, Bacterial, Mice, Inbred BALB C, Multidisciplinary, biology, Glanders, Antibodies, Monoclonal, General Chemistry, Gene Expression Regulation, Bacterial, biology.organism_classification, medicine.disease, Virology, 3. Good health, Bacterial vaccine, 030104 developmental biology, Bacterial Vaccines, Female
Abstract

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an l-rhamnose into a 6-deoxy-l-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide–CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm., Melioidosis and glanders are multifaceted infections caused by gram-negative bacteria. Here, the authors synthesize a series of oligosaccharides that mimic the lipopolysaccharides present on the pathogens’ surface and use them to develop novel glycoconjugates for vaccine development.

Published
2017

7. Unraveling the Interaction between the LPS O-Antigen ofBurkholderia anthinaand the 5D8 Monoclonal Antibody by Using a Multidisciplinary Chemical Approach, with Synthesis, NMR, and Molecular Modeling Methods

Author

Inga Nilsson, Ángeles Canales, Jesús Jiménez-Barbero, Rosa Lanzetta, Roberta Marchetti, Alba Silipo, Antonio Molinaro, Dana E. Reed, Christian Vogel, David P. AuCoin, Marchetti, Roberta, Angeles, Canale, Lanzetta, Rosa, Inga, Nilsson, Christian, Vogel, Dana E., Reed, David P., Aucoin, Jesús Jiménez, Barbero, Molinaro, Antonio, and Silipo, Alba

Subjects
Lipopolysaccharides, Models, Molecular, Magnetic Resonance Spectroscopy, Molecular model, Burkholderia, Stereochemistry, medicine.drug_class, Molecular Dynamics Simulation, Disaccharides, Monoclonal antibody, Biochemistry, Chemical synthesis, Epitope, Molecular recognition, medicine, Molecular Biology, biology, Chemistry, Organic Chemistry, Antibodies, Monoclonal, O Antigens, Nuclear magnetic resonance spectroscopy, biology.organism_classification, Carbohydrate Sequence, Molecular Medicine, Burkholderia anthina, Trisaccharides, Two-dimensional nuclear magnetic resonance spectroscopy
Abstract

The interaction between the O-chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide-specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8-bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr-NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O-antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O-chain have been determined.

Published
2013

8. A Peptidoglycan-Remodeling Enzyme Is Critical for Bacteroid Differentiation in Bradyrhizobium spp. During Legume Symbiosis

Author

Daniel Gargani, Djamel Gully, Clémence Chaintreuil, Peter Mergaert, Katia Bonaldi, Joël Fardoux, Cédric Grangeteau, Roberta Marchetti, Eric Giraud, Nico Nouwen, Antonio Molinaro, Phuong Nguyen, Laboratoire des symbioses tropicales et méditerranéennes (UMR LSTM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Center for Chronobiology, Division of Biological Sciences, University of California [San Diego] (UC San Diego), University of California-University of California, Vin Aliment Microbiologie et Stress (VAlMiS), Procédés Alimentaires et Microbiologiques (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Procédés Alimentaires et Microbiologiques [Dijon] (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC), Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Intéractions Plantes-Bactéries (PBI), Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ANR-13-BSV7-0013,BugsInACell,Accommodation intracellulaire des bactéries symbiotiques fixatrices d'azote(2013), University of California (UC)-University of California (UC), University of Naples Federico II = Università degli studi di Napoli Federico II, Laboratoire des symbioses tropicales et méditerranéennes ( LSTM ), Centre de Coopération Internationale en Recherche Agronomique pour le Développement ( CIRAD ) -Université Montpellier 1 ( UM1 ) -Institut National de la Recherche Agronomique ( INRA ) -Université Montpellier 2 - Sciences et Techniques ( UM2 ) -Université de Montpellier ( UM ) -Institut national d’études supérieures agronomiques de Montpellier ( Montpellier SupAgro ), Biologie et Génétique des Interactions Plante-Parasite ( BGPI ), Centre de Coopération Internationale en Recherche Agronomique pour le Développement ( CIRAD ) -Institut National de la Recherche Agronomique ( INRA ) -Centre international d'études supérieures en sciences agronomiques ( Montpellier SupAgro ) -Institut national d’études supérieures agronomiques de Montpellier ( Montpellier SupAgro ), University of California [San Diego] ( UC San Diego ), Vin Aliment Microbiologie et Stress ( VAlMiS ), Procédés Alimentaires et Microbiologiques ( PAM ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Procédés Alimentaires et Microbiologiques [Dijon] ( PAM ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté ( UBFC ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté ( UBFC ), Intéractions Plantes-Bactéries ( PBI ), Département Microbiologie ( Dpt Microbio ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Gully, Djamel, Gargani, Daniel, Bonaldi, Katia, Grangeteau, Cédric, Chaintreuil, Clémence, Fardoux, Joël, Nguyen, Phuong, Marchetti, Roberta, Nouwen, Nico, Molinaro, Antonio, Mergaert, Peter, and Giraud, Eric

Subjects
0301 basic medicine, Physiology, [SDV]Life Sciences [q-bio], Mutant, nodosité racinaire, chemistry.chemical_compound, Bacteroides, Bradyrhizobium, Photosynthesis, Photosynthèse, Différenciation cellulaire, 2. Zero hunger, food and beverages, Fabaceae, General Medicine, Polyploïdie, Code génétique, Rhizobium, Symbiosi, F60 - Physiologie et biochimie végétale, Symbiose, Bacterial Protein, Peptidoglycan, Biology, Microbiology, 03 medical and health sciences, Photosynthesi, Bacterial Proteins, Symbiosis, Peptidase, Binding Sites, [ SDV ] Life Sciences [q-bio], Binding Site, P34 - Biologie du sol, Aeschynomene, Gene Expression Regulation, Bacterial, biology.organism_classification, 030104 developmental biology, chemistry, Enzyme, Mutation, Agronomy and Crop Science, Bacteria
Abstract

International audience; In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs called nodules on their roots. These nodules house nitrogen-fixing bacteroids that are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization, and other morphological changes. Here, we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules, indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of plant immune responses by the invading bacteria. In addition to exopolysaccharides, capsular polysaccharides, and lipopolysaccharides, whose role during symbiosis is well defined, our work demonstrates an essential role in symbiosis for yet another rhizobial envelope component, the peptidoglycan layer.

Published
2016

9. NMR analysis of the binding mode of two fungal endo β-1,4-mannanases from GH5 and GH26 families

Author

Alba Silipo, Jean-Guy Berrin, Roberta Marchetti, Marie Couturier, Antonio Molinaro, Shah Ali Ul Qader, Department of Chemical Sciences, Università degli studi di Napoli Federico II, Biodiversité et Biotechnologie Fongiques (BBF), École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), The Karachi Institute of Biotechnology and Genetic Engineering, University of Karachi, Marchetti, Roberta, Silipo, Alba, Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)-École Centrale de Marseille (ECM), University of Naples Federico II = Università degli studi di Napoli Federico II, Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM), Berrin, Jean Guy, Couturier, Marie, Ul Qader, Shah Ali, and Molinaro, Antonio

Subjects
Models, Molecular, 0301 basic medicine, Magnetic Resonance Spectroscopy, digestion enzymatique, Stereochemistry, [SDV]Life Sciences [q-bio], spectroscopie rmn, Biochemistry, Podospora anserina, Cell wall, 03 medical and health sciences, lignocellulose, podospora anserina, Podospora, Mannosidases, Carbohydrate Conformation, Glycoside hydrolase, glycoside hydrolase, Physical and Theoretical Chemistry, Binding site, Mode of action, Binding Sites, biology, Chemistry, Organic Chemistry, biology.organism_classification, 030104 developmental biology, manno-oligosaccharide, cristallographie rayon x, Carbohydrate conformation, paroi cellulaire végétale
Abstract

The enzymatic digestion of the main components of lignocellulosic biomass, including plant cell wallmannans, constitutes a fundamental step in the renewable biofuel production, with great potential benefit in the industrial field. Despite several reports of X-ray structures of glycoside hydrolases, how polysaccharides are [br/]specifically recognized and accommodated in the enzymes binding site still remains a pivotal matter of research. Within this frame, NMR spectroscopic techniques provide key binding information, complementing and/or enhancing the structural view by X-ray crystallography. Here we provide deep insights into the binding mode of two [i]endo-β[/i]-1,4 mannanases from the coprophilous ascomycete[i] Podospora anserina[/i], [i]Pa[/i]Man26A and [i]Pa[/i]Man5A, involved in the hydrolysis of plant cell wall mannans and heteromannans. The investigation at a molecular level of the interaction between the wild-type enzymes and inactive mutants with manno-oligosaccharides, revealed a different mode of action among the two glycoside hydrolases most likely due to the presence of the additional and peculiar −4 subsite in the [i]Pa[/i]Man26A binding pocket.

Published
2016

10. RHA-P: Isolation, expression and characterization of a bacterial α-l-rhamnosidase from Novosphingobium sp. PP1Y

Author

Eugenio Notomista, Alberto Di Donato, Nicola Ventimiglia, Federica De Lise, Roberta Marchetti, Roberto Vinciguerra, Vincenzo Tarallo, Antonio Molinaro, Valeria Cafaro, Annabella Tramice, Elio Pizzo, Leila Birolo, Francesca Mensitieri, Viviana Izzo, DE LISE, Federica, Mensitieri, Francesca, Tarallo, Vincenzo, Ventimiglia, Nicola, Vinciguerra, Roberto, Tramice, Annabella, Marchetti, Roberta, Pizzo, Eliodoro, Notomista, Eugenio, Cafaro, Valeria, Molinaro, Antonio, Birolo, Leila, DI DONATO, Alberto, and Izzo, Viviana

Subjects
0301 basic medicine, Glycosyl hydrolases, Rhamnose, "Novosphingobium sp. PP1Y", 030106 microbiology, Bioengineering, Novosphingobium sp. PP1Y, medicine.disease_cause, Biochemistry, Catalysis, "Biotransformation", 03 medical and health sciences, Hydrolysis, chemistry.chemical_compound, medicine, Glycoside hydrolase, "α-l-Rhamnosidases", "Flavonoids", "Novosphingobium sp. PP1Y", "Glycosyl hydrolases", "Biotransformation", Escherichia coli, Biotransformation, chemistry.chemical_classification, Flavonoids, biology, Process Chemistry and Technology, Neohesperidin dihydrochalcone, Glycosidic bond, Alfa-l-Rhamnosidases, Flavonoids, Novosphingobium sp. PP1, YGlycosyl hydrolases, Biotransformation, biology.organism_classification, "Glycosyl hydrolases", ?-L-Rhamnosidases, 030104 developmental biology, Enzyme, chemistry, "Flavonoids", Bacteria, "α-l-Rhamnosidases"
Abstract

α- l -Rhamnosidases (α-RHAs) are a group of glycosyl hydrolases of biotechnological potential in industrial processes, which catalyze the hydrolysis of α- l -rhamnose terminal residues from several natural compounds. A novel α–RHA activity was identified in the crude extract of Novosphingobium sp. PP1Y, a marine bacterium able to grow on a wide range of aromatic polycyclic compounds. In this work, this α-RHA activity was isolated from the native microorganism and the corresponding orf was identified in the completely sequenced and annotated genome of strain PP1Y. The coding gene was expressed in Escherichia coli, strain BL21(DE3), and the recombinant protein, rRHA-P, was purified and characterized as an inverting monomeric glycosidase of ca. 120 kDa belonging to the GH106 family. A biochemical characterization of this enzyme using pNPR as substrate was performed, which showed that rRHA-P had a moderate tolerance to organic solvents, a significant thermal stability up to 45 °C and a catalytic efficiency, at pH 6.9, significantly higher than other bacterial α-RHAs described in literature. Moreover, rRHA-P was able to hydrolyze natural glycosylated flavonoids (naringin, rutin, neohesperidin dihydrochalcone) containing α- l -rhamnose bound to β- d -glucose with either α-1,2 or α-1,6 glycosidic linkages. Data presented in this manuscript strongly support the potential use of RHA-P as a biocatalyst for diverse biotechnological applications.

Published
2016

11. Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes

Author

Daniel Gargani, Gerardino D'Errico, Djamel Gully, Hae In Lee, Joël Fardoux, Michelangelo Parrilli, Otto Holst, Domenico Garozzo, Nicolas Busset, Antonio Molinaro, Gargi Kulkarni, Giuseppe Vitiello, Regina Engel, Woo Suk Chang, Rosa Lanzetta, Clémence Chaintreuil, Luisa Sturiale, Herman Moll, Dianne K. Newman, Angelo Palmigiano, Alba Silipo, Luigi Paduano, Eric Giraud, Roberta Marchetti, Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant’Angelo, Università degli studi di Napoli Federico II, Laboratoire des symbioses tropicales et méditerranéennes (UMR LSTM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Consiglio Nazionale delle Ricerche (CNR), Biologie et Génétique des interactions Plantes-parasites pour la Protection Intégrée, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), University of Texas at Arlington, Partenaires INRAE, Division of Biology and Biological Engineering, Howard Hughes Medical Institute (HHMI), Leibniz-Center for Medicine and Biosciences, Chonbuk National University, French national research agency ANR-SESAM-2010-BLAN-170801 ANR-BugsInaCell-13-BSV7-0013-02, Italian Ministry of Education, Universities and Research 2009J98Z3_001 BJ23MN_007 L9SH3K RBNE08HWLZ, Mizutani Foundation for Glycoscience, NASA NNX12AD93G, Howard Hughes Medical Institute, ANR-13-BSV7-0013,BugsInACell,Accommodation intracellulaire des bactéries symbiotiques fixatrices d'azote(2013), University of Naples Federico II = Università degli studi di Napoli Federico II, National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR), Silipo, Alba, Vitiello, Giuseppe, Djamel, Gully, Luisa, Sturiale, Clemence, Chaintreuil, Joel, Fardoux, Daniel, Gargani, Hae In, Lee, Gargi, Kulkarni, Nicolas, Busset, Marchetti, Roberta, Angelo, Palmigiano, Herman, Moll, Regina, Engel, Lanzetta, Rosa, Paduano, Luigi, Parrilli, Michelangelo, Woo Suk, Chang, Otto, Holst, Dianne K., Newman, Domenico, Garozzo, D'Errico, Gerardino, Eric, Giraud, and Molinaro, Antonio

Subjects
[SDV.SA]Life Sciences [q-bio]/Agricultural sciences, Lipopolysaccharide, General Physics and Astronomy, F62 - Physiologie végétale - Croissance et développement, identification, mechanism, agrobacterium, bacteria, gene, fatty-acid, mass-spectrometry, photosynthetic bradyrhizobia, lipopolysaccharide, absolute-configuration, Plant Root Nodulation, Lipid A, chemistry.chemical_compound, F01 - Culture des plantes, Bradyrhizobium, 0303 health sciences, Liposome, Vegetal Biology, Multidisciplinary, biology, Strain (chemistry), Molecular Structure, Fabaceae, Aeschynomène, Agricultural sciences, Biochemistry, Bactérie gram négatif, lipids (amino acids, peptides, and proteins), Bacterial outer membrane, Root Nodules, Plant, Symbiose, Stress, Cyclase, General Biochemistry, Genetics and Molecular Biology, Microbiology, 03 medical and health sciences, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Symbiosis, Physiologie, 030304 developmental biology, Bactériologie, 030306 microbiology, Membrane cellulaire, General Chemistry, biology.organism_classification, Triterpenes, chemistry, Biologie végétale, Sciences agricoles, Bacteria
Abstract

International audience; Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intra-cellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.

Published
2014

12. X-raystructural studies of the entire extracellular region of the serine/threonine kinase PrkC from Staphylococcus aureus

Author

Daniela Marasco, Flavia Squeglia, Rita Berisio, Roberta Marchetti, Antonio Molinaro, Alessia Ruggiero, Ruggiero, Alessia, Squeglia, Flavia, Marasco, Daniela, Marchetti, Roberta, Molinaro, Antonio, and Berisio, Rita

Subjects
Staphylococcus aureus, Protein Conformation, Peptidoglycan, Bacillus subtilis, Biology, Crystallography, X-Ray, Biochemistry, Serine, Cell wall, muropeptidi, Protein structure, Bacterial Proteins, Extracellular, Penicillin-Binding Proteins, Protein Interaction Domains and Motifs, Threonine, Molecular Biology, Protein Kinase C, Serine/threonine-specific protein kinase, Kinase, Cell Biology, biology.organism_classification, Recombinant Proteins, raggi X, tubercolosi, Dimerization
Abstract

Bacterial serine/threonine kinases modulate a wide number of cellular processes. The serine/threonine kinase PrkC from the human pathogen Staphylococcus aureus was also shown to induce germination of Bacillus subtilis spores, in response to cell wall muropeptides. The presence of muropeptides in the bacterial extracellular milieu is a strong signal that the growing conditions are promising. In the present paper, we report the X-ray structure of the entire extracellular region of PrkC from S. aureus. This structure reveals that the extracellular region of PrkC, EC-PrkC, is a linear modular structure composed of three PASTA (penicillin binding-associated and serine/threonine kinase-associated) domains and an unpredicted C-terminal domain, which presents the typical features of adhesive proteins. Using several solution techniques, we also found that EC-PrkC shows no tendency to dimerize even in the presence of high concentrations of muropeptides. X-ray structural results obtained in the present study provide molecular clues into the mechanism of muropeptide-induced PrkC activation.

Published
2011

13. Chemical basis of peptidoglycandiscrimination by PrkC, a key kinase involved in bacterial resuscitation from dormancy

Author

Antonio Molinaro, Rita Berisio, Alba Silipo, Rosa Lanzetta, Maxim V. Petoukhov, Daniela Marasco, Flavia Squeglia, Alessia Ruggiero, Roberta Marchetti, Jonathan Dworkin, Squeglia, F, Marchetti, Roberta, Ruggiero, A, Lanzetta, Rosa, Marasco, Daniela, Dworkin, J, Petoukhov, M, Molinaro, Antonio, Berisio, R, and Silipo, Alba

Subjects
Models, Molecular, Protein Conformation, Bacillus subtilis, Plasma protein binding, Peptidoglycan, Protein Serine-Threonine Kinases, Biochemistry, Catalysis, Substrate Specificity, Cell wall, chemistry.chemical_compound, Colloid and Surface Chemistry, Protein structure, Nuclear Magnetic Resonance, Biomolecular, biology, Kinase, Cell growth, General Chemistry, biology.organism_classification, PASTA domain, Protein Structure, Tertiary, chemistry, Mutation, Protein Binding
Abstract

Bacterial Ser/Thr kinases modulate a wide number of cellular processes. In Bacillus subtilis , the Ser/Thr kinase PrkC has been shown to induce germination of bacterial spores in response to DAP-type but not Lys-type cell wall muropeptides. Muropeptides are a clear molecular signal that growing conditions are promising, since they are produced during cell wall peptidoglycan remodeling associated with cell growth and division of neighboring bacteria. However, whether muropeptides are able to bind the protein physically and how the extracellular region is able to distinguish the two types of muropeptides remains unclear. Here we tackled the important question of how the extracellular region of PrkC (EC-PrkC) senses muropeptides. By coupling NMR techniques and protein mutagenesis, we exploited the structural requirements necessary for recognition and binding and proved that muropeptides physically bind to EC-PrkC through DAP-moiety-mediated interactions with an arginine residue, Arg500, belonging to the protein C-terminal PASTA domain. Notably, mutation of this arginine completely suppresses muropeptide binding. Our data provide the first molecular clues into the mechanism of sensing of muropeptides by PrkC.

Published
2011
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