Author: "Pathogenèse bactérienne et réponses cellulaires (PBRC)" / Topic: [sdv.mp.bac]life sciences [q-bio]/microbiology and parasitology/bacteriology - Searchworks@Jio Institute Digital Library Search Results

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27 results on '"Pathogenèse bactérienne et réponses cellulaires (PBRC)"'

1. Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa

Author: Paul Briaud, Sylvie Elsen, Karen Moreau, Anne Doléans-Jordheim, François Vandenesch, Sylvère Bastien, Laura Camus, Pathogénie des Staphylocoques – Staphylococcal Pathogenesis (StaPath), 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), 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), Institut des Agents Infectieux [Lyon] (IAI), Hospices Civils de Lyon (HCL), Elsen, Sylvie, 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Staphylococcus aureus, Cystic Fibrosis, Context (language use), intercation, medicine.disease_cause, Microbiology, Cystic fibrosis, Article, Transcriptome, 03 medical and health sciences, chemistry.chemical_compound, medicine, Bacteriology, Humans, Pseudomonas Infections, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Pseudomonas aeruginosa, Catabolism, Acetoin, coexistence, Staphylococcal Infections, biology.organism_classification, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, chemistry, acetoin catabolism, Biofilms, Microbial Interactions, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacteria
Abstract: In the context of infection, Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated, particularly in cystic fibrosis (CF) patients. Within lungs, the two pathogens exhibit a range of competitive and coexisting interactions. In the present study, we explored the impact of S. aureus on the physiology of P. aeruginosa in the context of coexistence. Transcriptomic analyses showed that S. aureus significantly and specifically affects the expression of numerous genes involved in P. aeruginosa carbon and amino acid metabolism. In particular, 65% of the strains presented considerable overexpression of the genes involved in the acetoin catabolic (aco) pathway. We demonstrated that acetoin is (i) produced by clinical S. aureus strains, (ii) detected in sputa from CF patients, and (iii) involved in P. aeruginosa’s aco system induction. Furthermore, acetoin is catabolized by P. aeruginosa, a metabolic process that improves the survival of both pathogens by providing a new carbon source for P. aeruginosa and avoiding the toxic accumulation of acetoin on S. aureus. Due to its beneficial effects on both bacteria, acetoin catabolism could testify to the establishment of trophic cooperation between S. aureus and P. aeruginosa in the CF lung environment, thus promoting their persistence.
Published: 2020
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2. Chimeric Protein–Protein Interface Inhibitors Allow Efficient Inhibition of Type III Secretion Machinery and Pseudomonas aeruginosa Virulence

Author: Rossimiriam Pereira de Freitas, Yung-Sing Wong, Aline Thomas, Sophie Plé, Younes Bouzidi, Antoine Fortuné, Caroline Barette, Flaviane Francisco Hilário, Ina Attrée, Marie-Odile Fauvarque, Tuan-Dung Ngo, Eric Faudry, 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), Département de pharmacochimie moléculaire (DPM ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Universidade Federal de Minas Gerais [Belo Horizonte] (UFMG), Universidade Federal de Ouro Preto (UFOP), Associations 'Vaincre la mucoviscidose' and 'Gregory Lemarchand', AVIESAN T3SS (ANR PRP1.4), GIS-IBiSA and ChemBioFrance, Froggy platform of the CIMENT infrastructure (https://ciment.ujf-grenoble.fr), which is supported by the Rhône-Alpes region (GRANT CPER07_13 CIRA), 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), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-LABX-0056,OSUG@2020,Innovative strategies for observing and modelling natural systems(2010), ANR-10-EQPX-0029,EQUIP@MESO,Equipement d'excellence de calcul intensif de Mesocentres coordonnés - Tremplin vers le calcul petaflopique et l'exascale(2010), Faudry, Eric, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, Pneumonie hémorragique à Pseudomonas aeruginosa : étude de nouvelles stratégies de virulence - - HemoPseudo2015 - ANR-15-CE11-0018 - AAPG2015 - VALID, Grenoble, une chimie bio-motivée - - ARCANE2011 - ANR-11-LABX-0003 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Innovative strategies for observing and modelling natural systems - - OSUG@20202010 - ANR-10-LABX-0056 - LABX - VALID, Equipements d'excellence - Equipement d'excellence de calcul intensif de Mesocentres coordonnés - Tremplin vers le calcul petaflopique et l'exascale - - EQUIP@MESO2010 - ANR-10-EQPX-0029 - EQPX - VALID, 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], Protein subunit, 030106 microbiology, Virulence, hybrid molecules, medicine.disease_cause, Type three secretion system, Microbiology, 03 medical and health sciences, medicine, antivirulence, Secretion, biology, protein−protein interface inhibitors, Chemistry, Toxin, Pseudomonas aeruginosa, bacterial resistance, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Fusion protein, Type III secretion system, 030104 developmental biology, Infectious Diseases, click chemistry, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacteria
Abstract: International audience; Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen naturally resistant to many common antibiotics and acquires new resistance traits at an alarming pace. Targeting the bacterial virulence factors by an antivirulence strategy, therefore, represents a promising alternative approach besides antibiotic therapy. The Type III secretion system (T3SS) of P. aeruginosa is one of its main virulence factors. It consists of more than 20 proteins building a complex syringe-like machinery enabling the injection of toxin into host cells. Previous works showed that disrupting interactions between components of this machinery efficiently lowers the bacterial virulence. Using automated target-based screening of commercial and in-house libraries of small molecules, we identified compounds inhibiting the protein-protein interaction between PscE and PscG, the two cognate chaperones of the needle subunit PscF of P. aeruginosa T3SS. Two hits were selected and assembled using Split/Mix/Click chemistry to build larger hybrid analogues. Their efficacy and toxicity were evaluated using phenotypic analysis including automated microscopy and image analysis. Two nontoxic hybrid leads specifically inhibited the T3SS and reduced the ex vivo cytotoxicity of bacteria and their virulence in Galleria mellonella.
Published: 2019
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3. MagC is a NplC/P60‐like member of the $\alpha$‐2‐macroglobulin Mag complex of $Pseudomonas\ aeruginosa$ that interacts with peptidoglycan

Author: Ina Attrée, Andréa Dessen, Daniel M. Trindade, Carlos Contreras-Martel, Pauline Macheboeuf, Samira Zouhir, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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), Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), 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)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Groupe Pathogenèse Bactérienne et Réponses Cellulaires / Bacterial Pathogenesis and Cellular Responses Group (IBS-PBRC), 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)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Operon, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Biophysics, Peptidoglycan, Calorimetry, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Genetics, medicine, Amino Acid Sequence, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Protease, Sequence Homology, Amino Acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Pseudomonas aeruginosa, 030302 biochemistry & molecular biology, Isothermal titration calorimetry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Pregnancy-Associated alpha 2-Macroglobulins, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Macroglobulin, chemistry, Macroglobulins, Ultracentrifugation, Function (biology), Protein Binding
Abstract: International audience; Bacterial $\alpha$-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In $Pseudomonas\ aeruginosa$, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.
Published: 2021
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4. 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
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5. 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
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6. The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates in Pseudomonas aeruginosa

Author: Michel Ragno, Charles M. Stopford, Eric Faudry, Arne Rietsch, Alexandra Kraut, Caroline Perdu, Bakhos Jneid, Tuan Dung Ngo, Yohann Couté, Julia Novion Ducassou, Ina Attrée, Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-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 (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)-Institut National de la Santé et de la Recherche Médicale (INSERM), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), 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), Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, ANR PRP1.4 AVIESAN T3SS, Association Vaincre la Mucoviscidose, Association Grégory Lemarchal, 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), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Virulence Factors, ATPase, Microscale thermophoresis, Type three secretion system, Protein–protein interaction, Substrate Specificity, 03 medical and health sciences, 0302 clinical medicine, Bacterial Proteins, Structural Biology, Type III Secretion Systems, Bacteria pathogenesis, Secretion, Protein Interaction Maps, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, 030304 developmental biology, Adenosine Triphosphatases, 0303 health sciences, Protein-Protein interaction, biology, Mass spectrometry, Chemistry, Effector, Type 3 Secretion System, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Cytoplasm, Chaperone (protein), Pseudomonas aeruginosa, biology.protein, 030217 neurology & neurosurgery, Molecular Chaperones
Abstract: International audience; Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa .
Published: 2020
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7. Inflammasome activation by <scp> Pseudomonas aeruginosa ' </scp> s <scp>ExlA</scp> pore‐forming toxin is detrimental for the host

Author: Benoit Gallet, Vincent Deruelle, Stéphanie Bouillot, Philippe Huber, Ina Attrée, Stéphane Pont, Christine Moriscot, 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 de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Lipopolysaccharide, Inflammasomes, Interleukin-1beta, Immunology, Virulence, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, medicine.disease_cause, Microbiology, Mice, 03 medical and health sciences, chemistry.chemical_compound, Leukocidins, Interleukin-1alpha, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Virology, Type III Secretion Systems, medicine, Animals, Pseudomonas Infections, Secretion, Lung, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Inflammation, 0303 health sciences, Pore-forming toxin, 030306 microbiology, Pseudomonas aeruginosa, Toxin, Macrophages, Pyroptosis, Receptors, Interleukin-1, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Inflammasome, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Peptide Fragments, Neutrophil Infiltration, chemistry, Cytokines, medicine.drug
Abstract: During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase-1, which in turn triggers macrophage pyroptosis and IL-1β/IL-18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore-forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase-11 activation. Surprisingly, previous studies indicated that a T3SS-induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS-negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore-forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll-like receptors, and thus enhanced the expression of inflammatory proteins including pro-IL-1β and TNF-α. However, mature-IL-1β and IL-18 were undetectable in wild-type mice, suggesting that ExlA failed to effectively activate caspase-1. Nevertheless, caspase-1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA-induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome-dependent process.
Published: 2020
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8. Species-specific recruitment of transcription factors dictates toxin expression

Author: Trouillon, Julian, Sentausa, Erwin, Ragno, Michel, Robert-Genthon, Mylène, Lory, Stephen, Attrée, Ina, Elsen, Sylvie, 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), Harvard Medical School [Boston] (HMS), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and ATTREE, Ina
Subjects: Base Sequence, Virulence, Gene regulation, Chromatin and Epigenetics, Bacterial Toxins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Gene Expression Regulation, Bacterial, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Repressor Proteins, Bacterial Proteins, Species Specificity, A549 Cells, Pseudomonas, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Operon, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Promoter Regions, Genetic, ComputingMilieux_MISCELLANEOUS, Protein Binding, Transcription Factors
Abstract: Tight and coordinate regulation of virulence determinants is essential for bacterial biology and involves dynamic shaping of transcriptional regulatory networks during evolution. The horizontally transferred two-partner secretion system ExlB–ExlA is instrumental in the virulence of different Pseudomonas species, ranging from soil- and plant-dwelling biocontrol agents to the major human pathogen Pseudomonas aeruginosa. Here, we identify a Cro/CI-like repressor, named ErfA, which together with Vfr, a CRP-like activator, controls exlBA expression in P. aeruginosa. The characterization of ErfA regulon across P. aeruginosa subfamilies revealed a second conserved target, the ergAB operon, with functions unrelated to virulence. To gain insights into this functional dichotomy, we defined the pan-regulon of ErfA in several Pseudomonas species and found ergAB as the sole conserved target of ErfA. The analysis of 446 exlBA promoter sequences from all exlBA+ genomes revealed a wide variety of regulatory sequences, as ErfA- and Vfr-binding sites were found to have evolved specifically in P. aeruginosa and nearly each species carries different regulatory sequences for this operon. We propose that the emergence of different regulatory cis-elements in the promoters of horizontally transferred genes is an example of plasticity of regulatory networks evolving to provide an adapted response in each individual niche.
Published: 2020
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9. The human NAIP-NLRC4-inflammasome senses the Pseudomonas aeruginosa T3SS inner-rod protein

Author: Marion Thepaut, Laura Monlezun, B. Guery, Eric Faudry, Eric Kipnis, Rodrigue Dessein, Teddy Grandjean, Anne Boucher, Université de Lille, 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), and Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV)
Subjects: 0301 basic medicine, Inflammasomes, THP-1 Cells, Interleukin-1beta, medicine.disease_cause, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Type three secretion system, Mice, 0302 clinical medicine, NLRC4, Type III Secretion Systems, Immunology and Allergy, Toll-like receptor, biology, Chemistry, Caspase 1, NOD-like receptor, Inflammasome, General Medicine, Neuronal Apoptosis-Inhibitory Protein, Innate Immunity, Salmonella enterica, Pseudomonas aeruginosa, Intercellular Signaling Peptides and Proteins, medicine.drug, Immunology, Microbiology, 03 medical and health sciences, Pathogen-associated microbial pattern, medicine, Animals, Humans, Pseudomonas Infections, Nod-like receptor, Macrophages, Calcium-Binding Proteins, Pathogen-Associated Molecular Pattern Molecules, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immunity, Innate, Toll-Like Receptor 2, CARD Signaling Adaptor Proteins, Toll-Like Receptor 4, 030104 developmental biology, bacteria, NAIP, Carrier Proteins, 030215 immunology
Abstract: While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIP-NLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1β maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components.
Published: 2017
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10. Baseplate Component TssK and Spatio-Temporal Assembly of T6SS in Pseudomonas aeruginosa

Author: David Liebl, Mylène Robert-Genthon, Viviana Job, Valentina Cogoni, Ina Attrée, 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), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Microbiology (medical), molecular nanomachine, Cell, Phosphatase, lcsh:QR1-502, baseplate assembly, T4 bacteriophage, medicine.disease_cause, Microbiology, lcsh:Microbiology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Inner membrane, PPPA, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Type VI secretion system, Original Research, 0303 health sciences, biology, 030306 microbiology, Pseudomonas aeruginosa, Chemistry, Kinase, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, medicine.anatomical_structure, T6SS, bacterial competition, Bacteria
Abstract: The Gram-negative bacteria use the contractile multi-molecular structure, called the Type VI Secretion System (T6SS) to inject toxic products into eukaryotic and prokaryotic cells. In this study, we use fluorescent protein fusions and time-lapse microscopy imaging to study the assembly dynamics of the baseplate protein TssK in Pseudomonas aeruginosa T6SS. TssK formed transient higher-order structures that correlated with dynamics of sheath component TssB. Assembly of peri-membrane TssK structures occurred de novo upon contact with competing bacteria. We show that this assembly required presence of TagQ-TagR envelope sensors, activity of PpkA kinase and anchoring to the inner membrane via TssM. Disassembly and repositioning of TssK component was dependent on PppA phosphatase and indispensable for repositioning and deployment of the entire contractile apparatus toward a new target cell. We also show that TssE is necessary for correct elongation and stability of TssB-sheath, but not for TssK assembly. Therefore, in P. aeruginosa, assembly of the TssK-containing structure relays on the post-translational regulatory envelope module and acts as spatio-temporal marker for further recruitment and subsequent assembly of the contractile apparatus.
Published: 2019
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11. Stories from the wall: Envelope-associated functions in bacteria. Focus on cell wall synthesis, protein secretion & signal transduction machineries

Author: Maillard, Antoine P., MAILLARD, Antoine, Groupe Pathogenèse Bactérienne et Réponses Cellulaires / Bacterial Pathogenesis and Cellular Responses Group (IBS-PBRC), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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)-Centre National de la Recherche Scientifique (CNRS)-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), Communauté Université Grenoble Alpes, Christelle Breton, 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), 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), 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: Structure and function, Paroi bactérienne, Relations structure fonction, Facteurs sigma ECF, SecYEG-DF-YajC, Bacterial cell wall, Signalisation transmembranaire, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacterial envelope, Cobalt nickel resistance, Transmembrane signaling, Factor essential for expression of methicillin resistance, Enveloppe bactérienne, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein secretion, Exolysin, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Sécrétion protéique, Sigma factors
Published: 2019

12. Insertion sequences drive the emergence of a highly adapted human pathogen

Author: Pauline Basso, Ina Attree, Gwendoline Bellement, Sylvie Elsen, Yohann Couté, Annie Adrait, Stephen Lory, Alice Berry, Erwin Sentausa, 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), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Harvard Medical School [Boston] (HMS), 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: Proteomics, antibiotic resistance, Virulence Factors, Virulence, Human pathogen, Hemorrhage, adaptation, Moths, medicine.disease_cause, Microbiology, Transcriptome, 03 medical and health sciences, Antibiotic resistance, Bacterial Proteins, medicine, Animals, Humans, Pseudomonas Infections, Insertion sequence, Gene, Phylogeny, 030304 developmental biology, Genetics, 0303 health sciences, biology, Whole Genome Sequencing, 030306 microbiology, Pseudomonas aeruginosa, Gene Expression Profiling, bacterial infection, General Medicine, Gene Expression Regulation, Bacterial, Genomics, Pneumonia, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Microbe-niche Interactions: Host Adaptation, Disease Models, Animal, DNA Transposable Elements, Mobile genetic elements, Bacteria, Genome, Bacterial, multiomics, Research Article
Abstract: Pseudomonas aeruginosa is a highly adaptive opportunistic pathogen that can have serious health consequences in patients with lung disorders. Taxonomic outliers of P. aeruginosa of environmental origin have recently emerged as infectious for humans. Here, we present the first genome-wide analysis of an isolate that caused fatal haemorrhagic pneumonia. In two clones, CLJ1 and CLJ3, sequentially recovered from a patient with chronic pulmonary disease, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to the antibiotics used to combat the infection. Comparative genome, proteome and transcriptome analyses revealed that this ISL3-family insertion sequence disrupted the genes for flagellar components, type IV pili, O-specific antigens, translesion polymerase and enzymes producing hydrogen cyanide. Seven-fold more insertions were detected in the later isolate, CLJ3, than in CLJ1, some of which modified strain susceptibility to antibiotics by disrupting the genes for the outer-membrane porin OprD and the regulator of β-lactamase expression AmpD. In the Galleria mellonella larvae model, the two strains displayed different levels of virulence, with CLJ1 being highly pathogenic. This study revealed insertion sequences to be major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both its virulence and its resistance to antimicrobials, and explains how this bacterium adapts from the environment to a human host.
Published: 2018
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13. cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

Author: Kook Han, Julian Trouillon, Stephen Lory, Mylène Robert-Genthon, Michel Ragno, Alice Berry, Ina Attrée, Sylvie Elsen, 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), Department of Microbiology and Immunology, and Harvard Medical School [Boston] (HMS)
Subjects: 0301 basic medicine, Cyclic AMP Receptor Protein, Operon, [SDV]Life Sciences [q-bio], Bacterial Toxins, 030106 microbiology, Mutant, Virulence, TPS, Biology, medicine.disease_cause, Microbiology, Virulence factor, Cell Line, Type three secretion system, exolysin, 03 medical and health sciences, Bacterial Proteins, cAMP, Cyclic AMP, medicine, Humans, Pseudomonas Infections, Secretion, Frameshift Mutation, Promoter Regions, Genetic, Vfr, Molecular Biology, Regulation of gene expression, Base Sequence, Pseudomonas aeruginosa, PA7 group, Gene Expression Regulation, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, gene regulation, Research Article, Adenylyl Cyclases
Abstract: The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.
Published: 2018
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14. Pore-forming activity of the $Pseudomonas\ aeruginosa$ type III secretion system translocon alters the host epigenome

Author: Charlotte Lombardi, Alain Filloux, Laurent Dortet, François Cretin, Andréa Dessen, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), 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), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Vaincre la Mucoviscidose RF20140501133, ANR project PRP1.4 T3SS, European Project: 654909,H2020,H2020-MSCA-IF-2014,T6SS-PSEUDO-LIP(2016), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Imperial College London, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Medical Research Council (MRC)
Subjects: 0301 basic medicine, EFFECTOR TARGETS, NF-KAPPA-B, Moths, medicine.disease_cause, Applied Microbiology and Biotechnology, Virulence factor, Epigenesis, Genetic, Type three secretion system, Histones, INFECTION, Type III Secretion Systems, MESH: Animals, MESH: Epigenesis, Genetic, PHOSPHORYLATION, MESH: Bacterial Proteins, Host cell membrane, MESH: Histones, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, MESH: Moths, EPITHELIAL-CELLS, Translocon, Cell biology, Histone Code, Larva, Host-Pathogen Interactions, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Life Sciences & Biomedicine, Microbiology (medical), GENES, Immunology, Microbiology, 03 medical and health sciences, MESH: Type III Secretion Systems, Bacterial Proteins, Genetics, medicine, Animals, Humans, Secretion, HISTONE H3, PROTEIN PHOSPHATASES, Antigens, Bacterial, Science & Technology, MESH: Humans, Cell Membrane, MESH: Host-Pathogen Interactions, Cell Biology, Epigenome, biochemical phenomena, metabolism, and nutrition, MAPK, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, MESH: Histone Code, A549 Cells, MESH: HeLa Cells, bacteria, MESH: A549 Cells, VI SECRETION, MESH: Larva, MESH: Antigens, Bacterial, HeLa Cells, MESH: Cell Membrane
Abstract: Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS ‘needle’ delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB–PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion. The pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon serves as a virulence factor to induce host epigenome modification and promote infection.
Published: 2018
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15. 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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16. 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
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17. Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

Author: Ina Attrée, Philippe Huber, Stéphanie Bouillot, Benoît Béganton, Emeline Reboud, Sabine Patot, 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), Pathogénie des Staphylocoques – Staphylococcal Pathogenesis (StaPath), 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), 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), 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)-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, Serratia, Cell, Cell Membranes, Toxicology, Pathology and Laboratory Medicine, Cell junction, Epithelium, Mice, Hemolysin Proteins, ADAM10 Protein, Animal Cells, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Medicine and Health Sciences, Toxins, lcsh:QH301-705.5, Inbred BALB C, Serratia marcescens, Mice, Inbred BALB C, Microscopy, Microscopy, Confocal, medicine.diagnostic_test, Virulence, Blotting, Cadherins, Transmembrane protein, Cell biology, Bacterial Pathogens, medicine.anatomical_structure, Medical Microbiology, Confocal, Pseudomonas aeruginosa, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, Cellular Structures and Organelles, Pathogens, Cellular Types, Anatomy, Western, Research Article, lcsh:Immunologic diseases. Allergy, Virulence Factors, Proteolysis, Immunology, Toxic Agents, Blotting, Western, Bacterial Toxins, Biology, Microbiology, 03 medical and health sciences, Enzyme activator, Enterobacteriaceae, Bacterial Proteins, Pseudomonas, Virology, Genetics, medicine, Cell Adhesion, Humans, Animals, Secretion, Molecular Biology, Microbial Pathogens, Bacteria, Cadherin, Host Cells, Organisms, Biology and Life Sciences, Endothelial Cells, Epithelial Cells, Cell Biology, Intracellular Membranes, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Enzyme Activation, Cytosol, 030104 developmental biology, Biological Tissue, lcsh:Biology (General), Parasitology, Calcium, lcsh:RC581-607, Gram-Negative Bacterial Infections, Viral Transmission and Infection
Abstract: Pore-forming toxins are potent virulence factors secreted by a large array of bacteria. Here, we deciphered the action of ExlA from Pseudomonas aeruginosa and ShlA from Serratia marcescens on host cell-cell junctions. ExlA and ShlA are two members of a unique family of pore-forming toxins secreted by a two-component secretion system. Bacteria secreting either toxin induced an ExlA- or ShlA-dependent rapid cleavage of E-cadherin and VE-cadherin in epithelial and endothelial cells, respectively. Cadherin proteolysis was executed by ADAM10, a host cell transmembrane metalloprotease. ADAM10 activation is controlled in the host cell by cytosolic Ca2+ concentration. We show that Ca2+ influx, induced by ExlA or ShlA pore formation in the plasma membrane, triggered ADAM10 activation, thereby leading to cadherin cleavage. Our data suggest that ADAM10 is not a cellular receptor for ExlA and ShlA, further confirming that ADAM10 activation occurred via Ca2+ signalling. In conclusion, ExlA- and ShlA-secreting bacteria subvert a regulation mechanism of ADAM10 to activate cadherin shedding, inducing intercellular junction rupture, cell rounding and loss of tissue barrier integrity., Author summary Pore-forming toxins are the most widespread toxins delivered by pathogenic bacteria and are required for full virulence. Pore-forming toxins perforate membranes of host cells for intracellular delivery of bacterial factors, for bacterial escape from phagosomes or in order to kill cells. Loss of membrane integrity, especially the plasma membrane, has broad implications on cell and tissue physiology. Here, we show that two members of a unique family of pore-forming toxins, secreted by Pseudomonas aeruginosa and Serratia marcescens, have the capacity to disrupt cell-cell junctions of epithelial and endothelial cells, hence breaching two major tissue barriers.
Published: 2017
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18. Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination

Author: François Cretin, Guillaume Golovkine, Emmanuel Lemichez, Philippe Huber, Benoit Gallet, Stéphanie Bouillot, Patrick Munro, Ina Attree, Guy Schoehn, Emeline Reboud, 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), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), This work was supported by institutional grants from the Commissariat à l’Energie Atomique, the Institut National de la Santé Et de la Recherche Médicale, the Université Grenoble-Alpes and the Centre National de la Recherche Scientifique, as well as the Laboratoire d’Excellence GRAL [grant number ANR-10-LABX-49-01 to ER] and SIGNALIFE [grant number ANR-11-LABX-28-01], Agence National de la Recherche [ANR-15-CE11-0018-01]. This work used the platforms of the Grenoble Instruct centre (ISBG, UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility is supported by the Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER, the Centre National de la Recherche Scientifique (CNRS), the Commissariat à l’Energie Atomique, the University of Grenoble, EMBL, and the GIS-Infrastrutures en Biologie Sante et Agronomie (IBISA)., Grenoble Instruct-ERIC Center (ISBG : UMS 3518 CNRS-CEA-UGA-EMBL), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-15-CE11-0018,HemoPseudo,Pneumonie hémorragique à Pseudomonas aeruginosa : étude de nouvelles stratégies de virulence(2015), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Huber, Philippe, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, Centres d'excellences - Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie - - SIGNALIFE2011 - ANR-11-LABX-0028 - LABX - VALID, Pneumonie hémorragique à Pseudomonas aeruginosa : étude de nouvelles stratégies de virulence - - HemoPseudo2015 - ANR-15-CE11-0018 - AAPG2015 - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), 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)
Subjects: 0301 basic medicine, Pore Forming Cytotoxic Proteins, Science, medicine.medical_treatment, Phagocytosis, Bacterial Toxins, MESH: Mice, Inbred BALB C, Bacteremia, Biology, medicine.disease_cause, MESH: Alveolar Epithelial Cells, Virulence factor, Article, Microbiology, Type three secretion system, 03 medical and health sciences, Mice, Immune system, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Animals, MESH: Animals, MESH: Bacteremia, MESH: Phagocytosis, MESH: Mice, Mice, Inbred BALB C, Multidisciplinary, Lung, MESH: Pore Forming Cytotoxic Proteins, Pseudomonas aeruginosa, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, 030104 developmental biology, Cytokine, medicine.anatomical_structure, MESH: Bacterial Toxins, Alveolar Epithelial Cells, MESH: Pseudomonas aeruginosa, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Medicine, Ectopic expression, Female, MESH: Female
Abstract: Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.
Published: 2017
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19. Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death

Author: Basso, Pauline, Wallet, Pierre, Elsen, Sylvie, Soleilhac, Emmanuelle, Henry, Thomas, Faudry, Eric, Attrée, Ina, 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), Inflammasome, Infections bactériennes et autoinflammation, Inflammasome, Bacterial Infections and Autoinflammation (I2BA), 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), Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre International de Recherche en Infectiologie - UMR (CIRI), 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), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), 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]), 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cell Death, Inflammasomes, Macrophages, Bacterial Toxins, Caspase 1, Interleukin-1beta, Apoptosis, Bone Marrow Cells, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Mice, Inbred C57BL, Mice, Bacterial Proteins, Pseudomonas, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Animals
Abstract: International audience; Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1β. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA+ P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages.
Published: 2017
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20. Defining Lipoprotein Localisation by Fluorescence Microscopy

Author: Didier Grunwald, Mylène Robert-Genthon, Ina Attrée, Maria Guillermina Casabona, 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), Pathogenèse bactérienne et réponses cellulaires (PBRC), 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), 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: 0301 basic medicine, Chemistry, fungi, 030106 microbiology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Fusion protein, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Green fluorescent protein, Transport protein, Cell biology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Fluorescence microscope, Secretion, Cell envelope, Bacterial outer membrane, mCherry, ComputingMilieux_MISCELLANEOUS
Abstract: In recent years it has become evident that lipoproteins play crucial roles in the assembly of bacterial envelope-embedded nanomachineries and in the processes of protein export/secretion. In this chapter we describe a method to determine their precise localisation, for example inner versus outer membrane, in Gram-negative bacteria using human opportunistic pathogen Pseudomonas aeruginosa as a model. A fusion protein between a given putative lipoprotein and the red fluorescent protein mCherry must be created and expressed in a strain expressing cytoplasmic green fluorescent protein (GFP). Then the peripheral localisation of the fusion protein in the cell can be examined by treating cells with lysozyme to create spheroplasts and monitoring fluorescence under a confocal microscope. Mutants in the signal peptide can be engineered to study the association with the membrane and efficiency of transport. This protocol can be adapted to monitor lipoprotein localisation in other Gram-negative bacteria.
Published: 2017
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21. Phenotype and toxicity of the recently discovered exlA -positive Pseudomonas aeruginosa strains collected worldwide

Author: Emeline, Reboud, Sylvie, Elsen, Stéphanie, Bouillot, Guillaume, Golovkine, Pauline, Basso, Katy, Jeannot, Ina, Attrée, Philippe, Huber, 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), Centre National de Référence de la Résistance aux Antibiotiques (CNR), Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), and Service de bactériologie [Besançon]
Subjects: Mice, Inbred BALB C, Virulence, Virulence Factors, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Chicory, Mice, Phenotype, Bacterial Proteins, Pseudomonas aeruginosa, Animals, Humans, Female, Pseudomonas Infections, Phylogeny
Abstract: International audience; We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.
Published: 2016
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22. ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

Author: Ariel J. Blocker, Stéphanie Bouillot, Caroline Perdu, Eric Faudry, Philippe Huber, Sylvie Elsen, Ina Attree, 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), University of Bristol [Bristol], IRTELIS Ph.D. program from CEA, and Faudry, Eric
Subjects: Male, Operon, Virulence Factors, Lipoproteins, Immunology, Virulence, Biology, medicine.disease_cause, Microbiology, Bacterial Proteins, medicine, Animals, Humans, Secretion, Pseudomonas Infections, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Bacterial Secretion Systems, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cells, Cultured, Regulator gene, Mice, Inbred BALB C, Pseudomonas aeruginosa, Endothelial Cells, Membrane Proteins, Molecular Pathogenesis, Survival Analysis, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Cell biology, Disease Models, Animal, Infectious Diseases, Membrane protein, Host cell cytoplasm, bacteria, Parasitology, Protein Multimerization, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacterial outer membrane
Abstract: Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa . Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.
Published: 2015
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23. VE-cadherin cleavage by LasB protease from Pseudomonas aeruginosa facilitates type III secretion system toxicity in endothelial cells

Author: Stéphanie Bouillot, Guillaume Golovkine, Ina Attree, Romé Voulhoux, Eric Faudry, Philippe Huber, 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), 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), This work was supported by grants from the Commissariat à l’Energie Atomique, the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique and Joseph Fourier University. Part of this work was also supported by the Alliance pour les sciences de la VIE et de la SANté program in infectiology and FINOVI foundation., Marinari, Isabelle, and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)
Subjects: Male, medicine.medical_treatment, Endothelial cells, Fluorescent Antibody Technique, Pathogenesis, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Mouse models, Type three secretion system, Mice, Molecular Cell Biology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Biology (General), Bacterial Secretion Systems, Cells, Cultured, Mice, Inbred BALB C, Tight junction, Metalloendopeptidases, Animal Models, Proteases, Cell disruption, Cadherins, Bacterial Pathogens, Cell biology, Host-Pathogen Interaction, medicine.anatomical_structure, Pseudomonas aeruginosa, Research Article, Endothelium, QH301-705.5, Blotting, Western, Immunology, Biology, Microbiology, Model Organisms, Secretion systems, Bacterial Proteins, Antigens, CD, Virology, Cell Adhesion, Genetics, medicine, Extracellular, Animals, Humans, Pseudomonas Infections, Secretion, Molecular Biology, Tight junctions, Protease, RC581-607, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Actins, Disease Models, Animal, Parasitology, Endothelium, Vascular, Immunologic diseases. Allergy, VE-cadherin, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: Infection of the vascular system by Pseudomonas aeruginosa (Pa) occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS) toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS) effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-typeswere sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE)-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349). VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E)-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells., Author Summary Pseudomonas aeruginosa (Pa) is a leading agent of nosocomial infections in humans, and clinical isolates are often multiresistant to antibiotics. As with most Gram-negative bacteria, Pa possesses a type III secretion system which consists of an injectisome through which the bacterium injects exotoxins inducing cytoskeleton collapse and apoptosis. Pa also delivers various toxins in the extracellular milieu by the type II secretion system, including the protease LasB. In order to disseminate throughout the body from the infection site and eventually reach the blood, the bacterium generally needs to cross the main barriers of the organism: the epithelium, the basal lamina and the vascular endothelium. Here we show that LasB specifically cleaves one main component of endothelial cell-to-cell junctions, the adhesive protein VE-cadherin, thus leading to junction disruption and endothelial barrier breakdown. VE-cadherin proteolysis also facilitates the action of type III exotoxins in endothelial cells. This cleavage mechanism is likely of major importance in Pa pathogenesis, as suggested by our bacterial dissemination experiments in mice.
Published: 2014
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24. Pore Formation by T3SS Translocators: Liposome Leakage Assay

Author: Caroline Perdu, Eric Faudry, Ina Attree, 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), 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: 0303 health sciences, Liposome, biology, 030302 biochemistry & molecular biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transport protein, Type three secretion system, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 03 medical and health sciences, chemistry.chemical_compound, Membrane, Dextran, chemistry, Cytoplasm, Chaperone (protein), biology.protein, Biophysics, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Guanidine, 030304 developmental biology
Abstract: International audience; Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore-the translocon-formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.
Published: 2013
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25. Current Fluctuation Analysis of the PopB and PopD Translocon Components of the Pseudomonas aeruginosa Type III Secretion System

Author: Ina Attree, Anne H. Delcour, Eric Faudry, Beau Wager, Tyler Wills, University of Houston, 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), and Faudry, Eric
Subjects: Lipid Bilayers, Biophysics, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Biology, Protein Refolding, Type three secretion system, 03 medical and health sciences, Mice, Bacterial Proteins, Animals, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Lipid bilayer, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Guanidine, 030304 developmental biology, Host cell membrane, 0303 health sciences, Antigens, Bacterial, 030306 microbiology, Macrophages, Osmolar Concentration, Membrane, Conductance, Membranes, Artificial, Hydrogen-Ion Concentration, Translocon, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Electrophysiological Phenomena, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Biochemistry, Ionic strength, Pseudomonas aeruginosa, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Porosity
Abstract: International audience; Pseudomonas aeruginosa is a major agent of hospital-acquired infections, and a pathogen of immunocompromised, cystic fibrosis and burn patients. It uses a type III secretion system for the injection of toxins directly into host cells, through a translocon assembled in the host cell membrane. The hydrophobic translocator subunits of this system, PopB and PopD, have membrane permeabilizing activity based on previous dye leakage experiments, but little is known about the mechanism of assembly and the pore properties of this translocon. Using electrophysiology, we have observed that an equimolar mixture of PopB and PopD induces current fluctuations in planar lipid bilayers, with a unitary conductance of 57 pS in 1 M KCl and numerous larger conductance levels. The activity depends on voltage magnitude and polarity, and increases with protein concentration and the duration of the voltage step. PopB alone is sufficient for producing current fluctuations. PopD rarely displays any transitions, but accelerates PopB onset of activity. The effects of pH, ionic strength, and lipid composition have also been explored. Our data provide new, to our knowledge, insights into the behavior of PopB and PopD by highlighting similarities with secreted pore-forming peptides, and by suggesting that PopB/PopD may form channels via the toroidal pore model. We believe that the events we report here represent the initial steps of insertion and assembly of these translocators in the membrane.
Published: 2013
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26. The V Antigen of Pseudomonas aeruginosa Is Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

Author: Jacqueline Chabert, Eric Faudry, Julien Goure, Alexandrine Pastor, Ina Attree, Andréa Dessen, 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), Groupe Pathogénie Bactérienne (IBS-PATBAC ), 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), 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)-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), Faudry, Eric, and Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Pore Forming Cytotoxic Proteins, Erythrocytes, Immunoprecipitation, Operon, Immunology, Mutant, Bacterial Toxins, Biology, Microbiology, Hemolysis, Green fluorescent protein, 03 medical and health sciences, Mice, Bacterial Proteins, medicine, Animals, Humans, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Actin, 030304 developmental biology, 0303 health sciences, Antigens, Bacterial, 030306 microbiology, Effector, Erythrocyte Membrane, Biological Transport, Gene Expression Regulation, Bacterial, medicine.disease, Molecular biology, Molecular Pathogenesis, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Infectious Diseases, Pseudomonas aeruginosa, NIH 3T3 Cells, Parasitology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.
Published: 2004
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27. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

Author: Diego Carriel, Julien Pérard, Walid A. Houry, Sandrine Ollagnier de Choudens, Sylvie Elsen, Eaazhisai Kandiah, Hélène Malet, Kaiyin Liu, Sze W. S. Chan, Irina Gutsche, Maria Bacia, 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), 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), 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), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Department of Biochemistry [University of Toronto], University of Toronto, Rhône-Alpes Region (CIBLE and FEDER), Fondation pour la Recherche Médicale (FRM), GIS-IBISA, CIHR MOP-130374, electron microscope facility (Polara electron microscope) is supported by the Rhône-Alpes Region (CIBLE and FEDER), Grenoble Alliance for Integrated Structural and Cell Biology (GRAL) PhD fellowship, Ontario Graduate Scholarship and the Life Sciences Award from the University of Toronto, National Sciences and Engineering Research Council of Canada (NSERC) Undergraduate Student Research Award, CIHR MOP-130374 to WA, ANR-12-JSV8-0002,acidstress,La réponse bactérienne au stress acide: analyse des relations structure-fonction du système de la décarboxylase à lysine(2012), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 647784,H2020,ERC-2014-CoG,Chap4Resp(2015), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-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), Institut de Chimie du CNRS (INC)-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)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Models, Molecular, 0301 basic medicine, Carboxy-Lyases, Lysine, Plasma protein binding, Biology, medicine.disease_cause, Article, 03 medical and health sciences, Enzyme activator, Catalytic Domain, Escherichia coli, medicine, Amino Acid Sequence, Peptide sequence, Adenosine Triphosphatases, Multidisciplinary, 030102 biochemistry & molecular biology, Lysine decarboxylase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Escherichia coli Proteins, Cryoelectron Microscopy, Hydrogen-Ion Concentration, Lyase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, AAA proteins, Enzyme Activation, 030104 developmental biology, Biochemistry, Protein Binding
Abstract: The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.
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27 results on '"Pathogenèse bactérienne et réponses cellulaires (PBRC)"'

1. Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa

2. Chimeric Protein–Protein Interface Inhibitors Allow Efficient Inhibition of Type III Secretion Machinery and Pseudomonas aeruginosa Virulence

3. MagC is a NplC/P60‐like member of the $\alpha$‐2‐macroglobulin Mag complex of $Pseudomonas\ aeruginosa$ that interacts with peptidoglycan

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

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

6. The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates in Pseudomonas aeruginosa

7. Inflammasome activation by <scp> Pseudomonas aeruginosa ' </scp> s <scp>ExlA</scp> pore‐forming toxin is detrimental for the host

8. Species-specific recruitment of transcription factors dictates toxin expression

9. The human NAIP-NLRC4-inflammasome senses the Pseudomonas aeruginosa T3SS inner-rod protein

10. Baseplate Component TssK and Spatio-Temporal Assembly of T6SS in Pseudomonas aeruginosa

11. Stories from the wall: Envelope-associated functions in bacteria. Focus on cell wall synthesis, protein secretion & signal transduction machineries

12. Insertion sequences drive the emergence of a highly adapted human pathogen

13. cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

14. Pore-forming activity of the $Pseudomonas\ aeruginosa$ type III secretion system translocon alters the host epigenome

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

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

17. Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

18. Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination

19. Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death

20. Defining Lipoprotein Localisation by Fluorescence Microscopy

21. Phenotype and toxicity of the recently discovered exlA -positive Pseudomonas aeruginosa strains collected worldwide

22. ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

23. VE-cadherin cleavage by LasB protease from Pseudomonas aeruginosa facilitates type III secretion system toxicity in endothelial cells

24. Pore Formation by T3SS Translocators: Liposome Leakage Assay

25. Current Fluctuation Analysis of the PopB and PopD Translocon Components of the Pseudomonas aeruginosa Type III Secretion System

26. The V Antigen of Pseudomonas aeruginosa Is Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

27. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

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