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51. In vivo proteome dynamics during early bovine myogenesis

Author

Bruno Meunier, Thibault Chaze, Christophe Chambon, Brigitte Picard, Catherine Jurie, Unité de Recherches sur les Herbivores (URH), Institut National de la Recherche Agronomique (INRA), and Qualité des Produits Animaux (QuaPA)

Subjects
Proteomics, proliferation, [SDV]Life Sciences [q-bio], Down-Regulation, Muscle Proteins, Apoptosis, Gestational Age, Biology, Muscle Development, Biochemistry, Myoblasts, 03 medical and health sciences, Fetus, Downregulation and upregulation, Pregnancy, In vivo, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Animals, Myocyte, [INFO]Computer Science [cs], [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Molecular Biology, Cell Proliferation, 030304 developmental biology, Genetics, 0303 health sciences, Cell growth, Myogenesis, 030302 biochemistry & molecular biology, Cell cycle, Up-Regulation, Cell biology, in vivo, Proteome, bos taurus, Cattle, Female, myogenesis, Isoelectric Focusing
Abstract

International audience; Myogenesis is a complex process of which the underlying mechanisms are conserved between species, including birds and mammals. Despite a good understanding of the stages of myogenesis, many of the mechanisms involved in the regulation of proliferation of the successive myoblast generations, the cellular transitions cell proliferation/alignment of myoblasts/fusion of myoblasts into myotubes/differentiation of myofibres and the control of total myofibre number still remain unknown. An in vivo proteomic analysis of the semitendinosus muscle from Charolais foetuses, at three specific stages of myogenesis (60, 110 and 180 days postconception), was conducted using 2-DE and MS. Expression profiles of more than 170 proteins were revealed and analysed using two way hierarchical clustering and statistical analysis. Our studies identify, for the first time, distinct proteins of varied biological functions and protein clusters with myogenic processes, such as the control of cell cycle activity and apoptosis, the establishment of cellular metabolism and muscle contractile properties and muscle cell reorganisation. These results are of fundamental interest to the field of myogenesis in general, and more specifically to the control of muscle development in meat producing animals.

Published
2008
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52. 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
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53. O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry

Author

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
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54. Alteration of the serum N-glycome of mice locally exposed to high doses of ionizing radiation

Author

Tony Lefebvre-Darroman, Eric Bey, Thibault Chaze, Patrick Gourmelon, Marie-Christine Slomianny, Jean-Claude Michalski, Fabien Milliat, Olivier Guipaud, Georges Tarlet, Marc Benderitter, CNRS, Université de Lille, Institut de Radioprotection et de Sûreté Nucléaire [IRSN], Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Laboratoire de Radiopathologie et de Thérapies Expérimentales (IRSN/PRP-HOM/SRBE/LRTE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Hôpital d'instruction des Armées Percy, PRP-HOM/SRBE/LRTE, Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), and Laboratoire de Radiopathologie et Thérapies Expérimentales [IRSN, Fontenay-aux-Roses] (PRP-HOM - SRBE)

Subjects
Male, Glycosylation, [SDV]Life Sciences [q-bio], Interleukin-1beta, Biochemistry, Analytical Chemistry, Ionizing radiation, Mice, chemistry.chemical_compound, 0302 clinical medicine, Electrophoresis, Gel, Two-Dimensional, Glycomics, Fucosylation, Skin, Mice, Inbred BALB C, Principal Component Analysis, 0303 health sciences, Blood Proteins, Blood proteins, 3. Good health, Radiation Injuries, Experimental, Carbohydrate Sequence, Liver, 030220 oncology & carcinogenesis, Burns, Adult, Glycan, Molecular Sequence Data, Biology, Proinflammatory cytokine, 03 medical and health sciences, Polysaccharides, Animals, Humans, Gamma Rays, Interleukin-6, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Gene Expression Regulation, Tumor Necrosis Factor-alpha, Protein Processing, Post-Translational, Polysaccharide, Molecular Biology, 030304 developmental biology, Research, Molecular biology, Glycome, chemistry, biology.protein
Abstract

International audience; Exposure of the skin to ionizing radiation leads to characteristic reactions that will often turn into a pathophysiological process called the cutaneous radiation syndrome. The study of this disorder is crucial to finding diagnostic and prognostic bioindicators of local radiation exposure or radiation effects. It is known that irradiation alters the serum proteome content and potentially post-translationally modifies serum proteins. In this study, we investigated whether localized irradiation of the skin alters the serum glycome. Two-dimensional differential in-gel electrophoresis of serum proteins from a man and from mice exposed to ionizing radiation showed that potential post-translational modification changes occurred following irradiation. Using a large-scale quantitative mass-spectrometry-based glycomic approach, we performed a global analysis of glycan structures of serum proteins from non-irradiated and locally irradiated mice exposed to high doses of γ-rays (20, 40, and 80 Gy). Non-supervised descriptive statistical analyses (principal component analysis) using quantitative glycan structure data allowed us to discriminate between uninjured/slightly injured animals and animals that developed severe lesions. Decisional statistics showed that several glycan families were down-regulated whereas others increased, and that particular structures were statistically significantly changed in the serum of locally irradiated mice. The observed increases in multiantennary N-glycans and in outer branch fucosylation and sialylation were associated with the up-regulation of genes involved in glycosylation in the liver, which is the main producer of serum proteins, and with an increase in the key proinflammatory serum cytokines IL-1β, IL-6, and TNFα, which can regulate the expression of glycosylation genes. Our results suggest for the first time a role of serum protein glycosylation in response to irradiation. These protein-associated glycan structure changes might signal radiation exposure or effects. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Published
2013
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55. Biological markers for meat tenderness of the three main French beef breeds using 2-DE and MS approach

Author

Jacques Lepetit, Brigitte Picard, Christophe Denoyelle, Bruno Meunier, Thibault Chaze, Christophe Chambon, Gilles Renand, Catherine Jurie, Laurent Journaux, Jean-François Hocquette, Sylvie Rousset, Unité Mixte de Recherches sur les Herbivores - UMR 1213 (UMRH), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Recherche Agronomique (INRA), Génétique Animale et Biologie Intégrative (GABI), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Qualité des Produits Animaux (QuaPA), Institut National de la Recherche Agronomique (INRA), MNE, Union Nationale des Coopératives Agricoles d'Elevage et d'Insémination Animale, Unité de Nutrition Humaine (UNH), Institut National de la Recherche Agronomique (INRA)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Clermont Université, Institut de l'Elevage, F. Toldra, LML Nollet, Unité Mixte de Recherche sur les Herbivores - UMR 1213 (UMRH), Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Union nationale des coopératives d’élevage et d’insémination animale (UNCEIA), Institut de l'élevage (IDELE), Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Université d'Auvergne - Clermont-Ferrand I (UdA)-Clermont Université-Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherches sur les Herbivores ( UMR 1213 Herbivores ), VetAgro Sup ( VAS ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Recherche Agronomique ( INRA ), Génétique Animale et Biologie Intégrative ( GABI ), Institut National de la Recherche Agronomique ( INRA ) -AgroParisTech, Qualité des Produits Animaux ( QUAPA ), Institut National de la Recherche Agronomique ( INRA ), Unité de Nutrition Humaine ( UNH ), and Institut National de la Recherche Agronomique ( INRA ) -Université d'Auvergne - Clermont-Ferrand I ( UdA ) -Clermont Université

Subjects
Longissimus thoracis muscle, musculoskeletal diseases, 0303 health sciences, Veterinary medicine, [ SDV ] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], 0402 animal and dairy science, 04 agricultural and veterinary sciences, 040201 dairy & animal science, Breed, Tenderness, 03 medical and health sciences, Meat tenderness, Geography, Marker status, medicine, Statistical analysis, medicine.symptom, 030304 developmental biology
Abstract

In order to identify new markers of beef tenderness, a proteomic analysis was performed on Longissimus thoracis muscle to compare two extreme groups in terms of meat tenderness, consisting of 10 animals each of the three main French beef breeds: Blond d’Aquitaine, Charolais, and Limousin. Animals were grouped on the basis of an index combining a tenderness score estimated by a trained panel and mechanical shear force measurement (Warner-Bratzler). The large number of available experimental animals (more than 3,300 young bulls) allowed animals with different meat tenderness but similar main muscle characteristics (fibers, collagen, and lipids) to be chosen. The muscle proteins of the extreme groups considered 24 h after slaughter were analyzed by two-dimensional electrophoresis, statistical analysis, and mass spectrometry. Potential markers of tenderness were suggested for each breed; their marker status varied according to the breed. Only α actin appeared to be a potential marker of tenderness in the three studied breeds. We focused particularly on different abundances of HSP27 and Troponin T fast isoforms between tenderness groups and according to breed.

Published
2013
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56. Analyse protéomique appliquée à l'identification de la composition en protéines d'une préparation de muscle 'crushed-muscle'

Author

Brigitte Picard, Christiane Barboiron, Ludivine Minassian, Thibault Chaze, Isabelle Cassar Malek, Muriel Bonnet, Unité de Recherches sur les Herbivores (URH), Institut National de la Recherche Agronomique (INRA), and ProdInra, Migration

Subjects
[SDV.SA]Life Sciences [q-bio]/Agricultural sciences, [SDV.SA] Life Sciences [q-bio]/Agricultural sciences, ComputingMilieux_MISCELLANEOUS
Abstract

National audience

Published
2008

57. Mapping of alkaline proteins in bovine skeletal muscle

Author

Julien Bouley, Christiane Barboiron, Thibault Chaze, Christophe Chambon, Brigitte Picard, Unité de Recherches sur les Herbivores (URH), Institut National de la Recherche Agronomique (INRA), and Qualité des Produits Animaux (QuaPA)

Subjects
Proteomics, TROPONIN, Proteome, Muscle Proteins, ALKALINE PROTEINS, Biochemistry, 03 medical and health sciences, Troponin T, Protein purification, medicine, Animals, Electrophoresis, Gel, Two-Dimensional, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Muscle, Skeletal, Molecular Biology, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, Two-dimensional gel electrophoresis, biology, 030302 biochemistry & molecular biology, Skeletal muscle, Hydrogen-Ion Concentration, MUSCLE, Troponin, medicine.anatomical_structure, Gene Expression Regulation, biology.protein, Cattle, SKELETAL MUSCLE, TWO-DIMENSIONAL GEL ELECTROPHORESIS, Function (biology)
Abstract

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.

Published
2006

58. The Interplay of Glycosaminoglycans and Cysteine Cathepsins in Mucopolysaccharidosis

Author

Alexis David, Thibault Chazeirat, Ahlame Saidi, Gilles Lalmanach, and Fabien Lecaille

Subjects
cysteine proteases, glycosaminoglycans, inhibition, mucopolysaccharidosis, proteolysis, Biology (General), QH301-705.5
Abstract

Mucopolysaccharidosis (MPS) consists of a group of inherited lysosomal storage disorders that are caused by a defect of certain enzymes that participate in the metabolism of glycosaminoglycans (GAGs). The abnormal accumulation of GAGs leads to progressive dysfunctions in various tissues and organs during childhood, contributing to premature death. As the current therapies are limited and inefficient, exploring the molecular mechanisms of the pathology is thus required to address the unmet needs of MPS patients to improve their quality of life. Lysosomal cysteine cathepsins are a family of proteases that play key roles in numerous physiological processes. Dysregulation of cysteine cathepsins expression and activity can be frequently observed in many human diseases, including MPS. This review summarizes the basic knowledge on MPS disorders and their current management and focuses on GAGs and cysteine cathepsins expression in MPS, as well their interplay, which may lead to the development of MPS-associated disorders.

Published
2023
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60. The c‐di‐ <scp>AMP</scp> ‐binding protein <scp>CbpB</scp> modulates the level of <scp>ppGpp</scp> alarmone in Streptococcus agalactiae

Author

Giovanni Covaleda‐Cortés, Ariel Mechaly, Terry Brissac, Heike Baehre, Laura Devaux, Patrick England, Bertrand Raynal, Sylviane Hoos, Myriam Gominet, Arnaud Firon, Patrick Trieu‐Cuot, Pierre Alexandre Kaminski, Biologie des Bactéries pathogènes à Gram-positif - Biology of Gram-Positive Pathogens, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hannover Medical School [Hannover] (MHH), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by grants from the French Government ‘Laboratory of Excellence - Integrative Biology of Emerging Infectious Diseases’ (LabEx IBEID, grant number ANR-10-LABX-62-IBEID to PTC), the Fondation pour la Recherche Médicale (FRM grant number DEQ20181039599 to PTC)., We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we thank the staff of beamlines PROXIMA-1 and PROXIMA-2A for assistance. The authors are grateful to the Staff of the Crystallography platform at the Institut Pasteur for robot-driven crystallization screenings. We acknowledge Eduard Baquero Salazar for his help during the early stage of this work. We also acknowledge the help of Mariette Matondo and Thibault Chaze from the Proteomics facility of Institut Pasteur., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
CbpB protein, Cyclic diadenosine monophosphate (c‐di‐AMP), [SDV]Life Sciences [q-bio], X‐ray crystallography & binding, Streptococcus, Cell Biology, Potassium homeostasis, CBS domain, Molecular Biology, Biochemistry
Abstract

International audience; Cyclic di-AMP is an essential signaling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Published
2023
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61. Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments

Author

Virginija Cvirkaite-Krupovic, Patrick Forterre, Yunfeng Yang, Yulong Shen, Mart Krupovic, Junfeng Liu, Fan Zhou, Pierre-Henri Commere, Virologie des archées - Archaeal Virology, Institut Pasteur [Paris], Shandong University, Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), This work was supported by l’Agence Nationale de la Recherche (#ANR-17-CE15-0005-01) and Ville de Paris Emergence(s) program (project MEMREMA) grants to MK, and European Research Council grant from the European Union’s Seventh Framework Program (FP/2007-2013)/Project EVOMOBIL-ERC Grant Agreement 340440 to PF. JL was partly supported through the PRESTIGE post-doctoral program from European Union’s Seventh Framework Programme, the National Key Research and Development Program of China (No. 2020YFA0906800), and the post-doctoral fellowship from the State Key Laboratory of Microbial Technology, Shandong University., We would like to thank Thibault Chaze and Mariette Matondo (Proteomics Platform, Institut Pasteur) as well as Thomas Cokelaer and Marc Monot (Biomics Platform, Institut Pasteur) for help with the proteomics and sequencing analyses, respectively. We are also grateful to the Ultrastructural BioImaging (UTechS UBI) unit of Institut Pasteur for access to electron microscopes. We gratefully acknowledge the UtechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging, ANR-10–INSB–04, Investments for the Future)., ANR-17-CE15-0005,ENVIRA,Remodelage de la membrane cytoplasmique par les virus enveloppés d'archées(2017), European Project: 340440,EC:FP7:ERC,ERC-2013-ADG,EVOMOBIL(2014), Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)

Subjects
Cell division, macromolecular substances, Microbiology, ESCRT, Article, Cell cycle phase, 03 medical and health sciences, Saccharolobus solfataricus, CRISPR, hyperthermophiles, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, biology, Endosomal Sorting Complexes Required for Transport, 030306 microbiology, Chemistry, ESCRT system, Nutrients, biology.organism_classification, Archaea, Sulfolobus, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Membrane protein, Proteome, Horizontal gene transfer, extracellular vesicles, Biogenesis, Extreme Environments
Abstract

International audience; Role of extracellular vesicles in extreme environments Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only a diverse proteome, enriched in membrane proteins, but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results indicate that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. We find that all components of the ESCRT apparatus are encapsidated into EVs. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. In particular, the active EV production appears to coincide with the expression patterns of ESCRT-III-1 and ESCRT-III-2, rather than ESCRT-III, suggesting a prime role of these proteins in EV budding. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.

Published
2021
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62. A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

Author

Mart Krupovic, David Prangishvili, Diana P. Baquero, Maryse Moya-Nilges, Martin Sachse, Anastasia D. Gazi, Stefan Schouten, Junfeng Liu, Christine Schmitt, Virologie des archées - Archaeal Virology, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Royal Netherlands Institute for Sea Research (NIOZ), This work was supported by l’Agence Nationale de la Recherche (Grant ANR-17-CE15-0005-01) and Emergence(s) project from Ville de Paris (to M.K.). D.P.B. was part of the Pasteur–Paris University International PhD Program, which has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant Agreement No. 665807. The Unit of Techology & Service Ultrastructural BioImaging is a member facility of France BioImaging (ANR-10-INSB-0004)., We would like to thank Thibault Chaze and Mariette Matondo (Proteomics Platform, Institut Pasteur) for help with the proteomics and Anchelique Mets (Royal Netherlands Institute for Sea Research) for support with lipid analysis. We are also grateful for the helpful discussions and support provided by Jacomine Krijnse-Locker., ANR-17-CE15-0005,ENVIRA,Remodelage de la membrane cytoplasmique par les virus enveloppés d'archées(2017), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), and Institut Pasteur [Paris]

Subjects
Cytoplasm, Electron Microscope Tomography, Viral protein, archaeal viruses, viruses, virus-associated pyramids, virus assembly, medicine.disease_cause, Virus, Lipothrixviridae, Sulfolobus, Viral Proteins, 03 medical and health sciences, Viral envelope, Escherichia coli, medicine, 030304 developmental biology, 0303 health sciences, Budding, Multidisciplinary, biology, 030306 microbiology, Saccharolobus, Virion, virus egress, Archaeal Viruses, Biological Sciences, biology.organism_classification, hyperthermophilic archaea, Cell biology, Host-Pathogen Interactions, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, cell lysis, Archaea
Abstract

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus–host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus–host interactions, we studied the life cycle of the enveloped, ∼2-μm-longSulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus–host interactions in Archaea.

Published
2021
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63. The Chlamydia effector CT622/TaiP 3 targets a non-autophagy related function of ATG16L1 4 5

Author

Hamaoui, Daniel, Cossé, Mathilde, Mohan, Jagan, Lystad, Alf Håkon, Wollert, Thomas, Subtil, Agathe, Biologie cellulaire de l'Infection microbienne - Cellular Biology of Microbial Infection, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Sorbonne Université (SU), Biochimie membranaire et transport - Membrane Biochemistry and Transport, Department of Molecular Medicine [Oslo], Institute of Basic Medical Sciences [Oslo], Faculty of Medicine [Oslo], University of Oslo (UiO)-University of Oslo (UiO)-Faculty of Medicine [Oslo], University of Oslo (UiO)-University of Oslo (UiO), This work was supported by a European Research Council (ERC) Starting Grant (NUChLEAR 282046), and by a Grant from the Pasteur-Weizmann Collaborative Research Funds. D.H. was funded by the ERC and by the Fondation pour la Recherche Médicale (Grant SPF20170938695)., We thank Dr. A. Simonsen for discussions, Dr. S. Lichtenthaler (TUM Munich, Germany) for the gift of anti-TMEM59 antibodies and TMEM59-HA plasmid, Dr. B. Goud (Institut Curie, France) and Dr. M. T. Damiani (Mendoza, Argentina) for supplying Rab constructs and anti-Rab6 antibody, Dr. G. Zhong (San Antonio, Texas) for anti-CT813 antibodies. We also thank Dr. E. Morel (INEM, France) for providing the GFP-ATG16L1 constructs, Dr. Laurent Boyer (Nice, France) for the NOD constructs, and Dr. P. Cossart (Institut Pasteur, France) for the TLR2 vector. We thank Thibault Chaze (Proteomics platform Institut Pasteur) for the analysis of the PD experiments., European Project: 282046,EC:FP7:ERC,ERC-2011-StG_20101109,NUCHLEAR(2012), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Collège doctoral [Sorbonne universités]

Subjects
autophagy, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], host-pathogen interactions, ATG16L1, Chlamydia trachomatis, intracellular traffic, Chlamydia, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

International audience; The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.

Published
2020
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64. Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments

Author

Edward H. Egelman, Fengbin Wang, Leticia C. Beltran, Zhangli Su, David Prangishvili, Tomasz Osinski, Diana P. Baquero, Mart Krupovic, Weili Zheng, University of Virginia [Charlottesville], Virologie des archées - Archaeal Virology, Institut Pasteur [Paris], This work was supported by NIH Grant R35GM122510 (E.H.E.). M.K. was supported by l’Agence Nationale de la Recherche Grant ANR-17-CE15-0005-01. D.P.B. is part of the Pasteur–Paris University International PhD Program, which has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant Agreement 665807., Imaging of SSRV1 was performed at the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under Contract HSSN261200800001E. Imaging of SIFV was done at the Molecular Electron Microscopy Core Facility at the University of Virginia, which is supported by the School of Medicine, We thank Thibault Chaze and Mariette Matondo (Pasteur Proteomics Platform) for help with the mass spectrometry analyses., ANR-17-CE15-0005,ENVIRA,Remodelage de la membrane cytoplasmique par les virus enveloppés d'archées(2017), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), University of Virginia, and Institut Pasteur [Paris] (IP)

Subjects
Archaeal Viruses, viruses, Genome, Viral, Genome, Virus, Sulfolobus, 03 medical and health sciences, chemistry.chemical_compound, Capsid, Viral envelope, Phylogeny, extremophiles, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, 030306 microbiology, Chemistry, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], DNA Viruses, Protein superfamily, Biological Sciences, Biological Evolution, hyperthermophilic archaea, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Viral evolution, DNA, Viral, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, cryo-EM, DNA, Sulfolobales, filamentous viruses, Extreme Environments
Abstract

International audience; Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homo-dimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homo-dimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenvel-oped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution. cryo-EM | extremophiles | hyperthermophilic archaea | filamentous viruses

Published
2020
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65. The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth

Author

Cossé, Mathilde, Barta, Michael, Fisher, Derek, Oesterlin, Lena, Niragire, Béatrice, Perrinet, Stéphanie, Millot, Gaël, Hefty, P Scott, Subtil, Agathe, Biologie cellulaire de l'Infection microbienne - Cellular Biology of Microbial Infection, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Kansas [Kansas City], Southern Illinois University [Carbondale] (SIU), Compartimentation et dynamique cellulaires (CDC), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), This work was supported by an ERC Starting Grant (NUChLEAR N°282046), the Institut Pasteur and the Centre National de la Recherche Scientifique. MB and associated research was supported by NIGMS P30 GM110761. PH was supported by NIAID R01AI079083. Work by DF was supported by the NIAID of the National Institutes of Health under Award Number 1R21AI115238-01., We are very grateful to Dr. C. Parsot for initially pointing to the labile N-terminal portion of CT622 as a putative chaperone binding site, and for reagents and advice, and to Sara Campos for technical help. We acknowledge the contribution of the Recombinant Proteins in Prokaryotic Cells platform. We thank Thibault Chaze (Proteomics platform) for analysis of the pull-down experiment. We are thankful to T. Hackstadt (Rocky Mountain Laboratories, USA) and G. Zhong (University of Texas, USA) for anti-TarP and anti-CT813 antibodies respectively., European Project: 282046,ERC,ERC-2011-StG_20101109,NUCHLEAR, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], effector proteins, host-pathogen interactions, structural biology, Chlamydia trachomatis, chaperone proteins, prenylation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], genetic manipulation
Abstract

International audience; Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.

Published
2018
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66. Spindle-shaped predators

Author

Sung-Keun Rhee, Joo-Han Gwak, Mario López-Pérez, Virginija Cvirkaite-Krupovic, Jang-Cheon Cho, Jong-Geol Kim, Woon-Jong Yu, Mart Krupovic, Francisco Rodriguez-Valera, So-Jeong Kim, Chungbuk National University, Korea Institute of Geoscience and Mineral Resources, Virologie des archées - Archaeal Virology, Institut Pasteur [Paris] (IP), Département de Microbiologie - Department of Microbiology, Universidad Miguel Hernández [Elche] (UMH), Inha University, This research was supported by National Research Foundation of Korea (NRF) grants (Mid-Career Researcher Program [NRF-2018R1A2B6008861], Basic Research Laboratory Program [NRF-2015R1A4A1041869], and C1 Gas Refinery Program [NRF-2015M3D3A1A01064881]) funded by the Ministry of Science, Information and Communication Technology, and Future Planning. M.K. was supported by a grant from l'Agence Nationale de la Recherche (#ANR-17-CE15-0005-01)., The authors would like to thank Thibault Chaze and Mariette Matondo (Pasteur Proteomics Platform) for help with the proteomics analyses. M.L.-P. was supported by a postdoctoral fellowship (Juan de la Cierva) from the Spanish Ministerio de Economía y Competitividad (IJCI-2017-34002). F.R.-V. was supported by grant CGL2016-76273-P (Agencia Estatal de Investigación/European Development Regional Fund [FEDER], EU), (cofounded with FEDER funds) from the Spanish Ministerio de Economía, Industria y Competitividad., ANR-17-CE15-0005,ENVIRA,Remodelage de la membrane cytoplasmique par les virus enveloppés d'archées(2017), and Institut Pasteur [Paris]

Subjects
Aquatic Organisms, Thaumarchaeota, MESH: Virus Replication, viruses, Nitrosopumilus, Biology, Virus Replication, Microbiology, Virus, MESH: Aquatic Organisms*/metabolism, Predation, 03 medical and health sciences, Marine bacteriophage, Ammonia, Host chromosome, MESH: Ammonia/metabolism, Bacteriophages, 14. Life underwater, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, General Immunology and Microbiology, 030306 microbiology, MESH: Aquatic Organisms*/virology, MESH: DNA, Archaeal*/genetics, MESH: Aquatic Organisms*/genetics, MESH: Archaea*/metabolism, Biological Sciences, MESH: Archaea*/genetics, biology.organism_classification, chronic infection, MESH: Bacteriophages/physiology, Archaea, viral predation, DNA, Archaeal, Infectious Diseases, Lytic cycle, Evolutionary biology, ammonia-oxidizing archaea, Viruses, spindle-shaped virus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Identification (biology), Bacterial virus, MESH: DNA, Archaeal*/metabolism, Oxidation-Reduction, MESH: Archaea*/virology
Abstract

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeota are ubiquitous in marine ecosystems and play a prominent role in carbon and nitrogen cycling. Previous studies have suggested that, like all microbes, thaumarchaea are infected by viruses and that viral predation has a profound impact on thaumarchaeal functioning and mortality, thereby regulating global biogeochemical cycles. However, not a single virus capable of infecting thaumarchaea has been reported thus far. Here we describe the isolation and characterization of three Nitrosopumilus spindle-shaped viruses (NSVs) that infect AOA and are distinct from other known marine viruses. Although NSVs have a narrow host range, they efficiently infect autochthonous Nitrosopumilus strains and display high rates of adsorption to their host cells. The NSVs have linear double-stranded DNA genomes of ∼28 kb that do not display appreciable sequence similarity to genomes of other known archaeal or bacterial viruses and could be considered as representatives of a new virus family, the “Thaspiviridae.” Upon infection, NSV replication leads to inhibition of AOA growth, accompanied by severe reduction in the rate of ammonia oxidation and nitrite reduction. Nevertheless, unlike in the case of lytic bacteriophages, NSV propagation is not associated with detectable degradation of the host chromosome or a decrease in cell counts. The broad distribution of NSVs in AOA-dominated marine environments suggests that NSV predation might regulate the diversity and dynamics of AOA communities. Collectively, our results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important mesophilic archaea.

Published
2019
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