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23 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. 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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6. 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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7. 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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8. An unusual community-acquired invasive and multi systemic infection due to ExoU-harboring Pseudomonas aeruginosa strain: Clinical disease and microbiological characteristics

Author: Muriel Fartoukh, Guillaume Voiriot, Michel Djibré, Charlotte Verdet, Stéphane Pont, François Cretin, Alexandre Elabbadi, Patrick Plésiat, CHU Tenon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), 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), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Service de réanimation médicale et infectieuse, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), Service de Réanimation et USC Médico-Chirurgicale [CHU Tenon], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), 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), Service des soins intensifs [CHU Tenon], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Tenon [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de Réanimation médico-chirurgicale [CHU Tenon], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Tenon [APHP], 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)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Hôpital Jean Minjoz, Centre Hospitalier Régional Universitaire [Besançon] (CHRU Besançon), Université Paris Diderot - Paris 7 (UPD7)-AP-HP - Hôpital Bichat - Claude Bernard [Paris]-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), and Service de réanimation et USC médico-chirurgicale [CHU Tenon]
Subjects: Male, 0301 basic medicine, Microbiology (medical), medicine.medical_treatment, 030106 microbiology, lcsh:QR1-502, Virulence, medicine.disease_cause, lcsh:Microbiology, Microbiology, 03 medical and health sciences, 0302 clinical medicine, Pseudomonas aeruginosa Infections, Bacterial Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Sepsis, medicine, Humans, Immunology and Allergy, Pseudomonas Infections, 030212 general & internal medicine, Invasive infection, Lung, General Immunology and Microbiology, Strain (chemistry), Pseudomonas aeruginosa, business.industry, Immunosuppression, General Medicine, Middle Aged, Clinical disease, Shock, Septic, Systemic Inflammatory Response Syndrome, 3. Good health, Community-Acquired Infections, Infectious Diseases, medicine.anatomical_structure, A549 Cells, business, Immunocompetence
Abstract: International audience; Community-acquired Pseudomonas aeruginosa infections are rare. Most cases involve patients either with underlying immunosuppression or structural chronic lung diseases. We report here an atypical case of a severe community-acquired invasive infection due to a hypervirulent ExoU-producing strain, in an immunocompetent patient.
Published: 2019
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9. 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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10. 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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11. Corrigendum: Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach

Author: Emeline Reboud, Guillaume Golovkine, 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), and Huber, Philippe
Subjects: 0301 basic medicine, Microbiology (medical), endothelium, Immunology, lcsh:QR1-502, bacterial virulence factors, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Microbiology, Cell junction, lcsh:Microbiology, 03 medical and health sciences, Multi target, bacterial secretion systems, medicine, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS, Chemistry, Pseudomonas aeruginosa, bacterial invasion, Epithelium, Cell biology, 030104 developmental biology, Infectious Diseases, medicine.anatomical_structure, intercellular junctions, epithelium
Abstract: International audience
Published: 2018
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12. 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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13. Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach

Author: Emeline Reboud, Philippe Huber, Guillaume Golovkine, 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 Huber, Philippe
Subjects: 0301 basic medicine, Microbiology (medical), endothelium, Endothelium, Mini Review, Immunology, lcsh:QR1-502, Virulence, bacterial virulence factors, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, medicine.disease_cause, Microbiology, Cell junction, lcsh:Microbiology, 03 medical and health sciences, Opportunistic pathogen, Multi target, bacterial secretion systems, medicine, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS, Pseudomonas aeruginosa, Correction, bacterial invasion, Epithelium, 030104 developmental biology, Infectious Diseases, medicine.anatomical_structure, intercellular junctions, epithelium
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen which uses a number of strategies to cross epithelial and endothelial barriers at cell–cell junctions. In this review, we describe how the coordinated actions of P. aeruginosa's virulence factors trigger various molecular mechanisms to disarm the junctional gate responsible for tissue integrity.
Published: 2018
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14. 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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15. 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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16. 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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17. Host cell surfaces induce a Type IV pili-dependent alteration of bacterial swimming

Author: Laurence Lemelle, Claire Burny, Philippe Huber, Guillaume Golovkine, Cédric Vaillant, Jean-François Palierne, Christophe Place, 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 de Sciences de la Terre (LST), Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon), Laboratoire Joliot Curie, École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique de l'ENS Lyon (Phys-ENS), École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure de Lyon (ENS de Lyon)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Huber, Philippe, and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Subjects: 0301 basic medicine, 030106 microbiology, Fimbria, Microbial metabolism, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Article, Virulence factor, Pilus, 03 medical and health sciences, Human Umbilical Vein Endothelial Cells, medicine, Humans, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, biology, Pseudomonas aeruginosa, Pathogenic bacteria, biology.organism_classification, Cell biology, On cells, 030104 developmental biology, Fimbriae, Bacterial, Host-Pathogen Interactions, human activities, Bacteria
Abstract: For most pathogenic bacteria, flagellar motility is recognized as a virulence factor. Here, we analysed the swimming behaviour of bacteria close to eukaryotic cellular surfaces, using the major opportunistic pathogen Pseudomonas aeruginosa as a model. We delineated three classes of swimming trajectories on both cellular surfaces and glass that could be differentiated by their speeds and local curvatures, resulting from different levels of hydrodynamic interactions with the surface. Segmentation of the trajectories into linear and curved sections or pause allowed us to precisely describe the corresponding swimming patterns near the two surfaces. We concluded that (i) the trajectory classes were of same nature on cells and glass, however the trajectory distribution was strikingly different between surface types, (ii) on cell monolayers, a larger fraction of bacteria adopted a swimming mode with stronger bacteria-surface interaction mostly dependent upon Type IV pili. Thus, bacteria swim near boundaries with diverse patterns and importantly, Type IV pili differentially influence swimming near cellular and abiotic surfaces.
Published: 2016
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18. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion

Author: Guillaume Golovkine, Eric Faudry, Philippe Huber, Sylvie Elsen, Stéphanie Bouillot, 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), CEA Nanobio, ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Huber, Philippe, and Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID
Subjects: 0301 basic medicine, Cell division, QH301-705.5, Immunology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Flagellum, Biology, Transfection, medicine.disease_cause, Microbiology, Cell junction, Cell Line, Madin Darby Canine Kidney Cells, Type three secretion system, 03 medical and health sciences, Dogs, Virology, Genetics, medicine, Animals, Humans, Pseudomonas Infections, Biology (General), Cytoskeleton, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Cellular Senescence, Microscopy, Confocal, 030102 biochemistry & molecular biology, Pseudomonas aeruginosa, Epithelial Cells, RC581-607, Immunohistochemistry, Epithelium, Cell biology, Intercellular Junctions, 030104 developmental biology, medicine.anatomical_structure, Parasitology, Immunologic diseases. Allergy, Cell aging, Cell Division, Research Article
Abstract: To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment., Author Summary In normal situations, the mucosae constitute efficient barriers against the invasion of opportunistic pathogens. The bacteria inducing nosocomial infections take advantage of pre-existing pathological situations to cross the epithelium and spread in deeper tissues. The conditions on the host side permitting transmigration and the combination of virulence factors used by the bacteria to transmigrate are mostly speculative. Here, we studied the transmigration process of Pseudomonas aeruginosa, a bacterium causing hospital-acquired acute and chronic infections. We found that bacteria exploits weakened cell-cell junctions of the epithelium, such as those generated at sites of cell division or when dying cells are extruded from the cell layer, to breach the cell layer, using specific virulence factors and motility appendages.
Published: 2016
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19. 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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20. Pharmacological Activation of Rap1 Antagonizes the Endothelial Barrier Disruption Induced by Exotoxins ExoS and ExoT of Pseudomonas aeruginosa

Author: Ina Attrée, Stéphanie Bouillot, 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), and Huber, Philippe
Subjects: medicine.medical_treatment, Bacterial Toxins, Telomere-Binding Proteins, Immunology, Enzyme Activators, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, medicine.disease_cause, Microbiology, Shelterin Complex, Type three secretion system, chemistry.chemical_compound, medicine, Humans, Protein kinase A, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, ADP Ribose Transferases, Cellular Microbiology: Pathogen-Host Cell Molecular Interactions, Forskolin, Activator (genetics), Effector, Pseudomonas aeruginosa, Colforsin, GTPase-Activating Proteins, Endothelial Cells, 3. Good health, Infectious Diseases, Cytokine, chemistry, Parasitology, Rap1
Abstract: Most clinical strains of Pseudomonas aeruginosa , a leading agent of nosocomial infections, are multiresistant to antibiotherapy. Because of the paucity of new available antibiotics, the investigation of strategies aimed at limiting the action of its major virulence factors has gained much interest. The type 3 secretion system of P. aeruginosa and its effectors are known to be major determinants of toxicity and are required for bacterial dissemination in the host. Bacterial transmigration across the vascular wall is considered to be an important step in the infectious process. Using human endothelial primary cells, we demonstrate that forskolin (FSK), a drug inducing cyclic AMP (cAMP) elevation in eukaryotic cells, strikingly reduced the cell retraction provoked by two type 3 toxins, ExoS and ExoT, found in the majority of clinical strains. Conversely, cytotoxicity of a strain carrying the type 3 effector ExoU was unaffected by FSK. In addition, FSK altered the capacity of two ExoS/ExoT strains to transmigrate across cell monolayers. In agreement with these findings, other drugs and a cytokine inducing the increase of cAMP intracellular levels have also protected cells from retraction. cAMP is an activator of both protein kinase A and EPAC, a GTPase exchange factor of Rap1. Using activators or inhibitors of either pathway, we show that the beneficial effect of FSK is exerted by the activation of the EPAC/Rap1 axis, suggesting that its protective effect is mediated by reinforcing cell-cell and cell-substrate adhesion.
Published: 2015
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21. Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis

Author: Arnaud Ducruix, Martin Picard, Yann Ferrandez, Houssain Benabdelhak, Pierre Falson, Philippe Benas, Gilles Phan, Nathalie Ulryck, Isabelle Broutin, Laura Monlezun, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-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), 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 de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'enzymologie et biochimie structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA), and broutin, isabelle
Subjects: [SDV]Life Sciences [q-bio], Clinical Biochemistry, Trimer, Palmitic Acids, medicine.disease_cause, Biochemistry, Analytical Chemistry, 03 medical and health sciences, medicine, Escherichia coli, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Pseudomonas aeruginosa, 030302 biochemistry & molecular biology, Membrane Transport Proteins, Native Polyacrylamide Gel Electrophoresis, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Drug Resistance, Multiple, 3. Good health, Multiple drug resistance, [SDV] Life Sciences [q-bio], Multiprotein Complexes, Electrophoresis, Polyacrylamide Gel, Efflux, Bacteria, Stoichiometry, Bacterial Outer Membrane Proteins, Protein Binding
Abstract: Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that β-octyl glucopyranoside (β-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.
Published: 2012
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22. Crystallization and preliminary X-ray analysis of Weissella viridescens FemX UDP-MurNAc-pentapeptide:l-alanine ligase

Author: Wladimir Sougakoff, Sabrina Biarrotte-Sorin, Didier Blanot, Jean Delettré, Michel Arthur, Antoine P. Maillard, Jean-Emmanuel Hugonnet, Claudine Mayer, Karine Blondeau, 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 de minéralogie, cristallographie de Paris (LMCP), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC), Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut 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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV]Life Sciences [q-bio], chemistry.chemical_element, Biology, medicine.disease_cause, Crystallography, X-Ray, 03 medical and health sciences, chemistry.chemical_compound, X-Ray Diffraction, Structural Biology, medicine, Escherichia coli, Peptide Synthases, 030304 developmental biology, MESH: Crystallization, chemistry.chemical_classification, Alanine, 0303 health sciences, DNA ligase, Bacteria, 030306 microbiology, MESH: Escherichia coli, Resolution (electron density), MESH: X-Ray Diffraction, MESH: Chromatography, Gel, General Medicine, MESH: Crystallography, X-Ray, MESH: Peptide Synthases, Matrix-assisted laser desorption/ionization, Crystallography, MESH: Bacteria, Enzyme, chemistry, MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Chromatography, Gel, Peptidoglycan, Crystallization, Selenium
Abstract: International audience; Synthesis of the cell-wall peptidoglycan of firmicutes involves a unique family of peptide-bond-forming enzymes that use amino-acyl-tRNAs as substrates and are referred to as Fem proteins as they are factors essential for methicillin resistance in Staphylococcus aureus. The FemX UDP-MurNAc-pentapeptide:l-alanine ligase of Weissella viridescens was overexpressed, purified and crystallized. Native data were collected to 1.7 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.03, b = 99.92, c = 45.84 A, beta = 116.02 degrees. The asymmetric unit contains one molecule. A selenium-derivative data set has been collected to 2.1 A resolution at the peak wavelength of the selenium absorption edge. Six strong selenium positions were visible in the anomalous Patterson map. Three additional weaker Se atoms have been identified by anomalous Fourier synthesis.
Published: 2002
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23. 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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23 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. Inflammasome activation by <scp> Pseudomonas aeruginosa ' </scp> s <scp>ExlA</scp> pore‐forming toxin is detrimental for the host

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

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

8. An unusual community-acquired invasive and multi systemic infection due to ExoU-harboring Pseudomonas aeruginosa strain: Clinical disease and microbiological characteristics

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

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

11. Corrigendum: Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach

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

13. Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach

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

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

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

17. Host cell surfaces induce a Type IV pili-dependent alteration of bacterial swimming

18. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion

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

20. Pharmacological Activation of Rap1 Antagonizes the Endothelial Barrier Disruption Induced by Exotoxins ExoS and ExoT of Pseudomonas aeruginosa

21. Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis

22. Crystallization and preliminary X-ray analysis of Weissella viridescens FemX UDP-MurNAc-pentapeptide:l-alanine ligase

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

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