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1. HIV-1 hijacks the cell extracellular matrix to spread collectively and efficiently between T lymphocytes

Author: Frédéric Tangy, Faroudy Boufassa, Alexandre Dufour, Jean-Christophe Olivo-Marin, M. Mesel-Lemoine, Gianfranco Pancino, Adeline Mallet, Asier Sáez-Cirión, M.-I. Thoulouze, A. Desrames, Annie David, Olivier Lambotte, Félix A. Rey, M. Caillet, P. Bomme, Hugo Mouquet, C. Inizan, K. Bourdic, Thibault Lagache, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU), Dengue et Arbovirose (URE-DA), Institut Pasteur de Nouvelle-Calédonie, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pointe-à-Pitre/Abymes [Guadeloupe] -Université des Antilles (UA)-Université Paris Cité (UPCité), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche en épidémiologie et santé des populations (CESP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Génomique virale et vaccination, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Régulation des Infections Rétrovirales, Réponse humorale aux pathogènes, Analyse d'images biologiques - Biological Image Analysis (BIA), Columbia University [New York], This work was funded by grants from l’AgenceNationale de Recherche sur le SIDA et les hépatites (ANRS), l’Institut Pasteur (PTR-445), as well asby institutional grants from Institut Pasteur and the Centre National de la Recherche Scientifique(CNRS). CI was supported by an Allocation de Recherche du Ministère de l'Education Nationale, de laRecherche et de la Technologie and by a bourse from the Fondation pour la Recherche Médicale. MCwas supported by a fellowship from AXA Research fund, AD by a fellowship from l’Institut Pasteur(PTR-445), and TL by a Roux Fellowship (Institut Pasteur). We gratefully acknowledge the financialsupport of the FranceBioImaging infrastructure network supported by the French National ResearchAgency (ANR-10–INSB–04, Investments for the future and ANR-10-LABX-62-IBEID) and theRégion Ile-de-France (program DIM-Malinf). The support of E. Mottez (Center for HumanImmunology, Institut Pasteur) and A.M. Rey-Cuillé (ANRS) is thankfully acknowledged., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)
Subjects: Infectivity, biology, Cell-Extracellular Matrix, [SDV]Life Sciences [q-bio], Cell, Human immunodeficiency virus (HIV), [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Cell biology, Extracellular matrix, medicine.anatomical_structure, In vivo, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, medicine, biology.protein, Viral spread, Antibody
Abstract: Collective transmission via structures containing several virions has recently emerged as a highly efficient mode of viral spread. Here, we demonstrate that HIV-1 spreads between T lymphocytes in the form of viral particles colonies that are concentrated and sheltered in an extracellular matrix (ECM) lattice enabling their collective transmission upon cell contacts. Intrinsically, ECM-clustered viruses infect T lymphocytes more efficiently than individual viral particles. They preserve HIV-1 transmission from antiretroviral treatment (ArT) and potent broadly neutralizing antibodies. We also show that collagen induced by HIV-1 infection controls the clustering of virions and their collective spread, thereby enhancing infectivity. CD4+ T cells from HIV-1-infected patients produce and transmit ECM-virus clusters, supporting that they could be involved in vivo. This study provides new insights into modes of HIV-1 transmission and identifies a novel fundamental role for collagen in this process. HIV-1 spread via ECM-virus clusters may have important implications for viral dissemination and persistence, including during therapy.
Published: 2021

2. Dirhamnolipids secreted from Pseudomonas aeruginosa modify anjpegungal susceptibility of Aspergillus fumigatus by inhibiting β1,3 glucan synthase activity

Author: Briard, Benoit, Rasoldier, Vero, Bomme, Perrine, Elaouad, Noureddine, Guerreiro, Catherine, Chassagne, Pierre, Muszkieta, Laetitia, Latgé, Jean-Paul, Mulard, Laurence, Beauvais, Anne, Aspergillus, Institut Pasteur [Paris] (IP), Microscopie ultrastructurale - Ultrapole (CITECH), Fundación MEDINA [Granada], Chimie des Biomolécules - Chemistry of Biomolecules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Research in the Aspergillus Unit was supported by the Association Vaincre La Mucoviscidose (RF20140501052/1/1/141)., Institut Pasteur [Paris], Fundación MEDINA, and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]
Subjects: Aspergillus fumigatus, Hyphae, Quorum Sensing, Chitin, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Cell Wall, Glucosyltransferases, Polysaccharides, Gene Expression Regulation, Fungal, Pseudomonas aeruginosa, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Original Article, Glycolipids
Abstract: International audience; Pseudomonas aeruginosa and Aspergillus fumigatus are the two microorganisms responsible for most of the chronic infections in cystic fibrosis patients. P. aeruginosa is known to produce quorum-sensing controlled rhamnolipids during chronic infections. Here we show that the dirhamnolipids secreted from P. aeruginosa (i) induce A. fumigatus to produce an extracellular matrix, rich in galactosaminogalactan, 1,8-dihydroxynaphthalene (DHN)-and pyo-melanin, surrounding their hyphae, which facilitates P. aeruginosa binding and (ii) inhibit A. fumigatus growth by blocking β1,3 glucan synthase (GS) activity, thus altering the cell wall architecture. A. fumigatus in the presence of diRhls resulted in a growth phenotype similar to that upon its treatment with antifungal echinocandins, showing multibranched hyphae and thicker cell wall rich in chitin. The diRhl structure containing two rhamnose moieties attached to fatty acyl chain is essential for the interaction with β1,3 GS; however, the site of action of diRhls on GS is different from that of echinocandins, and showed synergistic antifungal effect with azoles.
Published: 2017
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3. Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting

Author: Charles-Orszag, Arthur, Tsai, Feng-Ching, Bonazzi, Daria, Manriquez, Valeria, Sachse, Martin, Mallet, Adeline, Salles, Audrey, Melican, Keira, Staneva, Ralitza, Bertin, Aurélie, Millien, Corinne, Goussard, Sylvie, Lafaye, Pierre, Shorte, Spencer, Piel, Matthieu, Krijnse-Locker, Jacomine, Brochard-Wyart, Françoise, Bassereau, Patricia, Duménil, Guillaume, Pathogénèse des Infections vasculaires / Pathogenesis of Vascular Infections, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pierre-Gilles de Gennes pour la Microfluidique, Institut Curie [Paris], This work was supported by the French ministry for research and higher education (ACO), the Integrative Biology of Emerging Infectious Diseases (IBEID) laboratory of excellence (GD) and the VIP European Research Council starting grant (GD). We gratefully acknowledge the Imagopole—Citech of Institut Pasteur (Paris, France) as well as the France–BioImaging infrastructure network supported by the French National Research Agency (ANR-10–INSB–04, Investments for the Future), and the Région Ile-de-France (program Domaine d’Intérêt Majeur-Malinf) for the use of the Zeiss LSM 780 Elyra PS1 microscope and the Zeiss Auriga scanning electron microscope. The authors greatly acknowledge the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04), and the PICT-IBiSA Institut Curie (Paris, France). This work was supported by Institut Curie, Fondation pour la Recherche Médicale (FRM FDT20170437130) and Ecole Doctorale Frontières du Vivant (FdV)–Programme Bettencourt (RS), ERC StG grant STARLIN (DMV), Centre National de la Recherche Scientifique (CNRS), and P. B. and F.B.-W. belong to the CNRS consortium CellTiss, to the Labex CelTisPhyBio (ANR-11-LABX0038) and to Paris Sciences et Lettres (). F.C.T. was funded by the EMBO Long-Term fellowship (ALTF 1527-2014) and Marie Curie actions (H2020-MSCA-IF-2014, project membrane-ezrin-actin)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-LABX-0038,CelTisPhyBio,Des cellules aux tissus: au croisement de la Physique et de la Biologie(2011), European Project: 656442,H2020,H2020-MSCA-IF-2014,membrane-ezrin-actin(2016), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut Pasteur [Paris], Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Lafaye, pierre, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Des cellules aux tissus: au croisement de la Physique et de la Biologie - - CelTisPhyBio2011 - ANR-11-LABX-0038 - LABX - VALID, Membrane-ezrin-actin interactions mediated by ezrin binding proteins in reconstituted systems - membrane-ezrin-actin - - H20202016-01-01 - 2018-01-01 - 656442 - VALID, and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Science, Cell Membrane, Nanofibers, Mice, SCID, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Neisseria meningitidis, Models, Biological, Bacterial Adhesion, Article, Liposomes, Human Umbilical Vein Endothelial Cells, Wettability, Animals, Humans, lcsh:Q, Cell Surface Extensions, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, lcsh:Science
Abstract: The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term ‘one-dimensional’ membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection., Meningococci remodel the plasma membrane of host cells during infection. Here, Charles-Orszag et al. show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that they term ‘one-dimensional’ membrane wetting.
Published: 2018
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4. Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation

Author: Beaune, Gregory, Blanch-Mercader, Carles, Douezan, Stephane, Dumond, Julien, Gonzalez-Rodriguez, David, Cuvelier, Damien, Ondarçuhu, Thierry, Sens, Pierre, Dufour, Sylvie, Murrell, Michael, Charles-Orszag, Arthur, Tsai, Feng-Ching, Bonazzi, Daria, Manriquez, Valeria, Sachse, Martin, Mallet, Adeline, Salles, Audrey, Melican, Keira, Staneva, Ralitza, Bertin, Aurélie, Millien, Corinne, Goussard, Sylvie, Lafaye, Pierre, Shorte, Spencer, Piel, Matthieu, Krijnse-Locker, Jacomine, Brochard-Wyart, Françoise, Bassereau, Patricia, Duménil, Guillaume, Weimershaus, Mirjana, Mauvais, Francois-Xavier, Saveanu, Loredana, Adiko, Cézaire, Babdor, Joel, Abramova, Anastasia, Montealegre, Sebastian, Lawand, Myriam, Evnouchidou, Irini, Huber, Katharina Julia, Chadt, Alexandra, Zwick, Markus, Vargas, Pablo, Dussiot, Michaël, Lennon-Dumenil, Ana Maria, Brocker, Thomas, Al-Hasani, Hadi, Van Endert, Peter, National Institute for Materials Science (NIMS), Physico-Chimie-Curie (PCC), Institut Curie-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre d'élaboration de matériaux et d'études structurales (CEMES), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Biologie des Organismes et Ecosystèmes Aquatiques (BOREA), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Muséum national d'Histoire naturelle (MNHN)-Institut de Recherche pour le Développement (IRD)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université des Antilles (UA), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Institut Jacques Monod (IJM (UMR_7592)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Pathogénèse des Infections vasculaires, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Microscopie ultrastructurale (plate-forme), Institut Pasteur [Paris], Imagopole (CITECH), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Neuroscience, Karolinska Institutet [Stockholm], Institut Curie, SAFT [Bordeaux], Société des accumulateurs fixes et de traction (SAFT), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Microscopie ultrastructurale - Ultrapole (CITECH), Diabète de Type 1 : mécanismes et traitements immunologiques, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche sur l'Inflammation (CRI (UMR_S_1149 / ERL_8252 / U1149)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunité et cancer, Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Hémostase, bio-ingénierie et remodelage cardiovasculaires (LBPC), Université Paris 13 (UP13)-Université Paris Diderot - Paris 7 (UPD7)-Institut Galilée-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris], Université Paris Diderot - Paris 7 (UPD7)-Université Paris 13 (UP13)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Galilée, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Male, Endosome, Antigen presentation, Fc receptor, Kinesins, Endosomes, Receptors, Fc, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Microtubules, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Mice, Cross-Priming, Phagosomes, Animals, lcsh:QH301-705.5, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Phagosome, Innate immune system, biology, Chemistry, Histocompatibility Antigens Class I, Cross-presentation, Dendritic cell, Immunity, Innate, Cell biology, Toll-Like Receptor 4, Protein Transport, 030104 developmental biology, lcsh:Biology (General), rab GTP-Binding Proteins, biology.protein, Kinesin, Female
Abstract: Summary: Both cross-presentation of antigens by dendritic cells, a key pathway triggering T cell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells. : Weimershaus et al. identify a molecular complex that controls the intracellular trafficking along microtubules of antigens internalized by dendritic cells. They show that this trafficking is regulated by innate immune signals and regulates presentation of internalized antigens to T lymphocytes. Keywords: antigen presentation, cross-presentation, dendritic cell, MHC class I, endosome, small GTPase, kinesin, Rab14
Published: 2018
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5. Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes

Author: Thomas Krey, Ahmed Haouz, Mark A. Johnson, Félix A. Rey, Juliette Fedry, Gérard Pehau-Arnaudet, Pierre Legrand, Jennifer Forcina, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Hannover Medical School [Hannover] (MHH), Brown University, Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Protéopole, German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), ERC Advanced grant CelCelFus (grant number 340371) received by FAR. Recurrent funding from the CNRS received by FAR. Recurrent funding from the Institut Pasteur received by FAR. Allocation ministerielle pour l’Ecole Polytechnique AMX received by JFe. National Science Foundation (NSF) (grant number IOS-1353798). Received by MJ. National Science Foundation (NSF) (grant number IOS-1540019). Received by MJ. National Institutes of Health Training Grant (grant number #T32-GM007601). Received by JFo., and European Project: 340371,EC:FP7:ERC,ERC-2013-ADG,CELCELFUS(2014)
Subjects: Protein Structure Comparison, 0301 basic medicine, MESH: Sequence Homology, Amino Acid, [SDV]Life Sciences [q-bio], Cell Membranes, Arabidopsis, Chlamydomonas reinhardtii, MESH: Chlamydomonas, Membrane Fusion, Biochemistry, Macromolecular Structure Analysis, MESH: Arabidopsis, MESH: Genetic Variation, Biology (General), Flowering Plants, Fusion, General Neuroscience, Eukaryota, Plants, Biological Evolution, Transmembrane protein, Cell biology, MESH: Eukaryota, Experimental Organism Systems, Cellular Structures and Organelles, General Agricultural and Biological Sciences, Research Article, Protein Structure, QH301-705.5, Arabidopsis Thaliana, MESH: Biological Evolution, MESH: Carrier Proteins, Brassica, MESH: Arabidopsis Proteins, Viral Structure, Biology, Research and Analysis Methods, MESH: Membrane Fusion, Microbiology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Model Organisms, Plant and Algal Models, Virology, Vesicles, Molecular Biology, Sequence Homology, Amino Acid, General Immunology and Microbiology, Arabidopsis Proteins, Chlamydomonas, Organisms, Biology and Life Sciences, Membrane Proteins, Proteins, Genetic Variation, Lipid bilayer fusion, Cell Biology, biology.organism_classification, Fusion protein, MESH: Germ Cells, Germ Cells, 030104 developmental biology, Membrane protein, Liposomes, Helix, Carrier Proteins
Abstract: HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution., Author summary The fusion of gamete plasma membranes is the fundamental cellular event that brings two parental cells together to form a new individual, yet we know surprisingly little about this process at the molecular level. HAPLESS 2 (HAP2) is a conserved sperm plasma membrane protein that is essential for gamete fusion in a diverse array of eukaryotes. It was recently shown to share a common ancestor with viral proteins that drive fusion of the viral envelope with host membranes, but its mechanism of action remained elusive, since the reported structure did not resolve the proposed membrane interaction surface. Here, we report two new HAP2 structures revealing that HAP2 has evolved diverse membrane interaction surfaces. In the flowering plants, HAP2 uses an amphipathic helix that presents nonpolar residues to the target membrane; in trypanosomes, the membrane interaction surface comprises three shallow nonpolar loops.
Published: 2018
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6. Structure-Function Dissection of Pseudorabies Virus Glycoprotein B Fusion Loops

Author: Delphine Brun, Barbara G. Klupp, Melina Vallbracht, Gérard Pehau-Arnaudet, Ahmed Haouz, Pablo Guardado-Calvo, Félix A. Rey, Matteo Tassinari, Thomas C. Mettenleiter, Marija Backovic, Marie-Christine Vaney, Friedrich-Loeffler-Institut (FLI), Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], Cristallographie (Plateforme) - Crystallography (Platform), Work done by M.B., D.B., M.-C.V., P.G.-C., and F.A.R. was supported by Institut Pasteur and CNRS recurrent funding and the Pasteur-Weizmann/Servier International Prize (F.A.R., 2015). Work done by M.V., B.G.K., and T.C.M. was supported by DFG, grant Me 854/11-2., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Models, Molecular, Protein Conformation, [SDV]Life Sciences [q-bio], 030106 microbiology, Immunology, Plasma protein binding, Biology, Crystallography, X-Ray, Microbiology, 03 medical and health sciences, Protein structure, Viral envelope, Protein Domains, Viral Envelope Proteins, Virology, Lipid bilayer, Host cell membrane, Binding Sites, Lipid bilayer fusion, Virus Internalization, Fusion protein, Herpesvirus 1, Suid, Cell biology, Virus-Cell Interactions, 030104 developmental biology, Ectodomain, Insect Science, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Liposomes, Mutation, Protein Binding
Abstract: Conserved across the family Herpesviridae , glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses. IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established.
Published: 2017
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7. The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein

Author: Fedry, Juliette, Liu, Yanjie, Pehau-Arnaudet, Gerard, Pei, Jimin, Li, Wenhao, Tortorici, M Alejandra, Traincard, François, Meola, Annalisa, Bricogne, Gérard, Grishin, Nick, Snell, William, Rey, Felix, Krey, Thomas, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hannover Medical School [Hannover] (MHH), University of Texas Southwestern Medical Center, Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Global Phasing Ltd, Global Phasing, German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), F.A.R. acknowledges funding from the ERC Advanced grant project (340371) CelCelFus for this work, which also used general support from Institut Pasteur and CNRS. W.J.S. was supported by a grant from the NIH (GM56778) and acknowledges funding from the UTSW HI/HR Program. N.V.G. is funded, in part, by a grant from the NIH (GM094575) and the Welch Foundation (I-1505). J.F. benefitted from an Allocation ministérielle pour l'Ecole Polytechnique AMX., We thank Francis-André Wollmann and Sandrine Bujaldon for discussions and help with initial experiments, Fredrick Grinnell, Saikat Mukhopadhyay, Michael Henne, and Margaret Phillips of UT Southwestern for helpful discussions, the crystallization platform of the Pasteur Proteopole for technical help, Remi Fronzes for help with native PAGE experiments, Vincent Olieric and the staff of synchrotron beamlines PX-III at the Swiss Light Source, Proxima-1 and -2 at SOLEIL and ID29 and ID30-3 at the European Synchrotron Radiation Facility for help during data collection, members of the Rey and Snell labs for discussions, and M. Nilges and the Equipement d’excellence CACSICE for providing the Falcon II direct electron detector., European Project: 340371,EC:FP7:ERC,ERC-2013-ADG,CELCELFUS(2014), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]
Subjects: Plasmodium, crystal structure, transmission-blocking malaria vaccine, sexual reproduction, [SDV]Life Sciences [q-bio], membrane fusion, Protozoan Proteins, MESH: Sequence Alignment, MESH: Biological Evolution, MESH: Amino Acid Sequence, virus entry, Crystallography, X-Ray, gamete fusion, MESH: Chlamydomonas, Article, MESH: Recombinant Proteins, Protein Domains, lipid insertion, HAP2, Humans, Amino Acid Sequence, MESH: Membrane Fusion Proteins, MESH: Protozoan Proteins, Membrane Fusion Proteins, Plant Proteins, class II fusion protein, MESH: Plant Proteins, Biochemistry, Genetics and Molecular Biology(all), MESH: Plasmodium, Chlamydomonas, Eukaryota, MESH: Crystallography, X-Ray, Biological Evolution, Recombinant Proteins, MESH: Germ Cells, Germ Cells, Virus Diseases, MESH: Protein Domains, Sequence Alignment, Chlamydomonas reinhardtii
Abstract: Summary Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa—animals, plants, and protists (including important human pathogens like Plasmodium)—suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life., Graphical Abstract, Highlights • The primordial gamete fusogen HAP2 exhibits homology to class II viral fusion proteins • HAP2 inserts into the target gamete membrane via a hydrophobic fusion loop • HAP2 links virus entry into target cells and the origins of sexual reproduction • HAP2 is a sex-specific target for blocking fertilization in multiple kingdoms, Gamete fusion across eukaryotic branches uses an ancient factor homologous to viral fusion proteins.
Published: 2017
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8. Chikungunya Virus Replication in Salivary Glands of the Mosquito Aedes albopictus

Author: Christine Schmitt, Jacomine Krijnse Locker, Anubis Vega-Rúa, Isabelle Bonne, Anna-Bella Failloux, Arbovirus et Insectes Vecteurs - Arboviruses and Insect Vectors, Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), This study was funded by the Institut Pasteur, the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant No. ANR-10-LABX-62-IBEID). AVR was supported by the French Ministry of Superior Education and Research., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and Institut Pasteur [Paris] (IP)
Subjects: Saliva, replication, Aedes albopictus, viruses, salivary glands, lcsh:QR1-502, budding, MESH: Chikungunya virus/physiology, medicine.disease_cause, Virus Replication, Arbovirus, Virus, lcsh:Microbiology, MESH: Chikungunya virus/growth & development, Microscopy, Electron, Transmission, stomatognathic system, Aedes, Virology, parasitic diseases, transmission electron microscopy, medicine, Animals, MESH: Animals, Chikungunya, biology, MESH: Aedes/virology, Brief Report, fungi, MESH: Virus Replication, Virion, virus diseases, Midgut, MESH: Salivary Glands/virology, biology.organism_classification, medicine.disease, 3. Good health, Infectious Diseases, Viral replication, MESH: Virion/ultrastructure, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Microscopy, Electron, Transmission, Female, Chikungunya virus, MESH: Female
Abstract: International audience; Chikungunya virus (CHIKV) is an emerging arbovirus transmitted to humans by mosquitoes such as Aedes albopictus. To be transmitted, CHIKV must replicate in the mosquito midgut, then disseminate in the hemocele and infect the salivary glands before being released in saliva. We have developed a standardized protocol to visualize viral particles in the mosquito salivary glands using transmission electron microscopy. Here we provide direct evidence for CHIKV replication and storage in Ae. albopictus salivary glands.
Published: 2017
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9. A Novel Type of Polyhedral Viruses Infecting Hyperthermophilic Archaea

Author: David Prangishvili, Gérard Pehau-Arnaudet, Yoshizumi Ishino, Mart Krupovic, Ying Liu, Sonoko Ishino, Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Institut Pasteur [Paris], Kyushu University [Fukuoka], Microscopie ultrastructurale - Ultrapole (CITECH), This work was supported by the European Union's Horizon 2020 research and innovation program under grant agreement 685778, project VIRUS-X., We are grateful to E. V. Koonin and T. G. Senkevich for their help in collecting environmental samples from hot springs in Beppu, Japan, to M. Duchateau (Proteomics Platform, Institut Pasteur) for help with proteomics analyses, and to M. Nilges and the Equipex CACSICE for providing the Falcon II direct detector., European Project: 685778,H2020,H2020-LEIT-BIO-2015-1,Virus-X(2016), Institut Pasteur [Paris] (IP), and Kyushu University
Subjects: 0301 basic medicine, MESH: Sequence Analysis, DNA, MESH: Sequence Homology, Amino Acid, viruses, [SDV]Life Sciences [q-bio], virion structure, Genome, hyperthermophile, MESH: Gene Order, Gene Order, Genetics, biology, viral genome, Archaeal Viruses, MESH: DNA Viruses, Sulfolobus, Capsid, MESH: Sulfolobus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Virion, MESH: Genome, Viral, archaea, Immunology, Genome, Viral, MESH: Microscopy, Electron, Microbiology, Virus, 03 medical and health sciences, Open Reading Frames, Viral Proteins, MESH: Viral Structures, Virology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Viral Structures, Sequence Homology, Amino Acid, DNA Viruses, Virion, Sequence Analysis, DNA, MESH: Open Reading Frames, biology.organism_classification, MESH: Viral Proteins, Hyperthermophile, Open reading frame, Microscopy, Electron, 030104 developmental biology, Genetic Diversity and Evolution, Insect Science, Archaea
Abstract: Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales . The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated. IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, “Portogloboviridae.”
Published: 2017
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10. Assessing Vacuolar Escape of Listeria Monocytogenes

Author: Javier Pizarro-Cerdá, Spencer L. Shorte, Anne Danckaert, Martin Sachse, Théodore Grenier, Damien Balestrino, Jennifer Fredlund, Pascale Cossart, Nathalie Aulner, Jost Enninga, Juan J. Quereda, 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), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Laboratoire Microorganismes : Génome et Environnement (LMGE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Work in the laboratory of P.C. is supported by the Institut Pasteur (Unité des Interactions Bactéries-Cellules), the Institut National de la Santé et de la Recherche Médicale (Unité 604), the Institut National de la Recherche Agronomique (Unité Sous Contrat 2020), the Fondation Les Mousquetaires, and an European Research Council Advanced Grant (BacEpiCell 670823). The Imagopole is part of the FranceBioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, 'Investments for the Future') and is grateful to supports by Conseil de la Region Ile-de-France (program Sesame 2007, project Imagopole to S.S.) and from the Fondation Française pour la Recherche Médicale (FRM, Programme Grands Equipements to N.A.). J.E. is supported by the Institut Pasteur (PTR-460), by the Institut Pasteur Carnot-MIE program and by a European Research Council Starting Grant (RuptEffects 261166)., Collin M, We thank Servier Medical Art (http://www.servier.com/Powerpoint-image-bank) for providing drawings used in Fig. 1., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris], Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Université d'Auvergne - Clermont-Ferrand I (UdA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Dynamique des Interactions Hôte-Pathogène, ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-15-CE15-0017,StopBugEntry,Novel common host targets during bacterial invasion of humans(2015), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), vicente, marie-therese, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, and Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID
Subjects: 0301 basic medicine, Phagocytosis, media_common.quotation_subject, [SDV]Life Sciences [q-bio], 030106 microbiology, education, Vacuole, Biology, medicine.disease_cause, Microbiology, 03 medical and health sciences, Listeria monocytogenes, medicine, Fluorescence Resonance Energy Transfer, Internalization, Pathogen, health care economics and organizations, media_common, Correlative light/electron microcopy (CLEM), CCF4, Listeriolysin O, Biological Transport, Förster resonance energy transfer (FRET) microscopy, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, [SDV] Life Sciences [q-bio], Microscopy, Electron, 030104 developmental biology, Förster resonance energy transfer, Microscopy, Fluorescence, Phospholipases, Vacuoles, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: International audience; Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.
Published: 2016
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11. Eukaryotic-Like Virus Budding in Archaea

Author: Mart Krupovic, Petr Chlanda, Emmanuelle R. J. Quemin, Martin Sachse, Patrick Forterre, David Prangishvili, Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Institut Pasteur [Paris] (IP), National Institutes of Health [Bethesda] (NIH), Microscopie ultrastructurale - Ultrapole (CITECH), This work was funded by HHS | National Institutes of Health (NIH)(Intramural Research Program of the Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development), EC | European Research Council (ERC) (Project EVOMOBIL - ERC Grant Agreement no. 340440), Agence Nationale de la Recherche (ANR) (program BLANCproject EXAVIR), ANR-13-BSV3-0017,EXAVIR,Exit and assembly of hyperthermophilic archaeal viruses(2013), European Project: 340440,EC:FP7:ERC,ERC-2013-ADG,EVOMOBIL(2014), and Institut Pasteur [Paris]
Subjects: Archaeal Viruses, 0301 basic medicine, Electron Microscope Tomography, viruses, Fuselloviridae, Observation, Genome, Viral, Microbiology, ESCRT, Virus, 03 medical and health sciences, Viral envelope, Virology, Virus Release, Budding, biology, Eukaryota, Membrane budding, biology.organism_classification, Archaea, QR1-502, 3. Good health, Nucleoprotein, 030104 developmental biology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Abstract: Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission., IMPORTANCE The replication of enveloped viruses has been extensively studied in eukaryotes but has remained unexplored for enveloped viruses infecting bacteria and archaea. Here, we provide a sequential view on the assembly and egress of SSV1, a prototypic archaeal virus. The observed process is highly similar to the budding of eukaryotic enveloped viruses, including human immunodeficiency virus, influenza virus, and Ebola virus. The present study is the first to characterize such a phenomenon in archaeal cells, showing that membrane budding is not an exclusive feature of eukaryotic viruses. Our results provide significant insights into the biogenesis and architecture of unique, spindle-shaped virions that infect archaea. Furthermore, our findings open doors for future inquiries into (i) the evolution of the virus budding process, (ii) mechanistic details of virus-mediated membrane scission in Archaea, and (iii) elucidation of virus- and host-encoded molecular players responsible for archaeal membrane and surface remodeling.
Published: 2016
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12. A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes

Author: Zhang, Gaotian, Sachse, Martin, Prevost, Marie-Christine, Luallen, Robert J., Troemel, Emily R., Félix, Marie-Anne, Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), University of California [San Diego] (UC San Diego), University of California (UC), This work was supported by: Ecole Normale Supérieure, Centre National de la Recherche Scientifique, Inserm, Fondation pour la Recherche Médicale DEQ20150331704, Fondation Bettencourt-Schueller Coup d'Elan 2011, Fellowship from the China Scholarship Council, National Science Foundation Graduate Research fellowship, NIH R01GM114139 and Burroughs Wellcome Fund fellowship, and National Science Foundation Graduate Research Opportunities Worldwide. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], University of California, and Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS)
Subjects: Nematoda, Cell Membranes, Pathogenesis, Pathology and Laboratory Medicine, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, Polymerase Chain Reaction, Microsporidiosis, Medicine and Health Sciences, Nematode Infections, lcsh:QH301-705.5, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Phylogeny, Fungal Pathogens, [SDV.BA]Life Sciences [q-bio]/Animal biology, Animal Models, Experimental Organism Systems, Medical Microbiology, Host-Pathogen Interactions, Pathogens, Anatomy, Cellular Structures and Organelles, Research Article, lcsh:Immunologic diseases. Allergy, Mycology, [SDV.BID]Life Sciences [q-bio]/Biodiversity, Research and Analysis Methods, Microbiology, Model Organisms, Microscopy, Electron, Transmission, Virology, parasitic diseases, Parasitic Diseases, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Caenorhabditis elegans, Microbial Pathogens, Host Cells, fungi, Organisms, Biology and Life Sciences, Correction, Cell Biology, Invertebrates, Gastrointestinal Tract, lcsh:Biology (General), Microsporidia, Caenorhabditis, lcsh:RC581-607, Digestive System, Viral Transmission and Infection
Abstract: Microsporidia are fungi-related intracellular pathogens that may infect virtually all animals, but are poorly understood. The nematode Caenorhabditis elegans has recently become a model host for studying microsporidia through the identification of its natural microsporidian pathogen Nematocida parisii. However, it was unclear how widespread and diverse microsporidia infections are in C. elegans or other related nematodes in the wild. Here we describe the isolation and culture of 47 nematodes with microsporidian infections. N. parisii is found to be the most common microsporidia infecting C. elegans in the wild. In addition, we further describe and name six new species in the Nematocida genus. Our sampling and phylogenetic analysis further identify two subclades that are genetically distinct from Nematocida, and we name them Enteropsectra and Pancytospora. Interestingly, unlike Nematocida, these two genera belong to the main clade of microsporidia that includes human pathogens. All of these microsporidia are horizontally transmitted and most specifically infect intestinal cells, except Pancytospora epiphaga that replicates mostly in the epidermis of its Caenorhabditis host. At the subcellular level in the infected host cell, spores of the novel genus Enteropsectra show a characteristic apical distribution and exit via budding off of the plasma membrane, instead of exiting via exocytosis as spores of Nematocida. Host specificity is broad for some microsporidia, narrow for others: indeed, some microsporidia can infect Oscheius tipulae but not its sister species Oscheius sp. 3, and conversely some microsporidia found infecting Oscheius sp. 3 do not infect O. tipulae. We also show that N. ausubeli fails to strongly induce in C. elegans the transcription of genes that are induced by other Nematocida species, suggesting it has evolved mechanisms to prevent induction of this host response. Altogether, these newly isolated species illustrate the diversity and ubiquity of microsporidian infections in nematodes, and provide a rich resource to investigate host-parasite coevolution in tractable nematode hosts., Author Summary Microsporidia are microbial parasites that live inside their host cells and can cause disease in humans and many other animals. The small nematode worm Caenorhabditis elegans has recently become a convenient model host for studying microsporidian infections. In this work, we sample Caenorhabditis and other small nematodes and 47 associated microsporidian strains from the wild. We characterize the parasites for their position in the evolutionary tree of microsporidia and for their lifecycle and morphology. We find several new species and genera, especially some that are distantly related to the previously known Nematocida parisii and instead closely related to human pathogens. We find that some of these species have a narrow host range. We studied two species in detail using electron microscopy and uncover a new likely mode of exit from the host cell, by budding off the host cell plasma membrane rather than by fusion of a vesicle to the plasma membrane as in N. parisii. We also find a new species that infects the epidermis and muscles of Caenorhabditis rather than the host intestinal cells and is closely related to human pathogens. Finally, we find that one Nematocida species fails to elicit the same host response that other Nematocida species do. These new microsporidia open up many windows into microsporidia biology and opportunities to investigate host-parasite coevolution in the C. elegans system.
Published: 2016
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13. Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus

Author: Henry, Christine, Fontaine, Thierry, Heddergott, Christoph, Robinet, Pauline, Aimanianda, Vishukumar, Beau, Remi, Beauvais, Anne, Mouyna, Isabelle, Prevost, Marie-Christine, Fekkar, Arnaud, Zhao, Yanan, Perlin, David, Latgé, Jean-Paul, Aspergillus, Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), Service de parasitologie - mycologie [CHU Pitié-Salpétrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Rutgers, The State University of New Jersey [New Brunswick] (RU), Rutgers University System (Rutgers), This work was partly supported by grants of Aviesan, FRM, and Labex IBEID., Institut Pasteur [Paris] (IP), CHU Pitié-Salpêtrière [AP-HP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
Subjects: Mycelium, Virulence, Aspergillus fumigatus, Galactose, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Spores, Fungal, bacterial infections and mycoses, Mannosyltransferases, carbohydrates (lipids), Fungal Proteins, Mannans, Mice, Cell Wall, Gene Expression Regulation, Fungal, Host-Pathogen Interactions, Carbohydrate Conformation, Animals, Aspergillosis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], skin and connective tissue diseases, Mannose, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Gene Deletion
Abstract: International audience; The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.
Published: 2016
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14. Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes

Author: Andrea Disanza, Chiara Zurzolo, Giorgio Scita, Esthel Pénard, Diégo Cordero Cervantes, Elise Delage, Christine Schmitt, Sylvie Syan, Trafic membranaire et Pathogénèse, Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), IFOM, Istituto FIRC di Oncologia Molecolare (IFOM), Università degli Studi di Milano [Milano] (UNIMI), We gratefully acknowledge the kind financial support of the Institut Pasteur (Paris), the France–BioImaging infrastructure network supported by the French National Research Agency (ANR-10–INSB–04, Investments for the future), and the Région Ile-de-France (program DIM-Malinf ). We thank Dr. Jean-Yves Tinevez and Dr. Audrey Salles from Imagopole at Institut Pasteur for their help with microscopy imaging. We thank Dr. Sophie Novault from the Flow Cytometry Platform at Institut Pasteur for her help with flow cytometry experiments. We thank Dr. Fabrice De Chaumont from the Bioimage Analysis Unit at Institut Pasteur for his help with the software: ICY. E.D. is a recipient of the Bourse Carnot-Pasteur Maladies Infectieuses and the Bourse Pasteur-Roux, Institut Pasteur. This work is supported by research grants from the Agence Nationale de la Recherche (ANR-14-JPCD-0002-01 and ANR-16-CE16-0019-Neurotunn), AIC RECH COLLECT CYANOBAC, and Equipe FRM (Fondation Recherche Médicale) 2014 (DEQ20140329557) to C.Z., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-14-JPCD-0002,Neutargets,Targeting the propagation of pathogenic protein assemblies in neurodegenerative disease(2014), ANR-16-CE16-0019,Neurotunn,Role des nanotubes membranaires dans la propagation d'agrégats protéiques impliqués dans les maladie neurodégénératives(2016), Institut Pasteur [Paris] (IP), Università degli Studi di Milano = University of Milan (UNIMI), Delage, E., Cervantes, D. C., Penard, E., Schmitt, C., Syan, S., Disanza, A., Scita, G., and Zurzolo, C.
Subjects: 0301 basic medicine, Cell signaling, Green Fluorescent Proteins, Nerve Tissue Proteins, CDC42, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Cell Communication, Biology, Endocytosis, Green Fluorescent Protein, Article, EPS8, 03 medical and health sciences, Mice, Animals, Pseudopodia, cdc42 GTP-Binding Protein, Actin, Adaptor Proteins, Signal Transducing, Neurons, Multidisciplinary, Nanotubes, 030102 biochemistry & molecular biology, Endocytosi, Animal, Cell Membrane, Microfilament Proteins, Signal transducing adaptor protein, Brain, Microfilament Protein, Neuron, Phosphoproteins, Actins, Cell biology, Nanotube, Actin Cytoskeleton, 030104 developmental biology, Cell Adhesion Molecule, Phosphoprotein, Nerve Tissue Protein, Microscopy, Electron, Scanning, Filopodia, Cell Adhesion Molecules, Intracellular
Abstract: Tunneling Nanotubes (TNTs) are actin enriched filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens use TNT-like structures as “freeways” to propagate across cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia, differently from TNTs, are not able to mediate transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both these structures. We demonstrate that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and TNTs form through distinct molecular mechanisms. Our results further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion.
Published: 2016
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15. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

Author: Célia Fontana, Mathieu Picardeau, Nadia Benaroudj, Nathalie Michele Cachet, Olivier Gorgette, David Gasparini, Natalia Bomchil, Ambroise Lambert, Biologie des Spirochètes / Biology of Spirochetes, Institut Pasteur [Paris], Laboratoire Lyon Gerland, MERIAL, Centre de Recherche Clinique de Saint Vulbas, Microscopie ultrastructurale - Ultrapole (CITECH), This work was funded by the Institut Pasteur (http://www.pasteur.fr), FUI 14 (Fonds Unique Interministériel) 'COVALEPT', BPI France (http://www.bpifrance.fr/), Association Nationale de la Recherche et de la Technologie (http://www.anrt.asso.fr/), and Merial SAS France (http://www.merial.com). The funder provided support in the form of salaries for authors [CF, DG, NC, N. Benaroudj, MP, N. Bomchil, OG, AL] and research materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript., and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, lcsh:Medicine, Pathology and Laboratory Medicine, Cell morphology, Biochemistry, Medicine and Health Sciences, lcsh:Science, Leptospira, Mammals, MESH: Gene Expression Regulation, Bacterial, MESH: Genetic Complementation Test, Multidisciplinary, Virulence, biology, Bacterial Pathogens, 3. Good health, MESH: Leptospira interrogans/ultrastructure, Leptospira Interrogans, Cell Motility, MESH: Leptospira interrogans/physiology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Flagella, Medical Microbiology, Vertebrates, Hamsters, Cellular Structures and Organelles, Pathogens, Leptospira interrogans, Research Article, Pathogen Motility, MESH: Mutation, Virulence Factors, Motor Proteins, 030106 microbiology, Motility, Flagellum, MESH: Bacterial Proteins/genetics, Microbiology, Rodents, Motor protein, 03 medical and health sciences, Bacterial Proteins, Molecular Motors, Animals, MESH: Virulence/genetics, Microbial Pathogens, [SDV.GEN]Life Sciences [q-bio]/Genetics, Bacteria, Genetic Complementation Test, lcsh:R, Organisms, Biology and Life Sciences, Proteins, Cell Biology, Flagellar Motility, Gene Expression Regulation, Bacterial, Periplasmic space, MESH: Flagella/physiology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, MESH: Flagella/ultrastructure, Amniotes, Mutation, lcsh:Q
Abstract: International audience; Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.
Published: 2016
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16. GH16 and GH81 family β-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis

Author: Mouyna, Isabelle, Aimanianda, Vishukumar, Hartl, Lukas, Prévost, Marie-Christine, Sismeiro, Odile, Dillies, Marie-Agnes, Jagla, Bernd, Legendre, Rachel, Coppée, Jean-Yves, Latgé, Jean-Paul, Aspergillus, Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), Transcriptome et Epigénome (PF2), Sanofi (Aviesan project Aspergillus (BA P1-09), Institut Pasteur [Paris] (IP), Microscopie électronique (Plate-forme), and This research was funded by Sanofi (Aviesan project Aspergillus (BA P1-09).
Subjects: Glycosylation, Glycoside Hydrolases, Aspergillus fumigatus, Spores, Fungal, MESH: Glycosylation, [SDV.MP.MYC] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, MESH: Morphogenesis, Fungal Proteins, MESH: Cell Wall, MESH: Spores, Fungal, Cell Wall, MESH: Glycoside Hydrolases, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Carbohydrate Conformation, Morphogenesis, MESH: Fungal Proteins, MESH: Aspergillus fumigatus, MESH: Carbohydrate Conformation, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology
Abstract: International audience; The fungal cell wall is a rigid structure because of fibrillar and branched β-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on β-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-β-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo β-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.
Published: 2016
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17. Paenibacillus faecis sp. nov., isolated from human faeces

Author: Dominique Clermont, Isabelle Bonne, José-Carlos Fernandez, Christian Malosse, Richard J. Wheeler, Simonetta Gribaldo, Julia Chamot-Rooke, Ivo G. Boneca, Maïté Gomard, Chantal Bizet, Sylviane Hamon, Collection de l'Institut Pasteur (CIP), Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Spectrométrie de Masse structurale et protéomique, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Centre de Ressources biologiques de l'Institut Pasteur - Biological Resource Center of the Institut Pasteur (CRBIP), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Sequence Analysis, DNA, MESH: Vitamin K 2/chemistry, [SDV]Life Sciences [q-bio], Diaminopimelic Acid, MESH: Paenibacillus/isolation & purification, MESH: Nucleic Acid Hybridization, Feces, chemistry.chemical_compound, Cell Wall, RNA, Ribosomal, 16S, Genotype, Phylogeny, MESH: Phylogeny, chemistry.chemical_classification, Base Composition, 0303 health sciences, Phylogenetic tree, Strain (chemistry), MESH: Feces/microbiology, Fatty Acids, MESH: Diaminopimelic Acid/chemistry, Nucleic Acid Hybridization, food and beverages, Vitamin K 2, General Medicine, MESH: Paenibacillus/genetics, MESH: Paenibacillus/classification, 6. Clean water, Bacterial Typing Techniques, MESH: Vitamin K 2/analogs & derivatives, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, France, MESH: Fatty Acids/chemistry, Paenibacillus, DNA, Bacterial, MESH: Cell Wall/chemistry, Molecular Sequence Data, Peptidoglycan, Biology, Microbiology, MESH: Bacterial Typing Techniques, Cell wall, 03 medical and health sciences, MESH: Base Composition, Humans, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, MESH: Humans, MESH: Molecular Sequence Data, 030306 microbiology, Fatty acid, Sequence Analysis, DNA, 16S ribosomal RNA, biology.organism_classification, MESH: RNA, Ribosomal, 16S/genetics, MESH: DNA, Bacterial/genetics, MESH: France, chemistry, MESH: Peptidoglycan/chemistry, Bacteria
Abstract: A spore-forming, rod-shaped Gram-strain-positive bacterium, strain 656.84T, was isolated from human faeces in 1984. It contained anteiso-C15 : 0 as the major cellular fatty acid, meso-diaminopimelic acid was found in the cell wall peptidoglycan, the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and aminophospholipids as the major components, and the predominant menaquinone was MK-7. The DNA G+C content was 52.9 mol%. The results of comparative 16S rRNA gene sequence studies placed strain 656.84T within the genus Paenibacillus. Its closest phylogenetic relatives were Paenibacillus barengoltzii and Paenibacillus timonensis. Levels of DNA–DNA relatedness between strain 656.84T and Paenibacillus timonensis CIP 108005T and Paenibacillus barengoltzii CIP 109354T were 17.3 % and 36.8 %, respectively, indicating that strain 656.84T represents a distinct species. On the basis of phenotypic and genotypic results, strain 656.84T is considered to represent a novel species within the genus Paenibacillus, for which the name Paenibacillus faecis sp. nov. is proposed; the type strain is 656.84T ( = DSM 23593T = CIP 101062T).
Published: 2015
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18. Biogenesis and structure of a Type VI secretion membrane core complex

Author: Abdelrahim Zoued, Annick Dujeancourt, Laureen Logger, Rémi Fronzes, Benjamin Bardiaux, Eric Cascales, Van Son Nguyen, Silvia Spinelli, Aline Desmyter, Marie-Stéphanie Aschtgen, Eric Durand, Christian Cambillau, Gérard Pehau-Arnaudet, Alain Roussel, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060)., ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS) - Aix Marseille Université (AMU) - Institut National de la Recherche Agronomique (INRA), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], Bioinformatique structurale, This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060), We thank O. Francetic for providing anti-DglA and anti-OmpF antibodies. We thank E. Marza, P. Violinova Krasteva and H. Remaut for comments on the manuscript, and T. Mignot, M. Guzzo and L. Espinosa for advice about the fluorescence microscopy experiments and the statistical analyses. We also thank the members of the R.F. and E.C. research groups for discussions and suggestions, and R. Lloubès, J. Sturgis and A. Galinier for encouragement. We thank the ERSF and Soleil Synchrotron radiation facilities for beamline allocation, ANR-10-JCJC-1303, A Fun T6SS, Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-14-CE14-0006, B-War, Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), ANR-10-INBS-05-01/10-INBS-0005, FRISBI, Infrastructure Française pour la Biologie Structurale Intégrée(2010), CASCALES, ERIC, Jeunes Chercheuses et Jeunes Chercheurs - Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens - - A Fun T6SS2010 - ANR-10-JCJC-1303 - JCJC - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Appel à projets générique - Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI - - B-War2014 - ANR-14-CE14-0006 - Appel à projets générique - VALID, Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)
Subjects: Models, Molecular, Cytoplasm, MESH : Bacterial Secretion Systems, MESH : Escherichia coli/chemistry, MESH: Escherichia coli Proteins, MESH : Cytoplasm/chemistry, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Crystallography, X-Ray, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH : Multiprotein Complexes/biosynthesis, MESH: Protein Structure, Tertiary, Bacterial secretion, MESH : Membrane Proteins/biosynthesis, MESH: Lipopeptides, MESH : Escherichia coli Proteins/chemistry, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH : Cell Membrane/chemistry, "structure of a type VI", MESH : Membrane Proteins/chemistry, MESH : Escherichia coli Proteins/biosynthesis, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Escherichia coli Proteins, MESH: Periplasm, MESH : Escherichia coli/metabolism, MESH: Protein Subunits, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, Periplasm, MESH : Cytoplasm/metabolism, MESH: Membrane Proteins, Cell envelope, Bacterial outer membrane, Porosity, MESH : Lipopeptides/biosynthesis, MESH : Protein Structure, Tertiary, MESH: Models, Molecular, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH : Models, Molecular, MESH : Lipopeptides/chemistry, MESH: Microscopy, Electron, Biology, MESH : Protein Subunits/biosynthesis, Lipopeptides, 03 medical and health sciences, MESH: Porosity, Escherichia coli, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Electron microscopy, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], "Biogenesis", MESH : Cell Membrane/metabolism, "secretion membrane", MESH : Protein Subunits/chemistry, 030304 developmental biology, Type VI secretion system, X-ray crystallography, MESH : Periplasm/metabolism, MESH : Periplasm/chemistry, 030306 microbiology, MESH: Cytoplasm, Cell Membrane, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Periplasmic space, MESH: Multiprotein Complexes, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Microscopy, Electron, MESH : Multiprotein Complexes/chemistry, Protein Structure, Tertiary, Microscopy, Electron, Protein Subunits, Membrane protein, Multiprotein Complexes, Biophysics, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Porosity, MESH : Crystallography, X-Ray, Biogenesis, MESH: Cell Membrane
Abstract: International audience; Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine–glycine repeat protein G (VgrG) spike and punctures the prey’s cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)— TssJ, TssM and TssL—and present a structure of the fully assembled complex at 11.6 A ̊ resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct–TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.
Published: 2015
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19. Bioimage analysis of Shigella infection reveals targeting of colonic crypts

Author: Maryse Moya-Nilges, Giulia Nigro, Martin Sachse, Philippe J. Sansonetti, Ellen T. Arena, Jean-Yves Tinevez, François-Xavier Campbell-Valois, Katharina Nothelfer, Spencer L. Shorte, Benoit S. Marteyn, Pathogénie microbienne moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagerie Dynamique (Plate-Forme) (PFID), Institut Pasteur [Paris] (IP), Microscopie ultrastructurale - Ultrapole (CITECH), Collège de France - Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), E.T.A. was a Pasteur Foundation and Pasteur-Roux Fellow. F.-X.C.-V. was a Canadian Institutes of Health Research, European Molecular Biology Organization, Marie-Curie, and Fondation de la Recherche Médicale Fellow. P.J.S. is a Howard Hughes Medical Institute Senior International Research Scholar. This work was supported by the European Research Council (PS Advanced Grants 232798 and 339579), France Bioimaging Infrastructure, supported by the French National Research Agency ANR-10-INSB-04-01 Program Investments for the Future, and the Wellcome Trust., We thank Sèbastien Simard (Welcome Trust, London) for his contributions in OMERO and Pamela Schnupf and Claude Parsot for helpful discussions., European Project: 232798,EC:FP7:ERC,ERC-2008-AdG,HOMEOEPITH(2009), European Project: 339579,EC:FP7:ERC,ERC-2013-ADG,DECRYPT(2014), Tinevez, Jean-Yves, Homeostasis and rupture of the gut epithelium in the presence of commensals and pathogens - HOMEOEPITH - - EC:FP7:ERC2009-02-01 - 2013-07-31 - 232798 - VALID, Decrypting signals in the crypt. - DECRYPT - - EC:FP7:ERC2014-04-01 - 2019-03-31 - 339579 - VALID, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris], and Chaire Microbiologie et Maladies infectieuses
Subjects: Fluorescence-lifetime imaging microscopy, Colon, Confocal, [SDV]Life Sciences [q-bio], Crypt, Guinea Pigs, MESH: Intestinal Mucosa / microbiology, MESH: Shigella flexneri / pathogenicity, Biology, medicine.disease_cause, Microbiology, Shigella flexneri, MESH: Guinea Pigs, tissue microbiology, Intestinal mucosa, In vivo, medicine, Animals, Humans, Shigella, MESH: Animals, bioimage analysis, intestinal crypts, Intestinal Mucosa, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Dysentery, Bacillary, Multidisciplinary, MESH: Humans, biology.organism_classification, digestive system diseases, 3. Good health, Cell biology, MESH: Dysentery, Bacillary / pathology, [SDV] Life Sciences [q-bio], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, PNAS Plus, MESH: Colon / microbiology, Infectious agent, host–pathogen interactions
Abstract: International audience; Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent.
Published: 2015
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20. SUN proteins belong to a novel family of β-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis

Author: Christine Schmitt, Christophe d'Enfert, Audrey Nesseir, Anne Beauvais, Patrick England, Vishukumar Aimanianda, Rémi Beau, Arnaud Firon, Marie-Christine Prévost, Amandine Gastebois, Sophie Bachellier-Bassi, Jean-Paul Latgé, Isabelle Mouyna, Aspergillus, Institut Pasteur [Paris], Biologie et Pathogénicité fongiques, Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Microscopie ultrastructurale (plate-forme), Biochimie et Biophysique des Macromolécules (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from the European Commission (Galar Fungail 2, MRTN-CT-2003-504148, FINSysB, PITN-GA-2008-214004) (to C. d. E.) and the European Science Foundation (ESF) Fuminomics, Partenariat Sanofi-aventis-Aviesan (ITMO Maladies Infectieuses, Sanofi Aventis Research & Development TSU ID (SAR&D) TSU ID/Sanofi-Pasteur), and Agence Nationale de Recherches (ANR) (Grant 09-BLAN-0287) (to J. P. L.), ANR-09-BLAN-0287,REMODELE,Nouvelles voies métaboliques essentielles pour la biosynthèse de la paroi des champignons(2009), European Project: 214004,PEOPLE,FP7-PEOPLE-2007-1-1-ITN,FINSYSB(2008), Institut Pasteur [Paris] (IP), Biologie et Pathogénicité fongiques (BPF), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris], Biophysique des macromolécules et de leurs interactions, ANR Grant 09-BLAN-0287,ANR Grant 09-BLAN-0287,Grant 09-BLAN-0287, Institut National de la Recherche Agronomique (INRA) - Institut Pasteur [Paris], Microscopie ultrastructurale - Ultrapole (CITECH), Biophysique des macromolécules et leurs interactions, and Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS)
Subjects: Hyphal growth, MESH : Spores, Fungal, MESH : Molecular Sequence Data, Glycosylation, MESH: Sequence Homology, Amino Acid, Conidiation, Gene Expression, Oligosaccharides, Glycobiology and Extracellular Matrices, MESH: Amino Acid Sequence, Biochemistry, Aspergillus fumigatus, Cell Wall, Gene Expression Regulation, Fungal, Candida albicans, Morphogenesis, MESH : Fungal Proteins, skin and connective tissue diseases, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 0303 health sciences, integumentary system, MESH : Amino Acid Sequence, Hydrolysis, MESH : Glycosylation, Glucan Hydrolase Activity, SUN, MESH : Protein Binding, MESH : Glycoside Hydrolases, Spores, Fungal, MESH: Glycosylation, MESH : Sequence Homology, Amino Acid, Cell biology, [SDV] Life Sciences [q-bio], Aspergillus, CELL-WALL BIOGENESIS, MOTOR-LIKE DOMAIN, ASPERGILLUS-FUMIGATUS, CANDIDA-ALBICANS, SCHIZOSACCHAROMYCES-POMBE, SACCHAROMYCES-CEREVISIAE, BIOFILM FORMATION, BETA-GLUCOSIDASE, CHITIN SYNTHASES, GENE, MESH: Fungal Proteins, MESH: Aspergillus fumigatus, MESH: Gene Expression Regulation, Fungal, MESH: Hydrolysis, Protein Binding, CAZy, MESH: Gene Expression, Hypha, MESH : Candida albicans, Glycoside Hydrolases, Molecular Sequence Data, Hyphae, MESH: Glycoproteins, Biology, Microbiology, Cell wall, Fungal Proteins, 03 medical and health sciences, MESH : Hydrolysis, MESH: Hyphae, MESH: Spores, Fungal, MESH : Gene Expression Regulation, Fungal, MESH: Glycoside Hydrolases, MESH: Protein Binding, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, MESH : Protein Processing, Post-Translational, Glycoproteins, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, 030306 microbiology, MESH: Candida albicans, MESH : Oligosaccharides, Cell Biology, biology.organism_classification, MESH : Glycoproteins, [SDV.MP.MYC] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Yeast, MESH: Morphogenesis, MESH : Gene Expression, MESH : Morphogenesis, MESH : Aspergillus fumigatus, MESH: Protein Processing, Post-Translational, MESH : Hyphae, Carbohydrate-binding Protein, Protein Processing, Post-Translational, MESH: Oligosaccharides, Biogenesis
Abstract: International audience; BACKGROUND:SUN proteins are involved in yeast morphogenesis, but their function is unknown.RESULTS:SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated.CONCLUSION:Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity.SIGNIFICANCE:The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.
Published: 2013
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21. Crystal structure of glycoprotein C from a hantavirus virus in the post-fusion conformation

Author: Gérard Pehau-Arnaudet, Pablo Guardado-Calvo, Félix A. Rey, M. Alejandra Tortorici, Jean Luc Jestin, Scott A. Jeffers, Patrick England, Nicole D. Tischler, Eva Stettner, Jimena Pérez-Vargas, Eduardo A Bignon, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Molecular Virology Laboratory, Fundación Ciencia & Vida, Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Biophysique des macromolécules et leurs interactions, FAR and PGC acknowledge support the Infect-ERA IMI European network, program 'HantaHunt' and its coordinator, Professor Andreas Herrmann. FAR also received funding from the 'Integrative Biology of Emerging Infectious Diseases' Labex (Laboratoire d'Excellence ) grant N° ANR-10-LABX-62-IBEID (French Government's 'Investissements d'Avenir' program). ES was funded as 'Experienced Researche' (ER) by the Marie Curie Training Network 'Virus Entry' (Call:FP7-PEOPLE-2007-1-1-ITN. NDT received support from FONDECYT 1140050 and Basal PFB-16 grants from CONICYT (Chile). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 235649,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,VIRUS ENTRY(2009), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]
Subjects: RNA viruses, Retinal Ganglion Cells, 0301 basic medicine, Orthohantavirus, Orthobunyavirus, Protein Conformation, viruses, Cell Membranes, Andes virus, Pathology and Laboratory Medicine, medicine.disease_cause, Membrane Fusion, Physical Chemistry, Viral Envelope Proteins, Animal Cells, Bunyaviruses, Medicine and Health Sciences, Biology (General), Neurons, Crystallography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Physics, virus diseases, Condensed Matter Physics, 3. Good health, Chemistry, Medical Microbiology, Viral Pathogens, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viruses, Physical Sciences, Crystal Structure, Mathematisch-Naturwissenschaftliche Fakultät, Pathogens, Cellular Structures and Organelles, Cellular Types, Research Article, Hantavirus, Ganglion Cells, QH301-705.5, Viral protein, 030106 microbiology, Immunology, Viral Structure, Biology, Microbiology, Structure-Activity Relationship, 03 medical and health sciences, Viral entry, Virology, Genetics, medicine, Solid State Physics, Animals, Humans, ddc:610, Vesicles, Microbial Pathogens, Molecular Biology, Institut für Biochemie und Biologie, Hantaan virus, Glycoproteins, Hantavirus pulmonary syndrome, Biology and life sciences, Chemical Bonding, Organisms, Afferent Neurons, Lipid bilayer fusion, Hydrogen Bonding, Cell Biology, RC581-607, Surface Plasmon Resonance, Fusion protein, 030104 developmental biology, Cellular Neuroscience, Liposomes, Bunyavirus, Parasitology, Immunologic diseases. Allergy, viral fusion, Neuroscience
Abstract: Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens., Author Summary Hantaviruses belong to the Bunyaviridae family of enveloped viruses. This family englobes in total five established genera: Tospovirus (infecting plants), and Phlebovirus, Orthobunyavirus, Nairovirus and Hantavirus infecting animals, some of which cause serious disease in humans. An important characteristic of the hantaviruses is that they are not transmitted to humans by arthropod vectors, as those from the other genera, but by direct exposure to excretions from infected small mammals. As all enveloped viruses, they require the activity of a membrane fusogenic protein, Gc, for entry into their target cells. Our structural analysis of the hantavirus fusion protein Gc led to the identification of a conserved pattern of cysteines involved in disulfide bonds stabilizing the Gc fold. This motif is matched exclusively by all of the available bunyavirus Gc sequences in the database, with the notable exception of phlebovirus Gc, which appears closer in structure to the fusion proteins of other families of arthropod-borne viruses, such as the flaviviruses and alphaviruses. This analysis further suggests mechanistic similarities with hantaviruses in the fusion mechanism of viruses in the remaining three most closely related bunyavirus genera, which we propose belong to a new separate sub-class of fusion proteins with a multipartite membrane targeting region.
Published: 2016
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22. Emergence and Transmission of Arbovirus Evolutionary Intermediates with Epidemic Potential

Author: Kenneth A. Stapleford, Sreyrath Lay, Veasna Duong, Isabelle Bonne, Camilo Arias-Goeta, Christine Schmitt, Hervé Blanc, Anna-Bella Failloux, Stéphanie Beaucourt, Kathryn Rozen-Gagnon, Lark L. Coffey, Turkan Haliloglu, Ofer Isakov, Noam Shomron, Marco Vignuzzi, Antonio V. Bordería, Philippe Buchy, Nir Ben-Tal, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of California [Davis] (UC Davis), University of California (UC), Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Sackler Faculty of Medicine, Tel Aviv University (TAU), Arbovirus et Insectes Vecteurs - Arboviruses and Insect Vectors, Institut Pasteur [Paris] (IP), Boǧaziçi üniversitesi = Boğaziçi University [Istanbul], Microscopie ultrastructurale - Ultrapole (CITECH), This work was supported by the European Research Council (ERC Starting Grant No. 242719), by the Bill and Melinda Gates Foundation Grand Challenges Exploration Initiative, the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant ANR-10-LABX-62-IBEID), and the Israel-France High Council for Science and Technology Research 'Complexity in Biology' program. Salary for L.L.C. and K.A.S. was provided by the Region of Ile-de-France DIM program on Infectious, Parasitic or Nosocomial Emerging Diseases and the Philippe Foundation. Salary for A.V.B. was supported by the French National grant ANR-09-JCJC-0118-1. Salary for C.A.-G. was supported by the French Ministry of Superior Education and Research. N.B.-T. and T.H. were supported by NATO traveling grant CBP.MD.CLG 984340., We are grateful to Ngan Chantha, Huy Rekol, and the team of the National Malaria Centre in Cambodia for providing clinical specimens. We thank Christophe Paupy (IRD), Louis Lambrechts, and Paul Reiter (IP) for providing some mosquito strains., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-09-JCJC-0118,QuasispeciesVax(2009), European Project: 242719,EC:FP7:ERC,ERC-2009-StG,RNAVIRUSPOPDIVNVAX(2009), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of California, Tel Aviv University [Tel Aviv], Institut Pasteur [Paris], and Boğaziçi University [Istanbul]
Subjects: Cancer Research, Mutation rate, viruses, MESH: Chikungunya Fever, medicine.disease_cause, Virus Replication, Aedes, MESH: Arboviruses, MESH: Animals, MESH: Genetic Variation, Chikungunya, Epidemic strain, Genetics, Mammals, Transmission (medicine), virus diseases, MESH: Aedes, Viral Load, Biological Evolution, 3. Good health, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Host-Pathogen Interactions, MESH: Arbovirus Infections, Female, MESH: Viral Load, Cambodia, Chikungunya virus, MESH: Biological Evolution, MESH: Insect Vectors, Biology, Arbovirus Infections, Microbiology, Arbovirus, MESH: Mammals, Virus, Article, MESH: Mice, Inbred C57BL, Immunology and Microbiology(all), Virology, parasitic diseases, medicine, Animals, Humans, MESH: Saliva, Epidemics, Saliva, Molecular Biology, MESH: Epidemics, MESH: Humans, MESH: Cambodia, MESH: Host-Pathogen Interactions, MESH: Virus Replication, fungi, RNA, Genetic Variation, MESH: Chikungunya virus, medicine.disease, Insect Vectors, Mice, Inbred C57BL, Disease Models, Animal, Population bottleneck, Culicidae, Chikungunya Fever, Parasitology, MESH: Disease Models, Animal, MESH: Culicidae, MESH: Female, Arboviruses
Abstract: Comment in: "Toward an activist agenda for monitoring virus emergence."https://doi.org/10.1016/j.chom.2014.05.014; International audience; The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapit-ulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary tra-jectories can be predicted and studied in the short term before new variants displace currently circulating strains.
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23. The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution

Author: Michael Elbaum, Virginie Stévenin, Jacomine Krijnse-Locker, Katya Rechav, Jennifer Fredlund, Patricia Latour-Lambert, Jost Enninga, Adeline Mallet, Allon Weiner, José Carlos Santos, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Materials and Interfaces [Rehovot, Israël], Weizmann Institute of Science [Rehovot, Israël], Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], This work was funded by grants from the Pasteur‐Weizmann Foundation to J. E., by a fellowship from the Pasteur Foundation to J. F. by the FCT (SFRH/BD/51006/2010), and an Institut Pasteur PTR grant to J. C. S. by fellowships from the Pasteur‐Weizmann Foundation and the FRM to A. W., by a fellowship from the University Paris Diderot allocated by the ENS Cachan, Université Paris‐Saclay, and a grant from the FRM to V.S., and by grants from the ANR (StopBugEntry), and the European Research Council (Starting grant 'Rupteffects' and Consolidator grant 'EndoSubvert') to J. E., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), European Project: 261166,EC:FP7:ERC,ERC-2010-StG_20091118,RUPTEFFECTS(2011), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Salmonella, Membrane ruffling, intracellular trafficking, 030106 microbiology, Immunology, Vacuole, medicine.disease_cause, Microbiology, 03 medical and health sciences, Cytosol, Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Humans, Compartment (development), Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], host cell invasion, biology, Salmonella enterica, Epithelial Cells, biology.organism_classification, Cell biology, correlative light electron mciroscopy, Host-Pathogen Interactions, Salmonella Infections, Ultrastructure, macropinosomes, Intracellular, HeLa Cells
Abstract: International audience; Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.
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23 results on '"Microscopie ultrastructurale - Ultrapole (CITECH)"'

1. HIV-1 hijacks the cell extracellular matrix to spread collectively and efficiently between T lymphocytes

2. Dirhamnolipids secreted from Pseudomonas aeruginosa modify anjpegungal susceptibility of Aspergillus fumigatus by inhibiting β1,3 glucan synthase activity

3. Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting

4. Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation

5. Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes

6. Structure-Function Dissection of Pseudorabies Virus Glycoprotein B Fusion Loops

7. The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein

8. Chikungunya Virus Replication in Salivary Glands of the Mosquito Aedes albopictus

9. A Novel Type of Polyhedral Viruses Infecting Hyperthermophilic Archaea

10. Assessing Vacuolar Escape of Listeria Monocytogenes

11. Eukaryotic-Like Virus Budding in Archaea

12. A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes

13. Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus

14. Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes

15. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

16. GH16 and GH81 family β-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis

17. Paenibacillus faecis sp. nov., isolated from human faeces

18. Biogenesis and structure of a Type VI secretion membrane core complex

19. Bioimage analysis of Shigella infection reveals targeting of colonic crypts

20. SUN proteins belong to a novel family of β-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis

21. Crystal structure of glycoprotein C from a hantavirus virus in the post-fusion conformation

22. Emergence and Transmission of Arbovirus Evolutionary Intermediates with Epidemic Potential

23. The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution

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23 results on '"Microscopie ultrastructurale - Ultrapole (CITECH)"'

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