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1. Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

Author

Roberto Notario Manzano, Thibault Chaze, Eric Rubinstein, Esthel Penard, Mariette Matondo, Chiara Zurzolo, and Christel Brou

Subjects
tunneling nanotubes, tetraspanins, extracellular vesicles, Medicine, Science, Biology (General), QH301-705.5
Abstract

Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from the cytoplasm to the cytoplasm. Although they are important especially, in pathological conditions (e.g. cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused on two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct non-redundant functions: CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicles in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

Published
2024
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2. Using a multiomics approach to unravel a septic shock specific signature in skeletal muscle

Author

Baptiste Duceau, Michael Blatzer, Jean Bardon, Thibault Chaze, Quentin Giai Gianetto, Florence Castelli, François Fenaille, Lucie Duarte, Thomas Lescot, Christophe Tresallet, Bruno Riou, Mariette Matondo, Olivier Langeron, Pierre Rocheteau, Fabrice Chrétien, and Adrien Bouglé

Subjects
Medicine, Science
Abstract

Abstract Sepsis is defined as a dysregulated host response to infection leading to organs failure. Among them, sepsis induces skeletal muscle (SM) alterations that contribute to acquired-weakness in critically ill patients. Proteomics and metabolomics could unravel biological mechanisms in sepsis-related organ dysfunction. Our objective was to characterize a distinctive signature of septic shock in human SM by using an integrative multi-omics approach. Muscle biopsies were obtained as part of a multicenter non-interventional prospective study. Study population included patients in septic shock (S group, with intra-abdominal source of sepsis) and two critically ill control populations: cardiogenic shock (C group) and brain dead (BD group). The proteins and metabolites were extracted and analyzed by High-Performance Liquid Chromatography-coupled to tandem Mass Spectrometry, respectively. Fifty patients were included, 19 for the S group (53% male, 64 ± 17 years, SAPS II 45 ± 14), 12 for the C group (75% male, 63 ± 4 years, SAPS II 43 ± 15), 19 for the BD group (63% male, 58 ± 10 years, SAPS II 58 ± 9). Biopsies were performed in median 3 days [interquartile range 1–4]) after intensive care unit admission. Respectively 31 patients and 40 patients were included in the proteomics and metabolomics analyses of 2264 proteins and 259 annotated metabolites. Enrichment analysis revealed that mitochondrial pathways were significantly decreased in the S group at protein level: oxidative phosphorylation (adjusted p = 0.008); branched chained amino acids degradation (adjusted p = 0.005); citrate cycle (adjusted p = 0.005); ketone body metabolism (adjusted p = 0.003) or fatty acid degradation (adjusted p = 0.008). Metabolic reprogramming was also suggested (i) by the differential abundance of the peroxisome proliferator-activated receptors signaling pathway (adjusted p = 0.007), and (ii) by the accumulation of fatty acids like octanedioic acid dimethyl or hydroxydecanoic. Increased polyamines and depletion of mitochondrial thioredoxin or mitochondrial peroxiredoxin indicated a high level of oxidative stress in the S group. Coordinated alterations in the proteomic and metabolomic profiles reveal a septic shock signature in SM, highlighting a global impairment of mitochondria-related metabolic pathways, the depletion of antioxidant capacities, and a metabolic shift towards lipid accumulation. ClinicalTrial registration: NCT02789995. Date of first registration 03/06/2016.

Published
2022
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3. CYP450 core involvement in multiple resistance strains of Aedes aegypti from French Guiana highlighted by proteomics, molecular and biochemical studies.

Author

Yanouk Epelboin, Lanjiao Wang, Quentin Giai Gianetto, Valérie Choumet, Pascal Gaborit, Jean Issaly, Amandine Guidez, Thibaut Douché, Thibault Chaze, Mariette Matondo, and Isabelle Dusfour

Subjects
Medicine, Science
Abstract

Insecticide resistance is a worldwide threat for vector control around the world, and Aedes aegypti, the main vector of several arboviruses, is a particular concern. To better understand the mechanisms of resistance, four isofemale strains originally from French Guiana were isolated and analysed using combined approaches. The activity of detoxification enzymes involved in insecticide resistance was assayed, and mutations located at positions 1016 and 1534 of the sodium voltage-gated channel gene, which have been associated with pyrethroid resistance in Aedes aegypti populations in Latin America, were monitored. Resistance to other insecticide families (organophosphates and carbamates) was evaluated. A large-scale proteomic analysis was performed to identify proteins involved in insecticide resistance. Our results revealed a metabolic resistance and resistance associated with a mutation of the sodium voltage-gated channel gene at position 1016. Metabolic resistance was mediated through an increase of esterase activity in most strains but also through the shifts in the abundance of several cytochrome P450 (CYP450s). Overall, resistance to deltamethrin was linked in the isofemale strains to resistance to other class of insecticides, suggesting that cross- and multiple resistance occur through selection of mechanisms of metabolic resistance. These results give some insights into resistance to deltamethrin and into multiple resistance phenomena in populations of Ae. aegypti.

Published
2021
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4. The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli

Author

Gizem Özbaykal, Eva Wollrab, Francois Simon, Antoine Vigouroux, Baptiste Cordier, Andrey Aristov, Thibault Chaze, Mariette Matondo, and Sven van Teeffelen

Subjects
cell-wall insertion, single-enzyme dynamics, MreB cytoskeleton, bacterial morphogenesis, Rod complex, Medicine, Science, Biology (General), QH301-705.5
Abstract

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

Published
2020
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5. Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis.

Author

Benoit Besson, Justine Basset, Sandrine Gatellier, Hélène Chabrolles, Thibault Chaze, Véronique Hourdel, Mariette Matondo, Nathalie Pardigon, and Valérie Choumet

Subjects
Medicine, Science
Abstract

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

Published
2020
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6. Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

Author

Antoine Vigouroux, Baptiste Cordier, Andrey Aristov, Laura Alvarez, Gizem Özbaykal, Thibault Chaze, Enno Rainer Oldewurtel, Mariette Matondo, Felipe Cava, David Bikard, and Sven van Teeffelen

Subjects
peptidoglycan cell wall, cell envelope, cell-wall repair, CRISPRi, single-molecule tracking, penicillin-binding proteins, Medicine, Science, Biology (General), QH301-705.5
Abstract

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Published
2020
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7. Proteomic analysis of plasma extracellular vesicles reveals mitochondrial stress upon HTLV-1 infection

Author

Patricia Jeannin, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Olivier Gout, Antoine Gessain, and Philippe V. Afonso

Subjects
Medicine, Science
Abstract

Abstract Extracellular vesicles (EVs) can participate in intercellular communication and pathogenesis. EVs contain many cargos, including proteins, and the composition of EVs differs between cell-types and activation levels. Thus, plasma EVs can be used as a biomarker of systemic response to infection and/or disease progression. In this study, we aimed at describing alterations in the protein content of plasma EVs upon infection with the human T-lymphotropic retrovirus type 1 (HTLV-1). HTLV-1 is the etiological agent of a lymphoproliferative disease (ATL) and a series of inflammatory diseases, including a neurodegenerative inflammatory disease (HAM/TSP). We found that plasma EVs are more abundant and smaller in HTLV-1 asymptomatic carriers or HAM/TSP patients when compared to uninfected healthy donors. Moreover, EVs from HTLV-1 infected donors contain markers of metabolic and mitochondrial stress.

Published
2018
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8. Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316.

Author

Eric Morello, Adeline Mallet, Yoan Konto-Ghiorghi, Thibault Chaze, Michel-Yves Mistou, Giulia Oliva, Liliana Oliveira, Anne-Marie Di Guilmi, Patrick Trieu-Cuot, and Shaynoor Dramsi

Subjects
Medicine, Science
Abstract

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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9. Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

Author

Timothy Wai, Erminia Donnarumma, Christoph Maack, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Elodie Vimont, Etienne Kornobis, Michael Kohlhaas, Maryse Moya-Nilges, Biologie mitochondriale – Mitochondrial biology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University Clinic Würzburg, Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Plateforme de Spectrométrie de Masse Protéomique - Mass Spectrometry Proteomics Platform (MSPP), and Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Montpellier (UM)

Subjects
uncoupling, Programmed cell death, permeability transition pore, Cardiac fibrosis, Chemistry, oxidative phosphorylation, Cardiomyopathy, Oxidative phosphorylation, Mitochondrion, medicine.disease, Cell biology, mitochondria, Mitochondrial permeability transition pore, Knockout mouse, medicine, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Mitochondrial fission, cardiomyopathy
Abstract

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

Published
2021
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10. Impact of the gut microbiota on the m 6 A epitranscriptome of mouse cecum and liver

Author

Pascale Cossart, Marie-Agnès Dillies, Sabrina Jabs, Amine Ghozlane, Quentin Giai Gianetto, Vincent Guérineau, Christophe Bécavin, David Touboul, Alessandro Pagliuso, Giulia Spanò, Mariette Matondo, Marie-Anne Nahori, Thibault Chaze, Anne Biton, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), We thank Marion Bérard and the Gnotobiology platform of the Institut Pasteur for support in conducting gnotobiology experiments, the Pasteur Biomics platforms for Transcriptomics and Epigenetics and Metagenomics for providing RNA sequencing and 16S rRNA sequencing services, Cédric Fund, Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA, Carla Saleh and Olivier Dussurget for helpful discussions, Hugo Varet (Institut Pasteur Bionformatics and Statistics Hub) for advice concerning statistical analysis, Stevenn Volant for help with analysis of 16S sequencing data. This work was supported by grants to P.C.: European Research Council (ERC) Advanced Grant BacCellEpi (670823), ANR Investissement d’Avenir Programme (10-LABX-62-IBEID), ERANET-Infect-ERA PROANTILIS (ANR-13-IFEC-0004-02) and the Fondation Le Roch-Les Mousquetaires. S.J. was supported by a Danone Research fellowship and European Research Council (ERC) Advanced Grant BacCellEpi (670823)., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-13-IFEC-0004,PROANTILIS,Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes(2013), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Nahori, marie-anne, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, ERA-NET Infect-ERA - Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes - - PROANTILIS2013 - ANR-13-IFEC-0004 - IFEC - VALID, Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Adenosine, Methyltransferase, Science, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Biology, Gut flora, Methylation, digestive system, Article, General Biochemistry, Genetics and Molecular Biology, Microbiology, Transcriptome, Mice, 03 medical and health sciences, Cecum, 0302 clinical medicine, fluids and secretions, Gene expression, medicine, Animals, RNA, Messenger, lcsh:Science, Multidisciplinary, Bacteria, Methyltransferases, General Chemistry, biology.organism_classification, Commensalism, Gastrointestinal Microbiome, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], stomatognathic diseases, 030104 developmental biology, medicine.anatomical_structure, Liver, Female, lcsh:Q, Microbiome, 030217 neurology & neurosurgery, Akkermansia muciniphila
Abstract

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host., The intestinal microbiota modulates host physiology and gene expression via unclear mechanisms. Here, Jabs et al. show that variations in the gut microbiota correlate with N6-methyladenosine modifications of host mRNAs in the cecum and liver of mice.

Published
2020
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11. The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli

Author

Baptiste Cordier, Andrey Aristov, Eva Wollrab, Mariette Matondo, Gizem Özbaykal, Thibault Chaze, Antoine Vigouroux, Sven van Teeffelen, François Simon, Morphogénèse et Croissance microbiennes / Microbial Morphogenesis and Growth, Institut Pasteur [Paris], Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Biologie de Synthèse - Synthetic biology, Université Paris-Descartes, Sorbonne Paris Cité, 75014 Paris, France, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), H2020 European Research Council 679980French National Research Agency ANR-10-LABX-62-IBEIDVolkswagenRégion Ile-de-FranceCampus France, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 679980,H2020,ERC-2015-STG,RCSB(2016), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
genetic structures, QH301-705.5, MreB cytoskeleton, Science, infectious disease, bacterial morphogenesis, Curvature, medicine.disease_cause, MreB, General Biochemistry, Genetics and Molecular Biology, Cell wall synthesis, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Cell Wall, physics of living systems, Escherichia coli, medicine, Penicillin-Binding Proteins, Rod complex, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biology (General), single-enzyme dynamics, 030304 developmental biology, Microbiology and Infectious Disease, 0303 health sciences, General Immunology and Microbiology, Escherichia coli Proteins, General Neuroscience, microbiology, E. coli, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, chemistry, Cytoplasm, Direct binding, Biophysics, Medicine, Peptidoglycan, sense organs, cell-wall insertion, 030217 neurology & neurosurgery, Subcellular Fractions, Research Article
Abstract

International audience; Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

Published
2020
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12. Listeria monocytogenes Exploits Mitochondrial Contact Site and Cristae Organizing System Complex Subunit Mic10 To Promote Mitochondrial Fragmentation and Cellular Infection

Author

Mariette Matondo, Anna Spier, Fabrizia Stavru, Filipe Carvalho, Thibault Chaze, and Pascale Cossart

Subjects
listeria monocytogenes, Mitochondrion, Biology, medicine.disease_cause, Mitochondrial Dynamics, Microbiology, Host-Microbe Biology, Mitochondrial Proteins, 03 medical and health sciences, proteomics, Listeria monocytogenes, Virology, Organelle, medicine, Humans, Inner mitochondrial membrane, 030304 developmental biology, 0303 health sciences, mic10, MICOS complex, mitochondrial fission, 030302 biochemistry & molecular biology, Listeriolysin O, Membrane Proteins, micos complex, HCT116 Cells, QR1-502, Cell biology, Mitochondria, Up-Regulation, Mitochondrial fission, Intracellular, Research Article
Abstract

Pathogenic bacteria can target host cell organelles to take control of key cellular processes and promote their intracellular survival, growth, and persistence. Mitochondria are essential, highly dynamic organelles with pivotal roles in a wide variety of cell functions. Mitochondrial dynamics and function are intimately linked. Our previous research showed that Listeria monocytogenes infection impairs mitochondrial function and triggers fission of the mitochondrial network at an early infection stage, in a process that is independent of the presence of the main mitochondrial fission protein Drp1. Here, we analyzed how mitochondrial proteins change in response to L. monocytogenes infection and found that infection raises the levels of Mic10, a mitochondrial inner membrane protein involved in formation of cristae. We show that Mic10 is important for L. monocytogenes-dependent mitochondrial fission and infection of host cells. Our findings thus offer new insight into the mechanisms used by L. monocytogenes to hijack mitochondria to optimize host infection., Mitochondrial function adapts to cellular demands and is affected by the ability of the organelle to undergo fusion and fission in response to physiological and nonphysiological cues. We previously showed that infection with the human bacterial pathogen Listeria monocytogenes elicits transient mitochondrial fission and a drop in mitochondrion-dependent energy production through a mechanism requiring the bacterial pore-forming toxin listeriolysin O (LLO). Here, we performed quantitative mitochondrial proteomics to search for host factors involved in L. monocytogenes-induced mitochondrial fission. We found that Mic10, a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex, is significantly enriched in mitochondria isolated from cells infected with wild-type but not with LLO-deficient L. monocytogenes. Increased mitochondrial Mic10 levels did not correlate with upregulated transcription, suggesting a posttranscriptional mechanism. We then showed that Mic10 is necessary for L. monocytogenes-induced mitochondrial network fragmentation and that it contributes to L. monocytogenes cellular infection independently of MICOS proteins Mic13, Mic26, and Mic27. In conclusion, investigation of L. monocytogenes infection allowed us to uncover a role for Mic10 in mitochondrial fission.

Published
2020

13. Differentiation-dependent susceptibility of human muscle cells to Zika virus infection

Author

Gillian Butler-Browne, Jim Zoladek, Ingo Riederer, Quentin Giai Gianetto, Philippe Roingeard, Patricia Jeannin, Julien Burlaud-Gaillard, Pierre-Emmanuel Ceccaldi, Mariette Matondo, Valérie Choumet, Thibault Chaze, Vincent Legros, Vincent Mouly, Philippe V. Afonso, Antoine Gessain, Mariela-Natacha Gonzàlez, Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris], This work was supported by a Pasteur-Fiocruz grant. VL was supported by a fellowship from the 'Fondation pour la Recherche Médicale' (FRM), IR and MG by CNPq/Ciências sem Fronteiras program, CNPq/Brazilian National Institute of Science and Technology on Neuroimmunomodulation (INCT-NIM). FOCEM - Mercosur Structural Convergence Fund 03/11. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Pasteur [Paris] (IP), Matondo, Mariette, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), UMRS974, Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de Recherche en Myologie, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ)

Subjects
Proteomics, Pulmonology, RC955-962, Apoptosis, Pathology and Laboratory Medicine, Zika virus, 0302 clinical medicine, Medical Conditions, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Arctic medicine. Tropical medicine, MESH: Proteins, Chikungunya, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Zika Virus Infection, Stem Cells, MESH: Proteomics, 3. Good health, Medical Microbiology, Arboviral Infections, Viral Pathogens, Interferon Type I, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Public aspects of medicine, Cellular Types, MESH: Zika Virus, Microbiology, 03 medical and health sciences, Respiratory Disorders, MESH: Zika Virus Infection, Humans, Microbial Pathogens, MESH: Humans, Flaviviruses, MESH: Host-Pathogen Interactions, Public Health, Environmental and Occupational Health, Organisms, Biology and Life Sciences, Chikungunya Infection, Proteins, medicine.disease, Tropical Diseases, Virology, MESH: Cell Line, 030104 developmental biology, Biological Tissue, Interferon type I, Developmental Biology, 0301 basic medicine, RNA viruses, MESH: Cell Death, MESH: Interferon Type I, MESH: Muscle Cells, Viral Diseases, [SDV]Life Sciences [q-bio], Muscle Fibers, Skeletal, medicine.disease_cause, Virus Replication, Dengue fever, Myoblasts, Cytopathogenic Effect, Viral, Medicine and Health Sciences, Morphogenesis, Myocyte, Cytopathic effect, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Muscle Fibers, Skeletal, Cell Death, Myogenesis, Cell Differentiation, Muscle Differentiation, Infectious Diseases, Cell Processes, Viruses, Host-Pathogen Interactions, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Disease Susceptibility, Stem cell, RA1-1270, Pathogens, Anatomy, medicine.drug, Research Article, Neglected Tropical Diseases, MESH: Cell Differentiation, 030231 tropical medicine, Muscle Tissue, MESH: Disease Susceptibility, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Stem Cells, Biology, Cell Line, medicine, MESH: Myoblasts, Muscle Cells, MESH: Virus Replication, Cell Biology, Zika Virus, biology.organism_classification, MESH: Cytopathogenic Effect, Viral, Respiratory Infections
Abstract

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis., Author summary Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, and may be involved in the disease manifestation. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human muscle stem cells (myoblasts) that can be differentiated into differentiated muscle cells (myotubes), we found that myoblasts can be infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection. Infection induced the death of infected cells. Protein levels 24h and 48h post-infection indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, muscle stem cells being susceptible while differentiated muscle cells are resistant. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences during ZIKV infection.

Published
2020
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14. Class-A penicillin binding proteins do not contribute to cell shape but repair cell- wall defects

Author

Mariette Matondo, Enno R. Oldewurtel, Sven van Teeffelen, Antoine Vigouroux, Baptiste Cordier, Gizem Özbaykal, Felipe Cava, Andrey Aristov, Laura Alvarez, Thibault Chaze, David Bikard, Biologie de Synthèse - Synthetic biology, Institut Pasteur [Paris], Morphogénèse et Croissance microbiennes / Microbial Morphogenesis and Growth, Université Paris Descartes - Paris 5 (UPD5), Université Sorbonne Paris Cité (USPC), Department of Molecular Biology [Umeå], Umeå University, Université Paris Diderot - Paris 7 (UPD7), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the European Research Council (ERC) under the Europe Union’s Horizon 2020 research and innovation program [Grant Agreement No. (679980)], the French Government’s Investissement d’Avenir program Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID), the Mairie de Paris ‘Emergence(s)’ program, and the Volkswagen Foundation. Research in the Cava lab is supported by MIMS, the Knut and Alice Wallenberg Foundation (KAW), the Swedish Research Council and the Kempe Foundation., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 679980,H2020,ERC-2015-STG,RCSB(2016), European Project: 677823,H2020,ERC-2015-STG,CRISPAIR(2016), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Penicillin binding proteins, cell envelope, QH301-705.5, Science, infectious disease, Cell, Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy), medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Cell Wall, cell biology, medicine, Escherichia coli, Biology (General), Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci), 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Microbiology and Infectious Disease, General Immunology and Microbiology, 030306 microbiology, single-molecule tracking, penicillin-binding proteins, General Neuroscience, Escherichia coli Proteins, microbiology, E. coli, CRISPRi, General Medicine, Cell Biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, peptidoglycan cell wall, medicine.anatomical_structure, Enzyme, chemistry, Biophysics, cell-wall repair, Medicine, Peptidoglycan, sense organs, Cell envelope, Cell wall repair, Research Article
Abstract

International audience; Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Published
2020
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15. Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

Author

Andrey Aristov, Enno R. Oldewurtel, David Bikard, Baptiste Cordier, Thibault Chaze, Mariette Matondo, Gizem Özbaykal, Antoine Vigouroux, and Sven van Teeffelen

Subjects
0303 health sciences, 030306 microbiology, Cell, medicine.disease_cause, Bacterial cell structure, Cell biology, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, medicine.anatomical_structure, chemistry, Mechanical stability, medicine, Peptidoglycan, Cell shape, Escherichia coli, Function (biology), 030304 developmental biology
Abstract

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shapedEscherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

Published
2019
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16. RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

Author

Quentin Giai Gianetto, Didier Mazel, Sean Kennedy, Mariette Matondo, Thibault Chaze, Zeynep Baharoglu, Evelyne Krin, Sebastian Aguilar Pierlé, Veronica Negro, Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Work in the Mazel lab is funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525), the French National Research Agency (ANR Unibac ANR-17-CE13-0010-01), and the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases'(grant no. ANR-10-LABX-62-IBEID). V.N. was funded by fellowships from DIM Malinf 140051 (Conseil régional d’Île-de-France) and FRM grant DBF20160635736. S.A.P. was supported by a postdoctoral fellowship from the Roux Foundation (Institut Pasteur), the FRM grant DBF20160635736, and the ANR Unibac ANR-17-CE13-0010-01, We thank Christophe Thomas for his help with protein purification, Melis Asal and Ricardo Albino Camacho for their participation in strain constructions, and Julie Lambert for UV tests. We thank Micheline Fromont for the yeast strains and plasmids and for her help with the two-hybrid experiment. We are grateful to Bénédicte Michel for the gift of several E. coli strains. We thank Melanie Blokesch for the Vibrio cholerae WT strain used in this study. We thank Sébastien Fleurier for advice for TUNEL experiments., ANR-17-CE13-0010,UniBac,Etude de l'émergence de la résistance aux antibiotiques et de la diversification génétique chez les bactéries à l'échelle de cellules uniques.(2017), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA, Bacterial, Molecular Biology and Physiology, antibiotic resistance, DNA repair, DNA damage, [SDV]Life Sciences [q-bio], Microbial Sensitivity Tests, medicine.disease_cause, Microbiology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Virology, Escherichia coli, medicine, DNA Breaks, Double-Stranded, Vibrio cholerae, 030304 developmental biology, Adenosine Triphosphatases, RecBCD, 0303 health sciences, Microbial Viability, biology, 030306 microbiology, Chemistry, Escherichia coli Proteins, Aminoglycoside, biology.organism_classification, QR1-502, Anti-Bacterial Agents, 3. Good health, Homologous Recombination Pathway, Tobramycin, R-loop, R-Loop Structures, Bacteria, Research Article
Abstract

Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage., We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.

Published
2019
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17. CYP450 core involvement in multiple resistance strains of Aedes aegypti from French Guiana highlighted by proteomics, molecular and biochemical studies

Author

Jean Issaly, Thibaut Douché, Quentin Giai Gianetto, Mariette Matondo, Valérie Choumet, Yanouk Epelboin, Lanjiao Wang, Amandine Guidez, Pascal Gaborit, Thibault Chaze, Isabelle Dusfour, Unité d'entomologie médicale, Vectopôle Amazonien Emile Abonnenc [Cayenne, Guyane française], Institut Pasteur de la Guyane, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de la Guyane, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris], Département de Santé Globale - Department Global Health, This study was funded by the European Funds for Regional Development and CNES under FEDER 'CONTROLE', project n° Synergie: GY0010695 awarded to YE, LW, PG, JI, AG, and ID. This study was also funded by 'an Investissement d’Avenir grant of the Agence Nationale de la Recherche' (CEBA: ANR-10-LABX-25-01) in which the Institut Pasteur de la Guyane is partner awarded to YE, LW, PG, JI, AG, and ID., ANR-10-LABX-0025,CEBA,CEnter of the study of Biodiversity in Amazonia(2010), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
Proteomics, Insecticides, Proteome, Hydrolases, Oligonucleotides, Voltage-Gated Sodium Channels, Disease Vectors, Toxicology, Pathology and Laboratory Medicine, medicine.disease_cause, Mosquitoes, Biochemistry, Esterase, Insecticide Resistance, chemistry.chemical_compound, Medical Conditions, 0302 clinical medicine, Cytochrome P-450 Enzyme System, Aedes, Pyrethrins, Medicine and Health Sciences, Intestinal Mucosa, 2. Zero hunger, Genetics, 0303 health sciences, Mutation, Multidisciplinary, Organic Compounds, Esterases, Eukaryota, Agriculture, Organophosphates, French Guiana, Enzymes, 3. Good health, Insects, Chemistry, Infectious Diseases, Gene Knockdown Techniques, Inactivation, Metabolic, Physical Sciences, Insect Proteins, Medicine, Female, Agrochemicals, Detoxification, Research Article, Ethers, Genotype, Arthropoda, Death Rates, Science, 030231 tropical medicine, Aedes aegypti, Aedes Aegypti, Biology, Polymorphism, Single Nucleotide, 03 medical and health sciences, Population Metrics, Nitriles, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Gene, 030304 developmental biology, Population Biology, Resistance (ecology), Organic Chemistry, Organisms, Chemical Compounds, Biology and Life Sciences, Proteins, Cytochrome P450, biology.organism_classification, Invertebrates, Insect Vectors, Species Interactions, Deltamethrin, chemistry, Vector (epidemiology), Enzymology, biology.protein, Zoology, Entomology
Abstract

Insecticide resistance is a worldwide threat for vector control around the world, and Aedes aegypti, the main vector of several arboviruses, is a particular concern. To better understand the mechanisms of resistance, four isofemale strains originally from French Guiana were isolated and analysed using combined approaches. The activity of detoxification enzymes involved in insecticide resistance was assayed, and mutations located at positions 1016 and 1534 of the sodium voltage-gated channel gene, which have been associated with pyrethroid resistance in Aedes aegypti populations in Latin America, were monitored. Resistance to other insecticide families (organophosphates and carbamates) was evaluated. A large-scale proteomic analysis was performed to identify proteins involved in insecticide resistance. Our results revealed a metabolic resistance and resistance associated with a mutation of the sodium voltage-gated channel gene at position 1016. Metabolic resistance was mediated through an increase of esterase activity in most strains but also through the shifts in the abundance of several cytochrome P450 (CYP450s). Overall, resistance to deltamethrin was linked in the isofemale strains to resistance to other class of insecticides, suggesting that cross- and multiple resistance occur through selection of mechanisms of metabolic resistance. These results give some insights into resistance to deltamethrin and into multiple resistance phenomena in populations of Ae. aegypti.

Published
2021
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18. MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker

Author

Friederike Jönsson, Mariette Matondo, Dorothée Selimoglu-Buet, Benoit S. Marteyn, Bruno Baron, Naelle Lombion, Philippe J. Sansonetti, Jean-Yves Tinevez, Giulia Nigro, Thierry Lazure, Yves Marie Coïc, Valérie Lapierre, David J. Thornton, Katharina Nothelfer, Mark C. Anderson, Louise Injarabian, Thibault Chaze, Françoise Baleux, Ellen T. Arena, Eric Solary, Judith Souphron, Pascale Vonaesch, Caroline Ridley, Pathogénie microbienne moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme de Protéomique / Proteomics platform, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Chimie des Biomolécules - Chemistry of Biomolecules, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut de biochimie et génétique cellulaires (IBGC), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Laboratoire de thérapie cellulaire, Département de médecine oncologique [Gustave Roussy], Institut Gustave Roussy (IGR)-Institut Gustave Roussy (IGR), Hématopoïèse normale et pathologique, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), University of Manchester [Manchester], Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Institut Pasteur [Paris], AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Département d'Immunologie - Department of Immunology, Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), This work was supported by the Fondation Laurette Fugain (LF-2015-15) (B.S.M.) and ANR JCJC grants (ANR-17-CE15-0012) (B.S.M.) - (ANR-16-CE15-0012-01) (F.J.)., ANR-17-CE15-0012,NEUTROXIA,Effet de l'exposition des neutrophiles à l'oxygène sur leur activation et leur mort : une lame à double tranchant(2017), ANR-16-CE15-0012,PlanA,Rôle des plaquettes et leurs interactions dans les réactions anaphylactiques(2016), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), and Collège de France - Chaire Microbiologie et Maladies infectieuses

Subjects
0301 basic medicine, MESH: Inflammation, Clinical Biochemistry, Cell, Arthritis, MESH: Rabbits, Peptide, MESH: Neutrophils, Biochemistry, MESH: Shigella, MUB, Drug Discovery, MESH: Animals, chemistry.chemical_classification, MESH: Middle Aged, Lactoferrin, MESH: Peptides, imaging, neutrophil, MESH: Fluorescent Dyes, 3. Good health, lactoferrin, medicine.anatomical_structure, Molecular Medicine, biomarker, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Carbocyanines, medicine.symptom, MESH: Dysentery, Bacillary, Inflammation, Biology, MESH: Guinea Pigs, Guinea pig, 03 medical and health sciences, Immune system, MESH: Mice, Inbred C57BL, medicine, Secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, MESH: Mice, Pharmacology, MESH: Humans, MUB(40), MESH: Adult, medicine.disease, Molecular biology, MESH: Lactoferrin, 030104 developmental biology, chemistry, biology.protein, MESH: Biomarkers, MESH: Female
Abstract

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases. MUB40 is a universal and specific marker of mammalian neutrophils. MUB40 interacts with lactoferrin, stored in specific (β1), tertiary (β2), and secretome (γ) granules secreted upon stimulation. Neutrophil detection in inflamed or infected tissues is facilitated by MUB40 peptides conjugated to fluorophores. Neutrophilic inflammation detection may be envisaged by combining MUB40 with non-invasive imaging.

Published
2018
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19. Proteomic analysis of plasma extracellular vesicles reveals mitochondrial stress upon HTLV-1 infection

Author

Philippe V. Afonso, Antoine Gessain, Quentin Giai Gianetto, Olivier Gout, Mariette Matondo, Patricia Jeannin, Thibault Chaze, Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris], Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Fondation Ophtalmologique Adolphe de Rothschild [Paris], This work was supported by Ville de Paris [Programme Emergence(s)] and Ligue Contre le Cancer/Comité de Paris (RS18/75-5)., We would like to thank Camille Roesch (Izon) for her help in measuring the samples composition, Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology ( UTechS MSBio ), Centre de Bioinformatique, Biostatistique et Biologie Intégrative ( C3BI ), Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), and Institut Pasteur [Paris]-Institut Pasteur [Paris]

Subjects
Adult, Male, Proteomics, 0301 basic medicine, Science, Disease, Mitochondrion, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Mass Spectrometry, Article, Mitochondrial Proteins, Pathogenesis, Extracellular Vesicles, Young Adult, 03 medical and health sciences, Retrovirus, Stress, Physiological, immune system diseases, hemic and lymphatic diseases, Humans, Aged, Human T-lymphotropic virus 1, Multidisciplinary, biology, Proteins, virus diseases, Neurodegenerative Diseases, Middle Aged, biology.organism_classification, HTLV-I Infections, Lymphoproliferative Disorders, 3. Good health, Mitochondria, 030104 developmental biology, Viral infection, Carrier State, Immunology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Chromatography, Gel, Biomarker (medicine), Medicine, Female, HTLV-1 Infection, Lysosomes, Asymptomatic carrier, Biomarkers, Intracellular
Abstract

Extracellular vesicles (EVs) can participate in intercellular communication and pathogenesis. EVs contain many cargos, including proteins, and the composition of EVs differs between cell-types and activation levels. Thus, plasma EVs can be used as a biomarker of systemic response to infection and/or disease progression. In this study, we aimed at describing alterations in the protein content of plasma EVs upon infection with the human T-lymphotropic retrovirus type 1 (HTLV-1). HTLV-1 is the etiological agent of a lymphoproliferative disease (ATL) and a series of inflammatory diseases, including a neurodegenerative inflammatory disease (HAM/TSP). We found that plasma EVs are more abundant and smaller in HTLV-1 asymptomatic carriers or HAM/TSP patients when compared to uninfected healthy donors. Moreover, EVs from HTLV-1 infected donors contain markers of metabolic and mitochondrial stress.

Published
2018
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20. Serum Proteome Analysis for Profiling Predictive Protein Markers Associated with the Severity of Skin Lesions Induced by Ionizing Radiation

Author

Joëlle Vinh, Christophe Chambon, Jean-Philippe Peyrat, Marc Benderitter, Louis Hornez, Iman Haddad, Thibault Chaze, Olivier Guipaud, Radiobiologie et épidémiologie (DRPH/SRBE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Laboratoire d'Oncologie Moléculaire Humaine, Lille, France, Centre Oscat Lambré, Unité de Nutrition Humaine (UNH), Institut National de la Recherche Agronomique (INRA)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Clermont Université, Neurobiologie et diversité cellulaire (NDC), ESPCI ParisTech-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Oncologie Moléculaire Humaine, CRLCC Oscar Lambret, Laboratoire de Radiopathologie et Thérapies Expérimentales [IRSN, Fontenay-aux-Roses] (PRP-HOM - SRBE), Université d'Auvergne - Clermont-Ferrand I (UdA)-Clermont Université-Institut National de la Recherche Agronomique (INRA), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Centre Régional de Lutte contre le Cancer Oscar Lambret [Lille] (UNICANCER/Lille), Université Lille Nord de France (COMUE)-UNICANCER-Université Lille Nord de France (COMUE)-UNICANCER, Service de RadioBiologie et d'Epidémiologie (IRSN/DRPH/SRBE), Université de Lille-UNICANCER-Université de Lille-UNICANCER, and Laboratoire de Radiopathologie et de Thérapies Expérimentales (IRSN/PRP-HOM/SRBE/LRTE)

Subjects
Pathology, medicine.medical_specialty, skin, Necrosis, Erythema, medicine.medical_treatment, serum proteome, Clinical Biochemistry, lcsh:QR1-502, Biology, Proteomics, Biochemistry, Article, lcsh:Microbiology, Ionizing radiation, 03 medical and health sciences, 0302 clinical medicine, proteomics, Structural Biology, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, 2D-DIGE, biomarkers, cutaneous radiation syndrome, ionizing radiation, mass spectrometry, radiotherapy, SELDI-TOF, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Triage, Blood proteins, 3. Good health, Radiation therapy, Moist desquamation, 030220 oncology & carcinogenesis, medicine.symptom
Abstract

The finding of new diagnostic and prognostic markers of local radiation injury, and particularly of the cutaneous radiation syndrome, is crucial for its medical management, in the case of both accidental exposure and radiotherapy side effects. Especially, a fast high-throughput method is still needed for triage of people accidentally exposed to ionizing radiation. In this study, we investigated the impact of localized irradiation of the skin on the early alteration of the serum proteome of mice in an effort to discover markers associated with the exposure and severity of impending damage. Using two different large-scale quantitative proteomic approaches, 2D-DIGE-MS and SELDI-TOF-MS, we performed global analyses of serum proteins collected in the clinical latency phase (days 3 and 7) from non-irradiated and locally irradiated mice exposed to high doses of 20, 40 and 80 Gy which will develop respectively erythema, moist desquamation and necrosis. Unsupervised and supervised multivariate statistical analyses (principal component analysis, partial-least square discriminant analysis and Random Forest analysis) using 2D-DIGE quantitative protein data allowed us to discriminate early between non-irradiated and irradiated animals, and between uninjured/slightly injured animals and animals that will develop severe lesions. On the other hand, despite a high number of animal replicates, PLS-DA and Random Forest analyses of SELDI-TOF-MS data failed to reveal sets of MS peaks able to discriminate between the different groups of animals. Our results show that, unlike SELDI-TOF-MS, the 2D-DIGE approach remains a powerful and promising method for the discovery of sets of proteins that could be used for the development of clinical tests for triage and the prognosis of the severity of radiation-induced skin lesions. We propose a list of 15 proteins which constitutes a set of candidate proteins for triage and prognosis of skin lesion outcomes.

Published
2013
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21. 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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22. 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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23. 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

24. 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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