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1. An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

Author

Casabona, Maria, Silverman, Julie, Sall, Khady, Boyer, Frédéric, Couté, Yohann, Poirel, Jessica, Grunwald, Didier, Mougous, Joseph, Elsen, Sylvie, Attree, Ina, Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Microbiology, University of Washington [Seattle], Laboratoire d'Ecologie Alpine (LECA), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry]), Biothérapies des maladies génétiques et cancers, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Vaincre la mucoviscidose/NIH, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Université Joseph Fourier - Grenoble 1 (UJF)-Université Grenoble Alpes (UGA), ANTE-INSERM U836, équipe 4, Muscles et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Pathogénie bactérienne et réponses cellulaires, Roux-Buisson, Nathalie, and Université Joseph Fourier - Grenoble 1 (UJF)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)

Subjects
Molecular Sequence Data, Biological Transport, Gene Expression Regulation, Bacterial, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Protein Serine-Threonine Kinases, Phosphoric Monoester Hydrolases, Article, Pseudomonas aeruginosa, ATP-Binding Cassette Transporters, Amino Acid Sequence, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Bacterial Secretion Systems, Sequence Alignment, Gene Deletion, Bacterial Outer Membrane Proteins, Genome-Wide Association Study, Signal Transduction
Abstract

International audience; Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.

Published
2013

2. Metabotypes of Pseudomonas aeruginosa Correlate with Antibiotic Resistance, Virulence and Clinical Outcome in Cystic Fibrosis Chronic Infections

Author

Robert A. Quinn, Tuan-Dung Ngo, Claire Léger, Oriane Moyne, Ina Attree, Dalil Hannani, Samuel Terrier, Sarah Huot-Marchand, François Fenaille, Eric Faudry, Caroline Plazy, Florence Castelli, Julien Boccard, Bertrand Toussaint, Dominique J. Bicout, Audrey Le Gouellec, Boubou Camara, Benoit Cournoyer, Max Maurin, Translational Innovation in Medicine and Complexity / Recherche Translationnelle et Innovation en Médecine et Complexité - UMR 5525 (TIMC ), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Genève (UNIGE), Centre Hospitalier Universitaire [Grenoble] (CHU), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Translational microbial Evolution and Engineering (TIMC-TrEE), Université Grenoble Alpes (UGA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Michigan State University [East Lansing], Michigan State University System, Université de Genève = University of Geneva (UNIGE), Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and ATTREE, Ina

Subjects
0301 basic medicine, LC-HRMS, P. aeruginosa, Endocrinology, Diabetes and Metabolism, polyamines, 030106 microbiology, Ala-Glu-mesodiaminopimelate, lcsh:QR1-502, Virulence, medicine.disease_cause, Biochemistry, Cystic fibrosis, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Article, lcsh:Microbiology, cystic fibrosis, 03 medical and health sciences, Metabolomics, Antibiotic resistance, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Molecular Biology, Pathogen, ddc:615, Pseudomonas aeruginosa, business.industry, multiscale data analysis, medicine.disease, Phenotype, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, metabolomics, 3. Good health, 37 polyamines, 030104 developmental biology, Chronic lung infections, Immunology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, business
Abstract

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a &ldquo, metabotypes&rdquo, expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections

Published
2021

3. Bacterial behavior in human blood reveals complement evaders with some persister-like features

Author

Stéphane, Pont, Nathan, Fraikin, Yvan, Caspar, Laurence, Van Melderen, Ina, Attrée, François, Cretin, Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université libre de Bruxelles (ULB), Translational Innovation in Medicine and Complexity / Recherche Translationnelle et Innovation en Médecine et Complexité - UMR 5525 (TIMC ), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), Fondation Grenoble INP, Centre Hospitalier Universitaire [Grenoble] (CHU), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications Grenoble - UMR 5525 (TIMC-IMAG), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and ATTREE, Ina

Subjects
Acinetobacter baumannii, Physiology, Complement System, Bacteremia, Pathology and Laboratory Medicine, Biochemistry, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, White Blood Cells, Antibiotics, Animal Cells, Immune Physiology, Medicine and Health Sciences, Biology (General), Pathologie maladies infectieuses, Complement Activation, Immune System Proteins, Antimicrobials, Pseudomonas Aeruginosa, Drugs, Bacterial Infections, Body Fluids, Bacterial Pathogens, Klebsiella pneumoniae, Blood, Medical Microbiology, Anatomy, Pathogens, Cellular Types, Research Article, Lysis (Medicine), Burkholderia, QH301-705.5, Immune Cells, Immunology, Microbial Sensitivity Tests, Microbiology, Blood Plasma, Pseudomonas, Microbial Control, Tissue Repair, Escherichia coli, Humans, Pseudomonas Infections, Microbial Pathogens, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, Yersinia enterocolitica, Pharmacology, Blood Cells, Bacteria, Organisms, Biology and Life Sciences, Proteins, Complement System Proteins, Cell Biology, RC581-607, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immune System, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immunologic diseases. Allergy, Physiological Processes, Microbiologie et protistologie [bacteriol.virolog.mycolog.]
Abstract

Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named “evaders”. Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1–0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments., Author summary Blood infections by antibiotic resistant bacteria, notably Pseudomonas aeruginosa, are major concerns in hospital settings. The complex interplay between P. aeruginosa and the innate immune system in the context of human blood is still poorly understood. By studying the behavior of various P. aeruginosa strains in human whole blood and plasma, we showed that bacterial strains display different rate of tolerance to the complement system. Despite the complement microbicide activity, most bacteria withstand elimination through phenotypic heterogeneity creating a tiny (

Published
2020

4. CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei

Author

Stéphanie Bouillot, Ina Attree, Emmanuelle Soleilhac, Yann Wallez, Philippe Huber, Eric Faudry, Pathogénie bactérienne et réponses cellulaires, Centre National de la Recherche Scientifique (CNRS) - Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie du Cancer et de l'Infection (BCI - UMR S1036), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Université Grenoble Alpes (UGA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) - Université Grenoble Alpes [Saint Martin d'Hères] - Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Genetics and Chemogenomics (GenChem), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), AVIESAN T3SS (ANR PRP1.4), IBiSA, ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-15-CE11-0018,HemoPseudo,Pneumonie hémorragique à Pseudomonas aeruginosa : étude de nouvelles stratégies de virulence(2015), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Faudry, Eric, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, and Pneumonie hémorragique à Pseudomonas aeruginosa : étude de nouvelles stratégies de virulence - - HemoPseudo2015 - ANR-15-CE11-0018 - AAPG2015 - VALID

Subjects
0301 basic medicine, Programmed cell death, 030106 microbiology, Cell, lcsh:Medicine, Virulence, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Bacterial growth, Bacterial Physiological Phenomena, medicine.disease_cause, Article, Mice, 03 medical and health sciences, Live cell imaging, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Human Umbilical Vein Endothelial Cells, medicine, Animals, Humans, lcsh:Science, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Multidisciplinary, biology, 030306 microbiology, lcsh:R, Endothelial Cells, Pathogenic bacteria, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Molecular Imaging, 3. Good health, Cell biology, Eukaryotic Cells, 030104 developmental biology, medicine.anatomical_structure, NIH 3T3 Cells, lcsh:Q, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Ex vivo, Bacteria, HeLa Cells
Abstract

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Published
2018
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5. IFN-γ extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection

Author

Sacha Benaoudia, Sophia Djebali, Pierre Wallet, Angelina Provost, Pauline Basso, Igor Golovliov, Anders Sjöstedt, Etienne Meunier, Eric Faudry, Bénédicte F. Py, Fanny Michal, Omran Allatif, Brice Lagrange, Helena Lindgren, Amandine Mosnier, Amandine Martin, Petr Broz, Thomas Henry, Masahiro Yamamoto, Inflammasome, Infections bactériennes et autoinflammation, Inflammasome, Bacterial Infections and Autoinflammation (I2BA), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie de l'allergie cutanée et vaccination – Immunology of skin allergy and vaccination, Laboratory for Molecular Infection Medicine Sweden and Department of Clinical Microbiology, Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Immunité et lymphocytes cytotoxiques – Immunity and cytotoxic lymphocytes, Focal Area Infection Biology, Biozentrum, University of Basel (Unibas), Research Institute for Microbial Diseases [Osaka, Japan] (RIMD), Osaka University [Osaka], Inflammasome NLRP3 – NLRP3 Inflammasome, Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Male, Inflammasomes, Fluorescent Antibody Technique, Apoptosis, medicine.disease_cause, Pathology and Laboratory Medicine, Inbred C57BL, Biochemistry, White Blood Cells, Mice, 0302 clinical medicine, Animal Cells, Medicine and Health Sciences, Biology (General), Francisella, Francisella tularensis, Tularemia, Mice, Knockout, Immune System Proteins, Cell Death, Effector, Antimicrobials, Biochemistry and Molecular Biology, Drugs, Inflammasome, Flow Cytometry, 3. Good health, Cell biology, Bacterial Pathogens, Chemistry, Intracellular Pathogens, Cell Processes, Medical Microbiology, Gene Knockdown Techniques, Physical Sciences, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, Cellular Types, Pathogens, medicine.drug, Research Article, QH301-705.5, Immune Cells, Knockout, Immunology, Immunoblotting, Enzyme-Linked Immunosorbent Assay, Biology, Microbiology, 03 medical and health sciences, AIM2, Interferon-gamma, Immune system, GTP-Binding Proteins, Virology, Microbial Control, Genetics, medicine, Animals, Francisella novicida, Molecular Biology, Microbial Pathogens, Pharmacology, Blood Cells, Bacteria, Animal, Intracellular parasite, Macrophages, Chemical Compounds, Organisms, Biology and Life Sciences, Proteins, Cell Biology, RC581-607, Iodides, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, Disease Models, Antibacterials, Parasitology, Immunologic diseases. Allergy, Gram-Negative Bacterial Infections, Inflammasome complex, Biokemi och molekylärbiologi, 030215 immunology
Abstract

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cell-intrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-γ induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-γ priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-γ-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners., Author summary The cell-intrinsic immunity is defined as the mechanisms allowing a host cell infected by an intracellular pathogen to mount effective immune mechanisms to detect and eliminate pathogens without any help from other immune cells. In infected macrophages, the Guanylate Binding Proteins (GBPs) are immune proteins, induced at low levels in a cell autonomous manner by endogenous type I IFN or at high levels following IFN-γ production by innate and adaptive lymphocytes. The antibacterial activity of GBPs has been recently tightly linked to the inflammasomes. Inflammasomes are innate immune complexes leading to inflammatory caspases activation and death of the infected cell. Francisella novicida, a bacterium replicating in the macrophage cytosol, is closely related to F. tularensis, the agent of tularemia and is used as a model to study cytosolic immunity. GBPs contribute to F. novicida lysis within the host cytosol leading to DNA release and AIM2 inflammasome activation. In addition to their regulation of the AIM2 inflammasome, we identified that GBPs also control several other pyroptotic and apoptotic pathways activated in a hierarchical manner. Furthermore, we demonstrate that IFN-γ priming extends GBPs anti-microbial responses from the inflammasome-dependent control of cell death to an inflammasome-independent control of cytosolic bacterial replication. Our results, validated in a mouse model of tularemia, thus segregate the antimicrobial activities of inflammasomes and GBPs as well as highlight GBPs as the master effectors of IFN-γ-mediated cytosolic immunity.

Published
2017

6. A gacS deletion in Pseudomonas aeruginosa cystic fibrosis isolate CHA shapes its virulence

Author

Ina Attree, Khady Mayebine Sall, Maria Guillermina Casabona, Christophe Bordi, Sylvie Elsen, Sophie de Bentzmann, Philippe Huber, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), 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 Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
Molecular Sequence Data, DNA transcription, lcsh:Medicine, Virulence, Pathogenesis, Biology, Flagellum, Plasmid construction, medicine.disease_cause, Pathology and Laboratory Medicine, Microbiology, Virulence factor, Cystic fibrosis, Type three secretion system, Bacterial Proteins, medicine, Medicine and Health Sciences, Genetics, Amino Acid Sequence, Pathogen motility, lcsh:Science, Microbial Pathogens, Type VI secretion system, Regulation of gene expression, Multidisciplinary, Sequence Homology, Amino Acid, Virulence factors, Pseudomonas aeruginosa, lcsh:R, Biofilm, Biology and Life Sciences, Bacteriology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Polymerase chain reaction, Medical Microbiology, Genes, Bacterial, Biofilms, Host-Pathogen Interactions, Mutation, lcsh:Q, Gene expression, Gene Deletion, Research Article
Abstract

International audience; Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3' end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor.

Published
2014

7. Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB

Author

Jens Klockgether, Ina Attree, Colin F. Davenport, Oliver Ki Bezuidt, Burkhard Tümmler, Sylvie Elsen, Klinische Forschergruppe, Klinik für Pädiatrische Pneumologie, Allergologie und Neonatologie-Medizinische Hochschule Hannover (MHH), Bioinformatics and Computational Biology Unit, University of Pretoria [South Africa], Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Clinic for Paediatric Pneumology and Neonatology, BMC, Ed., Medizinische Hochschule Hannover (MHH)-Klinik für Pädiatrische Pneumologie, Allergologie und Neonatologie, Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
DNA, Bacterial, Lineage (genetic), Cystic Fibrosis, Clone (cell biology), Virulence, Single-nucleotide polymorphism, Genomics, Genome diversity, Biology, Polymorphism, Single Nucleotide, Genome, 03 medical and health sciences, INDEL Mutation, Species Specificity, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetic variation, Genetics, Humans, Microevolution, Habitat adaptation, Ecosystem, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Genetic Variation, Adaptation, Physiological, Clone Cells, Pseudomonas aeruginosa, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Female, Sequence Analysis, Genome, Bacterial, Research Article, Biotechnology
Abstract

Background Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. Results The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. Conclusions The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA.

Published
2013

8. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Author

Véronique Collin-Faure, Sylvie Elsen, Xavier Gidrol, Claudie Lemercier, Lemercier, Claudie, Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Commissariat à l'Energie Atomique et aux Energies Renouvelables (CEA), the Centre National de la Recherche Scientifique (CNRS) and the Université Joseph Fourier (UJF Grenoble)., Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)

Subjects
MESH: Signal Transduction, DNA Repair, MESH: ADP Ribose Transferases, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, Ataxia Telangiectasia Mutated Proteins, medicine.disease_cause, Histones, chemistry.chemical_compound, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Chromosome instability, DNA Breaks, Double-Stranded, H2AX, Phosphorylation, MESH: Ataxia Telangiectasia Mutated Proteins, MESH: Bacterial Proteins, ADP Ribose Transferases, MESH: DNA Repair, MESH: Histones, 0303 health sciences, biology, Kinase, Intracellular Signaling Peptides and Proteins, DNA double strand breaks, 3. Good health, [SDV] Life Sciences [q-bio], Histone, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Molecular Medicine, Signal transduction, Tumor Suppressor p53-Binding Protein 1, Infection, Signal Transduction, MESH: Cell Line, Tumor, DNA repair, Bacterial Toxins, HL-60 Cells, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microbiology, MESH: Chromosomal Instability, 03 medical and health sciences, Cellular and Molecular Neuroscience, Bacterial Proteins, [SDV.CAN] Life Sciences [q-bio]/Cancer, MESH: HL-60 Cells, Cell Line, Tumor, Chromosomal Instability, MESH: Intracellular Signaling Peptides and Proteins, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, Pharmacology, MESH: Humans, MESH: Phosphorylation, 030306 microbiology, Mutagenesis, JNK Mitogen-Activated Protein Kinases, Cell Biology, MESH: JNK Mitogen-Activated Protein Kinases, chemistry, MESH: Bacterial Toxins, ATM, biology.protein, DNA
Abstract

International audience; Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

Published
2013

9. Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB

Author

Friederike Ewald, Cathy Nguyen, Marie-Odile Fauvarque, Ina Attree, Virginie Calderon, Katy Jeannot, Sophie de Bentzmann, Christophe Bordi, Christophe S. Bernard, Didier Grunwald, Patrick Plésiat, Caroline Giraud, 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), Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de bactériologie, Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Technologies avancées pour le génôme et la clinique (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANTE-INSERM U836, équipe 4, Muscles et pathologies, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), French cystic fibrosis foundation, French Ministry of Research and Technology, Region Rhône-Alpes, European Project, Roux-Buisson, Nathalie, ERA-NET ADHRES 27481, INCOMING, Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Bourgogne Franche-Comté [COMUE] (UBFC), Theories and Approaches of Genomic Complexity (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherches en Technologies et Sciences pour le Vivant (IRTSV), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
MESH: Adhesins, Bacterial, Fimbria, Pathogenesis, medicine.disease_cause, MESH: Animals, Biology (General), MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Genomics, 3. Good health, Bacterial Pathogens, Host-Pathogen Interaction, Drosophila melanogaster, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Research Article, QH301-705.5, Immunology, Virulence, MESH: Biofilms, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Microbiology, Bacterial genetics, Cell Line, MESH: Drosophila melanogaster, MESH: Fimbriae, Bacterial, 03 medical and health sciences, Virology, medicine, Genetics, Animals, Secretion, Adhesins, Bacterial, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, 030306 microbiology, fungi, Biofilm, Bacteriology, RC581-607, MESH: Cell Line, Bacterial adhesin, Regulon, Biofilms, Fimbriae, Bacterial, Parasitology, Immunologic diseases. Allergy
Abstract

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections., Author Summary We unraveled that once the two-component system PprAB regulatory pathway is activated, Pseudomonas aeruginosa displays a unique hyper-biofilm phenotype due to a molecular signature combining a T1SS high molecular weight substrate, BapA, fimbriae of the chaperone-usher pathway, Type IVb pili and eDNA. Originally, this particular hyper-biofilm that is not strictly dependent on Psl or Pel exopolysaccharide (EPS) synthesis displays increased drug susceptibility, in contrary to previously reported biofilm lifestyle associated with increased resistance to antibiotics. PprB-dependent hyper-biofilm was also observed on intestinal mucosa of orally infected Drosophila flies in which it also displays a reduced capacity to cross the epithelial barrier from the intestinal lumen toward the hemolymph that consequently resulted in a reduced capacity to kill flies. Furthermore, constitutive activation of this PprB regulatory pathway triggers a reduced secretion of T3SS effectors which may account for the decreased virulence observed in epithelial and macrophage lineages and in acute Drosophila infections induced by septic injury. We appended in this study pieces of regulatory and molecular data that highlight the possibility to combat infections due to P. aeruginosa-biofilm with particular matrix.

Published
2012

10. Structural characterization and membrane localization of ExsB from the type III secretion system (T3SS) of Pseudomonas aeruginosa

Author

Eric Faudry, Andréa Dessen, Viviana Job, Ina Attree, Thierry Izoré, Caroline Perdu, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Conformation, Operon, Lipoproteins, Molecular Sequence Data, Yersinia, Crystallography, X-Ray, Models, Biological, Type three secretion system, 03 medical and health sciences, Maltose-binding protein, Bacterial Proteins, Structural Biology, Transcriptional regulation, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, 0303 health sciences, Sequence Homology, Amino Acid, biology, 030306 microbiology, Membrane Transport Proteins, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Biochemistry, Cytoplasm, Pseudomonas aeruginosa, biology.protein, Cell fractionation, Bacterial outer membrane, Sequence Alignment, Bacterial Outer Membrane Proteins
Abstract

International audience; Pseudomonas aeruginosa is an opportunistic human pathogen that employs a finely tuned type III secretion system (T3SS) to inject toxins directly into the cytoplasm of target cells. ExsB is a 15.6-kDa protein encoded in a T3SS transcription regulation operon that displays high sequence similarity to YscW, a lipoprotein from Yersinia spp. whose genetic neighborhood also involves a transcriptional regulator, and has been shown to play a role in the stabilization of the outer membrane ring of the T3SS. Here, we show that ExsB is expressed in P. aeruginosa upon induction of the T3SS, and subcellular fractionation studies reveal that it is associated with the outer membrane. The high-resolution crystal structure of ExsB shows that it displays a compact β-sandwich fold with interdependent β-sheets. ExsB possesses a large patch of basic residues that could play a role in membrane recognition, and its structure is distinct from that of MxiM, a lipoprotein involved in secretin stabilization in Shigella, as well as from those of Pil lipoproteins involved in pilus biogenesis. These results reveal that small lipoproteins involved in formation of the outer membrane secretin ring display clear structural differences that may be related to the different functions they play in these systems.

Published
2011

11. The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

Author

Michel Ragno, Carlos Roberto Felix, Christine Ebel, Keyla C. de Almeida, Bergmann Morais Ribeiro, Jaime M. Santana, Thiago S Gastardelo, Hugo de Almeida, Raquel S Negreiros, M. Lima, Teresa C.F. Assumpção, Izabela Marques Dourado Bastos, Gloria Cadavid-Restrepo, Eric Faudry, Department of Cell Biology, Universidade de Brasilia [Brasília] (UnB), Department of Bioscience, Universidad Nacional de Colombia, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Faculty of Ceilândia, Laboratory of Malaria and Vector Research, National Institutes of Health [Bethesda] (NIH), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), This work was supported by grants from CNPq, FINEP, FAP-DF, PRONEX, CAPES and UnB, Brazil., Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and BMC, Ed.

Subjects
Chagas disease, Cytoplasm, Trypanosoma cruzi, Molecular Sequence Data, lcsh:Animal biochemistry, Sequence alignment, Biochemistry, lcsh:Biochemistry, Patogênese, 03 medical and health sciences, Leucyl Aminopeptidase, Phylogenetics, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Drug Discovery, parasitic diseases, medicine, lcsh:QD415-436, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein Structure, Quaternary, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:QP501-801, Molecular Biology, Peptide sequence, Leucyl aminopeptidase, Phylogeny, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Drug discovery, Hydrolysis, Chagas, Doença de, medicine.disease, biology.organism_classification, Protein Transport, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Tripanossoma cruzi, Protein Multimerization, Trypanosomiasis, Sequence Alignment, Research Article
Abstract

Background Pathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease. Results The enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH. Conclusions LAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.

Published
2011

12. PtrA is a periplasmic protein involved in Cu tolerance in Pseudomonas aeruginosa

Author

Michel Ragno, Ina Attree, Sylvie Elsen, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Pathogénie bactérienne et réponses cellulaires, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Transcription, Genetic, chemistry.chemical_element, medicine.disease_cause, Microbiology, Type three secretion system, Periplasmic Proteins, 03 medical and health sciences, Bacterial Proteins, Drug Resistance, Bacterial, MESH: Drug Resistance, Bacterial, medicine, Molecular Biology, MESH: Bacterial Proteins, 030304 developmental biology, Molecular Biology of Pathogens, 0303 health sciences, MESH: Gene Expression Regulation, Bacterial, biology, 030306 microbiology, Pseudomonas aeruginosa, MESH: Transcription, Genetic, Gene Expression Regulation, Bacterial, Periplasmic space, biology.organism_classification, Copper, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Copper, Biochemistry, chemistry, Pseudomonadales, MESH: Pseudomonas aeruginosa, Bacteria, Pseudomonadaceae, MESH: Periplasmic Proteins
Abstract

In this work, we demonstrate that PtrA (U. H. Ha et al., Mol. Microbiol. 54:307–320, 2004) is a periplasmic protein, specifically synthesized in the presence of copper, that is involved in the tolerance of Pseudomonas aeruginosa to copper. Our biochemical and genetic analyses challenge its role in transcriptional inhibition of the type III secretion system.

Published
2011

13. Membrane targeting and pore formation by the type III secretion system translocon

Author

Pierre-Jean, Matteï, Eric, Faudry, Viviana, Job, Thierry, Izoré, Ina, Attree, Andréa, Dessen, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cytoplasm, MESH: Molecular Sequence Data, MESH: Biological Transport, MESH: Cytoplasm, Molecular Sequence Data, MESH: Hydrophobic and Hydrophilic Interactions, MESH: Sequence Alignment, Membrane Transport Proteins, Biological Transport, MESH: Amino Acid Sequence, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Membrane Transport Proteins, Bacterial Proteins, Gram-Negative Bacteria, MESH: Gram-Negative Bacteria, bacteria, lipids (amino acids, peptides, and proteins), Amino Acid Sequence, Hydrophobic and Hydrophilic Interactions, Sequence Alignment, MESH: Bacterial Proteins
Abstract

International audience; The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

Published
2011

14. Kinetic analysis of the regulation of the Na+/H+ exchanger NHE-1 by osmotic shocks

Author

Laurence Huc, Vincent Morello, Mary Rissel, Michel Ragno, Laurent Counillon, Mallorie Poët, Pierre Gounon, Xavier Tekpli, Nadir Djerbi, Jerome J. Lacroix, Dominique Lagadic-Gossmann, Transport ionique aspects normaux et pathologiques, Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie moléculaire et cellulaire (IPMC), Experimental Medicine and Toxicology, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Réponses aux Agents Infectieux et Chimiques (SeRAIC), Université de Rennes (UR), Physiological Genomics of the Eukaryotes, CNRS FRE3093 Transport ionique aspects normaux et pathologiques, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Gounon, Pierre, and Counillon, Laurent

Subjects
MESH: Signal Transduction, MESH: Hydrogen-Ion Concentration, Sodium-Hydrogen Exchangers, Allosteric regulation, MESH: Cricetinae, MESH: Microscopy, Fluorescence, MESH: Protein Isoforms, Models, Biological, Biochemistry, Cell Line, 03 medical and health sciences, MESH: Cation Transport Proteins, Cytosol, MESH: Cytosol, Osmotic Pressure, Cricetinae, Animals, Protein Isoforms, Osmotic pressure, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lipid bilayer, Cation Transport Proteins, 030304 developmental biology, 0303 health sciences, Sodium-Hydrogen Exchanger 1, MESH: Sodium-Hydrogen Antiporter, Osmotic concentration, MESH: Kinetics, Chemistry, 030302 biochemistry & molecular biology, MESH: Models, Biological, Cooperative binding, Fibroblasts, Hydrogen-Ion Concentration, MESH: Osmotic Pressure, MESH: Gene Expression Regulation, MESH: Cell Line, Kinetics, Sodium–hydrogen antiporter, Gene Expression Regulation, Microscopy, Fluorescence, MESH: Fibroblasts, Biophysics, lipids (amino acids, peptides, and proteins), Intracellular, Signal Transduction
Abstract

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H(2)O(2) formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H(2)O(2) dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization., NHE-1 is a ubiquitous, mitogen-activatable, mammalian Na+/H+ exchanger that maintains cytosolic pH and regulates cell volume. We have previously shown that the kinetics of NHE-1 positive cooperative activation by intracellular acidifications fit best with a Monod-Wyman-Changeux mechanism, in which a dimeric NHE-1 oscillates between a low- and a high-affinity conformation for intracellular protons. The ratio between these two forms, the allosteric equilibrium constant L0, is in favor of the low-affinity form, making the system inactive at physiological pH. Conversely the high-affinity form is stabilized by intracellular protons, resulting in the observed positive cooperativity. The aim of the present study was to investigate the kinetics and mechanism of NHE-1 regulation by osmotic shocks. We show that they modify the L0 parameter (865 +/- 95 and 3757 +/- 328 for 500 and 100 mOsM, respectively, vs 1549 +/- 57 in isotonic conditions).This results in an activation of NHE-1 by hypertonic shocks and, conversely, in an inhibition by hypotonic media. Quantitatively, this modulation of L0 follows an exponential distribution relative to osmolarity, that is, additive to the activation of NHE-1 by intracellular signaling pathways. These effects can be mimicked by the asymmetric insertion of amphiphilic molecules into the lipid bilayer. Finally, site-directed mutagenesis of NHE-1 shows that neither its association with membrane PIP2 nor its interaction with cortical actin are required for mechanosensation. In conclusion, NHE-1 allosteric equilibrium and, thus, its cooperative response to intracellular acidifications is extremely sensitive to modification of its membrane environment.

Published
2008

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