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1. Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Author: Serena Petracchini, Daniel Hamaoui, Anne Doye, Atef Asnacios, Florian Fage, Elisa Vitiello, Martial Balland, Sebastien Janel, Frank Lafont, Mukund Gupta, Benoit Ladoux, Jerôme Gilleron, Teresa M. Maia, Francis Impens, Laurent Gagnoux-Palacios, Mads Daugaard, Poul H. Sorensen, Emmanuel Lemichez, and Amel Mettouchi
Subjects: Science
Abstract: Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.
Published: 2022
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2. Correction: Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection.

Author: Flaviano S Martins, Guillaume Dalmasso, Rosa M E Arantes, Anne Doye, Emmanuel Lemichez, Patricia Lagadec, Veronique Imbert, Jean-François Peyron, Patrick Rampal, Jacques R Nicoli, and Dorota Czerucka
Subjects: Medicine, Science
Abstract: [This corrects the article DOI: 10.1371/journal.pone.0008925.].
Published: 2022
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3. Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation

Author: Caroline Stefani, David Gonzalez-Rodriguez, Yosuke Senju, Anne Doye, Nadia Efimova, Sébastien Janel, Justine Lipuma, Meng Chen Tsai, Daniel Hamaoui, Madhavi P. Maddugoda, Olivier Cochet-Escartin, Coline Prévost, Frank Lafont, Tatyana Svitkina, Pekka Lappalainen, Patricia Bassereau, and Emmanuel Lemichez
Subjects: Science
Abstract: Holes in endothelial barriers, called transendothelial cell macroapertures (TEMs), are predicted to be limited by line tension of unknown origin. Here the authors identify an actomyosin cable encircling TEMs and establish a role for ezrin in stabilising F-actin bundles, allowing their crosslinking by non-muscle myosin IIa.
Published: 2017
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4. EDIN-B Promotes the Translocation of Staphylococcus aureus to the Bloodstream in the Course of Pneumonia

Author: Johan Courjon, Patrick Munro, Yvonne Benito, Orane Visvikis, Coralie Bouchiat, Laurent Boyer, Anne Doye, Hubert Lepidi, Eric Ghigo, Jean-Philippe Lavigne, François Vandenesch, and Emmanuel Lemichez
Subjects: bacteremia, virulence factor, toxins, methicillin-resistant Staphylococcus aureus, Medicine
Abstract: It is crucial to define risk factors that contribute to host invasion by Staphylococcus aureus. Here, we demonstrate that the chromosomally encoded EDIN-B isoform from S. aureus contributes to the onset of bacteremia during the course of pneumonia. Deletion of edinB in a European lineage community-acquired methicillin resistant S. aureus (CA-MRSA) strain (ST80-MRSA-IV) dramatically decreased the frequency and magnitude of bacteremia in mice suffering from pneumonia. This deletion had no effect on the bacterial burden in both blood circulation and lung tissues. Re-expression of wild-type EDIN-B, unlike the catalytically inactive mutant EDIN-R185E, restored the invasive characteristics of ST80-MRSA-IV.
Published: 2015
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5. Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication.

Author: Onya Opota, Nils C Gauthier, Anne Doye, Colin Berry, Pierre Gounon, Emmanuel Lemichez, and David Pauron
Subjects: Medicine, Science
Abstract: Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.
Published: 2011
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6. Interaction of Saccharomyces boulardii with Salmonella enterica serovar Typhimurium protects mice and modifies T84 cell response to the infection.

Author: Flaviano S Martins, Guillaume Dalmasso, Rosa M E Arantes, Anne Doye, Emmanuel Lemichez, Patricia Lagadec, Veronique Imbert, Jean-François Peyron, Patrick Rampal, Jacques R Nicoli, and Dorota Czerucka
Subjects: Medicine, Science
Abstract: BACKGROUND: Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-kappaB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-kappaB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects. CONCLUSIONS: Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.
Published: 2010
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7. Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity

Author: Géraldine Gonfrier, Laurent Yvan-Charvet, Laurent Bailly, Julie Contenti, Christelle Pomares-Estran, David Chirio, Laurent Boyer, Céline Loubatier, Orane Visvikis, Michel Carles, Océane Dufies, Patrick Munro, Arnaud Jacquel, Cedric Torre, Sébastien Vitale, Anne Doye, Valérie Giordanengo, Johan Courjon, Stoyan Ivanov, Jean Dellamonica, Romain Lotte, Patrick Auberger, and Alexandre Robert
Subjects: 0301 basic medicine, Clinical Trials and Observations, Inflammasomes, Stimulation, 03 medical and health sciences, 0302 clinical medicine, Immune system, NLR Family, Pyrin Domain-Containing 3 Protein, medicine, Humans, Myeloid Cells, Prospective Studies, Prospective cohort study, Innate immune system, business.industry, SARS-CoV-2, Case-control study, COVID-19, Inflammasome, Hematology, Middle Aged, Peripheral, 030104 developmental biology, 030220 oncology & carcinogenesis, Case-Control Studies, Immunology, Biomarker (medicine), business, Biomarkers, medicine.drug
Abstract: Key Points Measurement of NLRP3 inflammasome activation in the blood of patients reveals an impaired immature neutrophil response in severe COVID-19. Inflammasome signature analysis in circulating myeloid cells allows COVID-19 patients to be stratified and predicts evolution., Dysregulated immune response is the key factor leading to unfavorable coronavirus disease 2019 (COVID-19) outcome. Depending on the pathogen-associated molecular pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing the NLRP3 response during severe acute respiratory syndrome coronavirus 2 infection in patients are lacking. We prospectively monitored caspase-1 activation levels in peripheral myeloid cells from healthy donors and patients with mild to critical COVID-19. The caspase-1 activation potential in response to NLRP3 inflammasome stimulation was opposed between nonclassical monocytes and CD66b+CD16dim granulocytes in severe and critical COVID-19 patients. Unexpectedly, the CD66b+CD16dim granulocytes had decreased nigericin-triggered caspase-1 activation potential associated with an increased percentage of NLRP3 inflammasome impaired immature neutrophils and a loss of eosinophils in the blood. In patients who recovered from COVID-19, nigericin-triggered caspase-1 activation potential in CD66b+CD16dim cells was restored and the proportion of immature neutrophils was similar to control. Here, we reveal that NLRP3 inflammasome activation potential differs among myeloid cells and could be used as a biomarker of a COVID-19 patient’s evolution. This assay could be a useful tool to predict patient outcome. This trial was registered at www.clinicaltrials.gov as #NCT04385017., Visual Abstract
Published: 2021

8. Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Author: Laurent Gagnoux, Serena Petracchini, Benoit Ladoux, Amel Mettouchi, Martial Balland, Mads Daugaard, Mukund Gupta, Emmanuel Lemichez, Francis Impens, Poul H. Sorensen, Sébastien Janel, Elisa Vitiello, Anne Doye, Frank Lafont, Florian Fage, Atef Asnacios, Teresa Mendes Maia, Daniel Hamaoui, Jerome Gilleron, Centre méditerranéen de médecine moléculaire (C3M), 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), Equipe labellisée Ligue contre le Cancer, Matière et Systèmes Complexes (MSC), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Cellular Microbiology and Physics of Infection Group [Lille] (CMPI), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), Universiteit Gent = Ghent University (UGENT), Institut de Biologie Valrose (IBV), 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)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Vancouver Prostate Centre [Vancouver General Hospital] (VPC), Vancouver General Hospital, University of British Columbia (UBC), Toxines bactériennes - Bacterial Toxins, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This study was supported by institutional INSERM and Institut Pasteur fundings, 'Investments for the Future' LabEx SIGNALIFE ANR-11-LABX-0028-01 and grants from the Ligue Nationale contre le Cancer (LNCC labellisation and RS20/75-63), Association pour la Recherche contre le Cancer (ARC PJA20191209650), EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union (PRC-5074) and FRM-Piraud prize to E.L., Bio-SPC 'Contrat Doctoral' PhD fellowship to S.P., LNCC PhD fellowship to D.H., the Vancouver Prostate Centre to M.D., and Team Finn and The Ride to Concur Cancer to P.H.S., Fundings from ANR to A.A. ('ImmunoMeca' ANR-12-BSV5-0007-01, 'Initiatives d’excellence' Idex ANR-11-IDEX-0005-02, and 'Labex Who Am I?' ANR-11-LABX-0071)and to F.L. (ANR-10-EQPX-04-01), and from FEDER (12001407) to F.L. B.L. acknowledges financial supports from the Mechanobiology Institute, the European Research Council under the European Union's 7th Framework Programme (FP7/2007-2013) / ERC n° 617233 and NUS-USPC collaborative program., ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-12-BSV5-0007,ImmunoMeca,Caractérisation du rôle de la mécanique dans l'immunité: vers un modèle intégré de l'activation des cellules T(2012), ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), ANR-11-LABX-0071,WHO AM I,Determinants de l'Identité : de la molécule à l'individu(2011), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 617233,EC:FP7:ERC,ERC-2013-CoG,DURACELL(2014), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université Nice Sophia Antipolis (... - 2019) (UNS), Matière et Systèmes Complexes (MSC (UMR_7057)), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), National University of Singapore (NUS), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University [Belgium] (UGENT), 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), and Institut Pasteur [Paris]-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: rac1 GTP-Binding Protein, Integrins, Cell division, Mechanotransduction, Ubiquitin-Protein Ligases, [SDV]Life Sciences [q-bio], Integrin, General Physics and Astronomy, RAC1, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Mechanotransduction, Cellular, General Biochemistry, Genetics and Molecular Biology, Extracellular matrix, Focal adhesion, Integrin signalling, 03 medical and health sciences, 0302 clinical medicine, Medicine and Health Sciences, Cyclin D1, 030304 developmental biology, Optineurin, 0303 health sciences, CNF1 toxin, Multidisciplinary, biology, Chemistry, Ubiquitination, Biology and Life Sciences, Cell migration, General Chemistry, Hostpathogen interaction, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, E3 ubiquitin ligase, biology.protein, Rac1 GTPase, Cell Division, 030217 neurology & neurosurgery, Tumour suppressor
Abstract: Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology. Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.
Published: 2022
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9. Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice

Author: Alexandre Gallerand, Arnaud Jacquel, Pedro Henrique Viana Saavedra, Cedric Torre, Paul Chaintreuil, Sandrine Marchetti, Alissa Majoor, Mohamed Lamkanfi, Raymond Ruimy, Céline Loubatier, Amaury Rey, Anne Doye, Laurent Boyer, Benedicte F. Py, Grégory Michel, Johan Courjon, Rodolphe Guinamard, Patrick Munro, Dorota Czerucka, Orane Visvikis, Océane Dufies, Els Verhoeyen, 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Centre Hospitalier Universitaire de Nice (CHU Nice), Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University [Belgium] (UGENT), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre Scientifique de Monaco (CSM), Laboratoire International Associé Réponse des Organismes et Populations face au Stress Environnemental - Université Côte d’Azur - Centre Scientifique de Monaco (LIA ROPSE), Université Côte d’Azur - Centre Scientifique de Monaco, Université Nice Sophia Antipolis (1965 - 2019) (UNS), Universiteit Gent = Ghent University (UGENT), 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), ANR-17-CE15-0001,AVI,Identification de l'inflammasome impliqué dans l'Immunité Anti-Virulence: Impact sur notre façon de combattre la bactériémie(2017), 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), and Guinamard, Rodolphe
Subjects: Microbiology (medical), Inflammasomes, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Immunology, Bacterial Toxins, Bacteremia, GTPase, Applied Microbiology and Biotechnology, Microbiology, Article, 03 medical and health sciences, Mice, PAK1, NLR Family, Pyrin Domain-Containing 3 Protein, Genetics, medicine, Escherichia coli, Animals, Phosphorylation, p21-activated kinases, Escherichia coli Infections, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, integumentary system, 030306 microbiology, Chemistry, Kinase, Escherichia coli Proteins, Pyroptosis, Pattern recognition receptor, Inflammasome, Cell Biology, Bacterial Load, Immunity, Innate, 3. Good health, Cell biology, rac GTP-Binding Proteins, [SDV] Life Sciences [q-bio], Cytokine, p21-Activated Kinases, medicine.drug, Signal Transduction
Abstract: Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin’s activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3–Nek7 interaction, inflammasome activation and IL-1β cytokine maturation. Furthermore, inhibition of the Pak–NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak–NLRP3 signalling axis in vivo during bacteraemia in mice. Here, the authors present the upstream pathway that controls the activation of the NLRP3 inflammasome during bacteraemia. The CNF1 toxin from Escherichia coli activates the Rho GTPase Rac2 and its activity is sensed by NLRP3, which is activated by a signalling cascade involving p21-activated kinases 1 and 2.
Published: 2021

10. Group-I PAKs-mediated phosphorylation of HACE1 at serine 385 regulates its oligomerization state and Rac1 ubiquitination

Author: Jérôme Boudeau, Amel Mettouchi, Serge Urbach, Orane Visvikis, Maria I. Acosta, Anne Doye, Emmanuel Lemichez, Yuen-Wai Ng, Anne Debant, Edward Manser, 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 Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Institute of Molecular and Cell Biology (IMCB), Epithelial Cell Biology Laboratory, Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), 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), and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Proteomics, Vascular Endothelial Growth Factor A, rac1 GTP-Binding Protein, Ubiquitin-Protein Ligases, Mutant, Bacterial Toxins, lcsh:Medicine, RAC1, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Article, Cell Line, Serine, 03 medical and health sciences, Ubiquitin, Human Umbilical Vein Endothelial Cells, Humans, Phosphorylation, p21-activated kinases, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, biology, Chemistry, Escherichia coli Proteins, lcsh:R, Ubiquitination, Cell biology, Ubiquitin ligase, Cytosol, 030104 developmental biology, p21-Activated Kinases, biology.protein, lcsh:Q, Protein Multimerization
Abstract: The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
Published: 2018

11. System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes

Author: Emmanuel Lemichez, Brenda A. Schulman, Zhenyue Hao, Sachdev S. Sidhu, Nan Li, Ryan Murchie, Wei Zhang, Maria A. Sartori, Kuen-Phon Wu, Jason Moffat, John R. Walker, Avinash Persaud, Yi Sheng, Alban Ordureau, Jicheng Hu, Chong Jiang, Manjeet Mukherjee, Kevin R. Brown, Yufeng Tong, Yanjun Li, Junjie Chen, Anne Doye, Peter Y. Mercredi, Daniela Rotin, Hari B. Kamadurai, J. Wade Harper, Donnelly Center Cellular and Biomolecular Research [Univ. Toronto], University of Toronto, St Jude Children's Research Hospital, Department of Cell Biology, Harvard Medical School [Boston] (HMS), Program in Cell Biology [Toronto], The Hospital for sick children [Toronto] (SickKids)-Department of Biochemistry [University of Toronto], University of Toronto-University of Toronto, Structural Genomics Consortium, The University of Texas M.D. Anderson Cancer Center [Houston], 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), University of York [York, UK], Campbell Family Institute for Breast Cancer Research, University Health Network, Department of Pharmacology and Toxicology [Toronto], Department of Molecular Genetics [Toronto], The Donnelly Centre for Cellular and Biomolecular Research [Toronto, ON, Canada] (CCBR), J.W.H. was supported by NIH (R37NS083524 and GM095567). A.O. was supported by an Edward R. and Anne G. Lefler Center postdoctoral fellowship. J.W.H. is a consultant for Millennium: the Takada Oncology Company and Biogen. D.R. holds a Canada Research Chair (Tier 1 in Biochemistry and Signal Transduction) and was supported by CIHR (MOP#130422). B.A.S. is an investigator of the Howard Hughes Medical Institute (HHMI) and was supported by ALSAC, NIH R37GM065930 and P30CA021765. NECAT and APS were supported by NIH P41 GM103403 and DOE Contract DE-AC02-06CH11357. W.Z. was supported by a CIHR postdoctoral fellowship. S.S.S. and J.M. were supported by CIHR (MOP#111149 and 136956). The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no. 115766), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust., and We thank members of the Sidhu and Schulman groups for helpful comments. We thank Andrew Vorobyov, Eva Chou, Yogesh Hooda, Jun Gu, and Aiping Dong for technical assistance. We are indebted to Pankaj Garg, Andreas Ernst, Abiodun Ogunjimi, Clare Jeon, Satra Nim, Frank Sicheri, and Hongrui Wang for reagents and advice.
Subjects: Models, Molecular, 0301 basic medicine, Subfamily, [SDV]Life Sciences [q-bio], MESH: Catalytic Domain, NEDD4, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], MESH: Ubiquitin/chemistry, MESH: Ubiquitin/metabolism, Madin Darby Canine Kidney Cells, MESH: Dogs, Ubiquitin, Cell Movement, Catalytic Domain, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], MESH: Animals, MESH: Cell Movement, chemistry.chemical_classification, MESH: Organoids/metabolism, biology, MESH: Ubiquitin-Protein Ligases/chemistry, Cell migration, Cell biology, MESH: Ubiquitin/genetics, Organoids, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Models, Molecular, MESH: HCT116 Cells, Ubiquitin-Protein Ligases, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Cell Line, 03 medical and health sciences, Dogs, Peptide Library, Animals, Humans, Molecular Biology, NEDD4L, DNA ligase, MESH: Humans, MESH: Organoids/cytology, MESH: Madin Darby Canine Kidney Cells, MESH: Ubiquitin-Protein Ligases/metabolism, Cell Biology, HCT116 Cells, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Cell Line, 030104 developmental biology, chemistry, biology.protein, MESH: Peptide Library, Genetic screen
Abstract: Comment in : A Billion Ubiquitin Variants to Probe and Modulate the UPS. [Mol Cell. 2016]; International audience; HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.
Published: 2016

12. GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism

Author: Laura Mondragón, Philippe Gaulard, Jaap G Neels, Camila Rubio-Patiño, Frédéric Coutant, Laurent Boyer, François Lemonnier, Florian Muller, Elodie Villa, Rana Mhaidly, Vahid Asnafi, Thierry Passeron, Gian Marco De Donatis, Jean-François Peyron, Nicole Fabien, Els Verhoeyen, Sandrine Marchetti, Pascal Dao, Laura Sormani, Marie Jacquin, Marie Tosolini, Jean-Ehrland Ricci, Emma Proïcs, Anne Doye, Laurent Genestier, Jozef P. Bossowski, Anthony Martin, Véronique Imbert, Frederic Luciano, Jean-Jacques Fournié, Rachid Benhida, Caroline Pons, Johanna Chiche, Centre méditerranéen de médecine moléculaire (C3M), 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 de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Nice (ICN), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Hospices Civils de Lyon (HCL), Centre méditérannéen de médecine moléculaire (C3M), 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), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Service d'immuno-hématologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [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), Physiopathologie de la survie et de la mort cellulaire et infection virale, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Institut National de la Santé et de la Recherche Médicale (INSERM), CCSD, Accord Elsevier, Université Nice Sophia Antipolis (... - 2019) (UNS), Université Nice Sophia Antipolis (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Universitaire du Cancer de Toulouse - Oncopole (IUCT Oncopole - UMR 1037), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Service d'immunologie [Centre Hospitalier Lyon Sud - HCL], Centre Hospitalier Lyon Sud [CHU - HCL] (CHLS), Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Immunogenomique et Inflammation, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Hospices Civils de Lyon (HCL), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Lyon (CRCL), Centre de Recherche en Cancérologie de Toulouse (CRCT), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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 Côte d'Azur (UCA), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Necker - Enfants Malades [AP-HP], Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-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)-IFR50-Université Nice Sophia Antipolis (... - 2019) (UNS), and Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)
Subjects: 0301 basic medicine, Male, Cancer Research, [SDV]Life Sciences [q-bio], T-Lymphocytes, Datasets as Topic, chemistry.chemical_compound, 0302 clinical medicine, Glyceraldehyde 3-phosphate dehydrogenase, ComputingMilieux_MISCELLANEOUS, biology, NF-kappa B, Middle Aged, 3. Good health, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Oncology, 030220 oncology & carcinogenesis, Gene Knockdown Techniques, Female, Signal Transduction, Genetically modified mouse, Angioimmunoblastic T-cell lymphoma, Lineage (genetic), T cell, [SDV.CAN]Life Sciences [q-bio]/Cancer, Mice, Transgenic, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Protein Serine-Threonine Kinases, Lymphoma, T-Cell, 03 medical and health sciences, stomatognathic system, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, medicine, Animals, Humans, Cell Lineage, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Protein Kinase Inhibitors, Aged, NF-κB, medicine.disease, Lymphoma, Disease Models, Animal, 030104 developmental biology, HEK293 Cells, chemistry, Immunoblastic Lymphadenopathy, biology.protein, Cancer research, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating), Function (biology)
Abstract: GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.
Published: 2018

13. EDIN-B Promotes the Translocation of Staphylococcus aureus to the Bloodstream in the Course of Pneumonia

Author: Hubert Lepidi, Coralie Bouchiat, Jean-Philippe Lavigne, Johan Courjon, Eric Ghigo, Patrick Munro, Anne Doye, Laurent Boyer, Orane Visvikis, Emmanuel Lemichez, François Vandenesch, Y. Benito, 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), Pathogénie des Staphylocoques – Staphylococcal Pathogenesis (StaPath), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR48, INSB-INSB-Centre National de la Recherche Scientifique (CNRS), Virulence bactérienne et maladies infectieuses (VBMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), 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 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), and Institut des sciences biologiques (INSB-CNRS)-Institut des sciences biologiques (INSB-CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Staphylococcus aureus, Health, Toxicology and Mutagenesis, Virulence, lcsh:Medicine, Bacteremia, Biology, methicillin-resistant Staphylococcus aureus, Toxicology, medicine.disease_cause, Staphylococcal infections, virulence factor, Virulence factor, Article, Microbiology, 03 medical and health sciences, Mice, Bacterial Proteins, medicine, Human Umbilical Vein Endothelial Cells, Pneumonia, Bacterial, Animals, Humans, 030304 developmental biology, 0303 health sciences, Mice, Inbred BALB C, Lung, 030306 microbiology, lcsh:R, toxins, Staphylococcal Infections, medicine.disease, bacterial infections and mycoses, Methicillin-resistant Staphylococcus aureus, Virology, 3. Good health, Pneumonia, Disease Models, Animal, medicine.anatomical_structure, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Bacterial Translocation, Female, Gene Deletion
Abstract: International audience; It is crucial to define risk factors that contribute to host invasion by Staphylococcus aureus. Here, we demonstrate that the chromosomally encoded EDIN-B isoform from S. aureus contributes to the onset of bacteremia during the course of pneumonia. Deletion of edinB in a European lineage community-acquired methicillin resistant S. aureus (CA-MRSA) strain (ST80-MRSA-IV) dramatically decreased the frequency and magnitude of bacteremia in mice suffering from pneumonia. This deletion had no effect on the bacterial burden in both blood circulation and lung tissues. Re-expression of wild-type EDIN-B, unlike the catalytically inactive mutant EDIN-R185E, restored the invasive characteristics of ST80-MRSA-IV.
Published: 2015

14. Contractile actin cables induced byBacillus anthracislethal toxin depend on the histone acetylation machinery

Author: Emmanuel Lemichez, Hannah G. Yevick, Anne Doye, Caroline Stefani, Orane Visvikis, Monica Rolando, Carmen Buchrieser, Amel Mettouchi, Patricia Bassereau, and Maria I. Acosta
Subjects: 0303 health sciences, biology, Actin remodeling, Arp2/3 complex, Cell Biology, Actin cytoskeleton, Actin cytoskeleton organization, Cell biology, Adherens junction, 03 medical and health sciences, 0302 clinical medicine, Trichostatin A, Structural Biology, biology.protein, medicine, MDia1, 030217 neurology & neurosurgery, Actin, 030304 developmental biology, medicine.drug
Abstract: It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.
Published: 2015

15. Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation

Author: Emilie Andrio, Emmanuel Lemichez, Jacqueline Cherfils, Poul H. Sorensen, Anne Doye, Daniel Hamaoui, Mads Daugaard, Raymond Ruimy, Frédéric Bost, Amel Mettouchi, Romain Lotte, 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), Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Laboratoire de Bactériologie [CHU Nice], Centre Hospitalier Universitaire de Nice (CHU Nice)-Hôpital Archet 2 [Nice] (CHU), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), University of British Columbia (UBC), Vancouver Prostate Centre [Vancouver General Hospital] (VPC), Vancouver General Hospital, BC Cancer Agency Research Centre (BCCRC), Signalisation moléculaire et obésité, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by INSERM, CNRS and fundings from the Ligue Nationale Contre le Cancer (LNCC, équipe labellisée), Association pour la Recherche sur le Cancer (ARC-SFI20111203659 & ARC-SFI20111203671), a fellowship LNCC to D.H., a PhD fellowship from the 'Fondation pour la Recherche Médicale' (FRM-FDM20150632804) to R.L., We are grateful to Melissa Sivaneson, Frédéric Reinier and Patrick Munro for technical help and fruitful discussions. We thank Orane Visvikis for critical reading of the manuscript., 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), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)
Subjects: Models, Molecular, rac1 GTP-Binding Protein, 0301 basic medicine, HECT domain, MESH: Ubiquitin-Protein Ligases/chemistry, Somatic cell, [SDV]Life Sciences [q-bio], MESH: Amino Acid Sequence, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], MESH: Structure-Activity Relationship, Ubiquitin, Neoplasms, MESH: Ubiquitin-Protein Ligases/genetics, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], MESH: Ubiquitin-Protein Ligases/metabolism, Ankyrin, Small GTPase, Genetics, chemistry.chemical_classification, Multidisciplinary, biology, 3. Good health, Ubiquitin ligase, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Protein Domains, MESH: Models, Molecular, Protein Binding, MESH: rac1 GTP-Binding Protein/metabolism, MESH: Mutant Proteins/chemistry, Ubiquitin-Protein Ligases, Protein domain, Mutation, Missense, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Article, Cell Line, MESH: Mutation, Missense/genetics, Structure-Activity Relationship, 03 medical and health sciences, Protein Domains, MESH: Neoplasms/genetics, MESH: Cell Proliferation, Humans, MESH: Protein Binding, Amino Acid Sequence, Cell Proliferation, MESH: Humans, Cell growth, Ubiquitination, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Cell Line, 030104 developmental biology, chemistry, Proteolysis, biology.protein, Mutant Proteins, MESH: Ubiquitination
Abstract: The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.
Published: 2017

16. cAMP Signaling by Anthrax Edema Toxin Induces Transendothelial Cell Tunnels, which Are Resealed by MIM via Arp2/3-Driven Actin Polymerization

Author: François Prodon, Patricia Bassereau, Stéphanie Torrino, David Gonzalez-Rodriguez, Frank Lafont, Juha Saarikangas, Michel Aurrand-Lions, Françoise Brochard, Sébastien Janel, Patrick Munro, Caroline Stefani, Emmanuel Lemichez, Madhavi P. Maddugoda, Pekka Lappalainen, Anne Doye, and Pierre L. Goossens
Subjects: Cancer Research, Endothelium, Bacterial Toxins, Cell, Vascular permeability, Biology, Microbiology, Actin-Related Protein 2-3 Complex, Polymerization, Anthrax, Mice, 03 medical and health sciences, 0302 clinical medicine, Immunology and Microbiology(all), Virology, Cyclic AMP, Human Umbilical Vein Endothelial Cells, medicine, Animals, Humans, Dewetting, Molecular Biology, Actin, 030304 developmental biology, Antigens, Bacterial, Mice, Inbred BALB C, 0303 health sciences, Microfilament Proteins, biology.organism_classification, Neoplasm Proteins, Cell biology, Bacillus anthracis, medicine.anatomical_structure, Membrane, Membrane curvature, Parasitology, 030217 neurology & neurosurgery, Signal Transduction
Abstract: Summary RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.
Published: 2011

17. Isoform-specific Interaction of C-RAF with Mitochondria

Author: Ilka Wittig, Ansgar Santel, Monica Rolando, Emmanuel Lemichez, Gilles Flatau, Ulf R. Rapp, Georg Krohne, Jochen Fueller, Antoine Galmiche, and Anne Doye
Subjects: Gene isoform, Mitochondrion, Biology, Biochemistry, Substrate Specificity, Serine, Mice, Organelle, Animals, Humans, c-Raf, Threonine, Mode of action, Molecular Biology, Cells, Cultured, Mice, Knockout, Mitogen-Activated Protein Kinase Kinases, Kinase, Cell Membrane, Cell Biology, Mitochondria, Cell biology, Isoenzymes, Proto-Oncogene Proteins c-raf, ras Proteins, Protein Binding
Abstract: The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria.
Published: 2008

18. Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin

Author: Vittorio Ricci, Nils C. Gauthier, Amanda Oldani, Pierre Gounon, Mireille Cormont, Teresa Gonzalez, Pascale Monzo, Patrice Boquet, and Anne Doye
Subjects: Endosome, Glycosylphosphatidylinositols, macromolecular substances, Endosomes, Endocytosis, Filamentous actin, Clathrin, Article, Bacterial Proteins, Humans, Cytoskeleton, Actin, Research Articles, Adaptor Proteins, Signal Transducing, biology, Helicobacter pylori, Vesicle, Cell Biology, bacterial infections and mycoses, digestive system diseases, Actins, Cell biology, Transport protein, Cytoskeletal Proteins, Protein Transport, biology.protein, bacteria, HeLa Cells
Abstract: Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP–enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852–4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs.In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.
Published: 2007

19. Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery

Author: Monica, Rolando, Caroline, Stefani, Anne, Doye, Maria I, Acosta, Orane, Visvikis, Hannah G, Yevick, Carmen, Buchrieser, Amel, Mettouchi, Patricia, Bassereau, Emmanuel, Lemichez, Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris]-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), Signalisation et pathologies (IFR50), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire de Nice (CHU Nice)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Immunology Program, Benaroya Research Institute at Virginia Mason, Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), INSERM, IHU Méditerranée Infection. The Institut Pasteur and the 'Fondation pour la Recherche Médicale' (FRM) grant no. DEQ20120323697., ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), 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), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cell Nucleus, Antigens, Bacterial, actin cytoskeleton, Light, Bacterial Toxins, Endothelial Cells, Acetylation, Adherens Junctions, Cell Communication, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Hydroxamic Acids, Actins, Histones, Gene Expression Regulation, Microscopy, Fluorescence, HDAC, Bacillus anthracis, Stress Fibers, Tensile Strength, Bacillus anthracis toxin, HAT, Human Umbilical Vein Endothelial Cells, gene expression, Humans, vascular permeability
Abstract: International audience; It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.
Published: 2015

20. Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors

Author: Pierre Gounon, Céline Pulcini, Patrick Munro, Luce Landraud, René L. Clément, Olivier Dussurget, Emmanuel Lemichez, Amel Mettouchi, Gilles Flatau, Anne Doye, Monica Rolando, Michel R. Popoff, Laurent Boyer, Lemichez, Emmanuel, Toxines bactériennes dans la relation hôtes-pathogènes, 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)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Centre Commun de Microscopie Appliquée (CCMA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Bactéries anaérobies et Toxines, Institut Pasteur [Paris] (IP), Biologie et physiopathologie cutanées : expression génique, signalisation et thérapie, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire central de bactériologie, Centre Hospitalier Universitaire de Nice (CHU Nice)-Hôpital l'Archet, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Nice Sophia Antipolis (... - 2019) (UNS), Institut Pasteur [Paris], Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA)
Subjects: RHOA, MESH: ADP Ribose Transferases, Epidermal cell differentiation, MESH: Research Support, Non-U.S. Gov't, MESH: Recombinant Proteins, 0302 clinical medicine, MESH: Staphylococcus aureus, Immunology and Allergy, MESH: Endothelial Cells, Transcellular, Non-U.S. Gov't, Cytoskeleton, MESH: Bacterial Proteins, Research Articles, Cells, Cultured, ADP Ribose Transferases, 0303 health sciences, Gene knockdown, Cultured, biology, 030302 biochemistry & molecular biology, Recombinant Proteins, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, medicine.anatomical_structure, RNA Interference, MESH: Cells, Cultured, Staphylococcus aureus, MESH: rhoA GTP-Binding Protein, Endothelium, Cells, Immunology, Molecular Sequence Data, MESH: RNA Interference, macromolecular substances, Research Support, Article, 03 medical and health sciences, Bacterial Proteins, medicine, Humans, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Actin, 030304 developmental biology, Basement membrane, MESH: Humans, MESH: Molecular Sequence Data, Endothelial Cells, Cell Biology, biology.protein, rhoA GTP-Binding Protein, 030217 neurology & neurosurgery
Abstract: The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.
Published: 2006

21. Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

Author: Gilles Flatau, Laurent Boyer, Jean-Louis Mege, Olivier Oregioni, Emmanuel Lemichez, Luce Landraud, Anne Doye, and Patrick Munro
Subjects: rho GTP-Binding Proteins, Proteasome Endopeptidase Complex, Bacterial Toxins, Inflammation, CDC42, Biology, Biochemistry, Virulence factor, Cell Line, Enzyme activator, Immune system, Escherichia coli, medicine, Humans, Molecular Biology, Escherichia coli Proteins, Monocyte, Chemotaxis, Cell Biology, Cell biology, Enzyme Activation, Endothelial stem cell, medicine.anatomical_structure, Gene Expression Regulation, Cytokines, medicine.symptom
Abstract: The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3alpha (MIP-3alpha) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3alpha, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.
Published: 2004

22. CNF1 Exploits the Ubiquitin-Proteasome Machinery to Restrict Rho GTPase Activation for Bacterial Host Cell Invasion

Author: Amel Mettouchi, Emmanuel Lemichez, René L. Clément, Gilles Flatau, Guillaume Bossis, Caroline Buisson-Touati, Anne Doye, Laurent Gagnoux, Patrice Boquet, Marc Piechaczyk, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)
Subjects: rho GTP-Binding Proteins, Proteasome Endopeptidase Complex, RHOA, Bacterial Toxins, Clostridium difficile toxin A, CDC42, GTPase, Biology, General Biochemistry, Genetics and Molecular Biology, Virulence factor, 03 medical and health sciences, 0302 clinical medicine, Ubiquitin, Multienzyme Complexes, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, cdc42 GTP-Binding Protein, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Cytotoxins, Biochemistry, Genetics and Molecular Biology(all), Escherichia coli Proteins, Molecular biology, rac GTP-Binding Proteins, Cell biology, Enzyme Activation, Cysteine Endopeptidases, Cytosol, Proteasome, biology.protein, cytotoxic necrotizing factor-1 uropathogenic escherichia-coli urinary-tract infections actin cytoskeleton factor type-1 protein toxin deamidation binding domain, rhoA GTP-Binding Protein, 030217 neurology & neurosurgery
Abstract: CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.
Published: 2002

23. Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin

Author: Silvia Pei, Patrice Boquet, and Anne Doye
Subjects: Recombinant Fusion Proteins, Bacterial Toxins, Lysine, Biological Transport, Active, Biology, Endocytosis, Microbiology, Cell Line, Cell membrane, Protein structure, Escherichia coli, medicine, Humans, Point Mutation, Cytotoxic T cell, Molecular Biology, DNA Primers, chemistry.chemical_classification, Diphtheria toxin, Base Sequence, Cytotoxins, Escherichia coli Proteins, Cell Membrane, Hydrogen-Ion Concentration, Protein Structure, Tertiary, Amino acid, Cytosol, medicine.anatomical_structure, Amino Acid Substitution, Biochemistry, chemistry
Abstract: The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine. Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.
Published: 2001

24. In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

Author: Michel Cohen-Tannoudji, Gérard Pehau-Arnaudet, Evelyne Ferrary, Lilia Boulouha, Charles Babinet, Daniel Louvard, Jean-Jacques Fontaine, Tereza Ruiz, Anne Doye, Frederic Jaisser, Sylvie Robine, Alexandre Lapillonne, Rafika Athman, Claude Antony, and Fatima El Marjou
Subjects: Male, Brush border, Polymers, macromolecular substances, Microfilament, digestive system, Mice, Intestinal mucosa, In vivo, Culture Techniques, actin-binding proteins, Animals, Intestinal Mucosa, microvilli, intestine, villin knockout, mouse, Actin, Mice, Knockout, biology, Dextran Sulfate, Microfilament Proteins, Fasting, Cell Biology, Colitis, Actin cytoskeleton, Actins, Cell biology, Mice, Inbred C57BL, Actin Cytoskeleton, Biochemistry, biology.protein, Calcium, Carbachol, Female, Original Article, Carrier Proteins, Villin, Gene Deletion, Intracellular
Abstract: Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.
Published: 1999

25. Transient Uptake and Storage of Serotonin in Developing Thalamic Neurons

Author: Christine Adelbrecht, Isabelle Seif, Cécile Lebrand, Salah El Mestikawy, Patricia Gaspar, Olivier Cases, Chantal Alvarez, and Anne Doye
Subjects: Serotonin, Time Factors, Neuroscience(all), Thalamus, Gene Expression, Sensory system, Nerve Tissue Proteins, Tritium, Rats, Sprague-Dawley, Glutamatergic, Mice, Antibody Specificity, Vesicular Biogenic Amine Transport Proteins, medicine, Animals, Neurons, Afferent, RNA, Messenger, Serotonin transporter, In Situ Hybridization, Cerebral Cortex, Serotonin Plasma Membrane Transport Proteins, Neurotransmitter Agents, Membrane Glycoproteins, biology, Raphe, General Neuroscience, Neuropeptides, Age Factors, Membrane Transport Proteins, Biological Transport, Immunohistochemistry, Rats, Vesicular monoamine transporter, Mice, Inbred C57BL, medicine.anatomical_structure, Cerebral cortex, Vesicular Monoamine Transport Proteins, biology.protein, Synaptic Vesicles, Carrier Proteins, Neuroscience
Abstract: Serotonin (5-HT) has been shown to affect the development and patterning of the mouse barrelfield. We show that the dense transient 5-HT innervation of the somatosensory, visual, and auditory cortices originates in the thalamus rather than in the raphe: 5-HT is detected in thalamocortical fibers and most 5-HT cortical labeling disappears after thalamic lesions. Thalamic neurons do not synthesize 5-HT but take up exogenous 5-HT through 5-HT high affinity uptake sites located on thalamocortical axons and terminals. 3H–5-HT injected into the cortex is retrogradely transported to thalamic neurons. In situ hybridization shows a transient expression of the genes encoding the serotonin transporter and the vesicular monoamine transporter in thalamic sensory neurons. In these glutamatergic neurons, internalized 5-HT might thus be stored and used as a “borrowed transmitter” for extraneuronal signaling or could exert an intraneuronal control on thalamic maturation.
Published: 1996
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26. Assessing ubiquitylation of Rho GTPases in mammalian cells

Author: Anne, Doye, Amel, Mettouchi, and Emmanuel, Lemichez
Subjects: rho GTP-Binding Proteins, Metals, Cricetinae, Blotting, Western, Ubiquitination, Animals, Proteins, Electrophoresis, Polyacrylamide Gel, CHO Cells, Transfection, Cells, Cultured, Protein Binding
Abstract: Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.
Published: 2011

27. Assessing Ubiquitylation of Rho GTPases in Mammalian Cells

Author: Emmanuel Lemichez, Anne Doye, and Amel Mettouchi
Subjects: RHOA, GTP', biology, Chemistry, Cell polarity, biology.protein, RAC1, CDC42, GTPase, Cytoskeleton, Cytokinesis, Cell biology
Abstract: Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cellular factors inducing both the exchange of GDP for GTP and the hydrolysis of GTP into GDP. Recent findings have unveiled another layer of complexity in the regulation of Rho proteins consisting in their ubiquitylation followed by their proteasomal degradation. Here, we describe how to assess the level of ubiquitylation of Rho proteins in cells, taking Rac1 as an example.
Published: 2011

28. Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion

Author: Patrick Lorès, Laurent Boyer, Daniel Bouvard, Gilles Flatau, Esteban Veiga, Pascale Cossart, Amel Mettouchi, Stéphanie Torrino, Monica Rolando, Emmanuel Lemichez, Anne Doye, Orane Visvikis, Marc Lemonnier, Gérard Gacon, Centre méditérannéen 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)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Université de Nice Sophia-Antipolis (UNSA), Université Joseph Fourier - Grenoble 1 (UJF), Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), conseil regional PACA, Conseil general des Alpes-Maritimes, INSERM, Agence Nationale de la Recherche [ANR A05135AS, R07120AA, R07113AS], Association pour la Recherche sur le Cancer [ARC 4906], Ligue Nationale contre le Cancer, Université Nice Sophia Antipolis (1965 - 2019) (UNS), and Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: rac1 GTP-Binding Protein, [SDV]Life Sciences [q-bio], BACTERIAL INVASION, medicine.disease_cause, Biochemistry, CYTOTOXIC NECROTIZING FACTOR-1, ACTIN DYNAMICS, Mice, Ubiquitin, Structural Biology, MAMMALIAN-CELLS, Cytotoxic T cell, Uropathogenic Escherichia coli, Internalization, media_common, 0303 health sciences, biology, Escherichia coli Proteins, 030302 biochemistry & molecular biology, Intracellular Signaling Peptides and Proteins, RHO GTPASES, Bacterial Infections, CNF1, Endocytosis, 3. Good health, Cell biology, FACTOR TYPE-1, CLATHRIN, MEMBRANE TRANSLOCATION, PROTEASOMAL DEGRADATION, Rac1, media_common.quotation_subject, P21 RHO, Bacterial Toxins, RAC1, ubiquitination, Clathrin, Microbiology, Cell Line, 03 medical and health sciences, Genetics, medicine, bacterial toxin, Animals, Humans, Molecular Biology, Escherichia coli, 030304 developmental biology, TOLLIP, Proteins, Cell Biology, Actin cytoskeleton, Tollip, biology.protein, UPEC
Abstract: International audience; Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.
Published: 2011

29. Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication

Author: Colin Berry, David Pauron, Onya Opota, Nils C. Gauthier, Pierre Gounon, Emmanuel Lemichez, Anne Doye, Interactions Biotiques et Santé Végétale, Institut National de la Recherche Agronomique (INRA), Centre méditérannéen 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)-Institut National de la Santé et de la Recherche Médicale (INSERM), School of Biosciences [Cardiff], Cardiff University, and Université de Nice Sophia-Antipolis (UNSA)
Subjects: DRUG EFFECTS, AUTOPHAGY, BACTERIAL TOXIN, TOXICITY, HOST PATHOGEN INTERACTIONS, BACILLUS SPHAERICUS, ANOPHELES GAMBIAE, CULEX PIPIENS, DIPTERA, lcsh:Medicine, Bacillus, Vacuole, Q1, medicine.disease_cause, Bacillus sphaericus, Phagosomes, lcsh:Science, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Pore-forming toxin, Multidisciplinary, Cell Biology/Cellular Death and Stress Responses, 3. Good health, Culex, toxine, Host-Pathogen Interactions, insecte vecteur, Microbiology/Cellular Microbiology and Pathogenesis, Intracellular, Cytoplasmic Vacuolation, Research Article, Endosome, Recombinant Fusion Proteins, Bacterial Toxins, Green Fluorescent Proteins, Bacillaceae Infections, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Transfection, Microbiology, métabolisme enzymatique, 03 medical and health sciences, Dogs, Cell Biology/Membranes and Sorting, vecteur de maladies, Anopheles, medicine, Animals, 030304 developmental biology, 030306 microbiology, Toxin, lcsh:R, Autophagy, fungi, alpha-Glucosidases, Cell Biology, biology.organism_classification, biopesticide, Vacuoles, lcsh:Q
Abstract: Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.
Published: 2011

30. alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells

Author: Laurent Boyer, Guerrino Meneguzzi, Eric Rubinstein, Laurence Cailleteau, Amel Mettouchi, Claude Boucheix, Anne Doye, Emmanuel Lemichez, Téni G. Ebrahimian, Soline Estrach, Raphaël Thyss, Jean-Sébastien Silvestre, Barbara Domange, and Nils Johnsson
Subjects: Neovascularization, Physiologic, Biology, CD49c, Tetraspanin 29, Collagen receptor, Focal adhesion, Mice, Tetraspanin, Antigens, CD, Animals, Humans, RNA, Small Interfering, Cells, Cultured, Cell Proliferation, Mice, Knockout, Focal Adhesions, Membrane Glycoproteins, Endothelial Cells, Cell Biology, Cell biology, Fibronectins, rac GTP-Binding Proteins, Endothelial stem cell, Fibronectin, Mice, Inbred C57BL, Protein Transport, Integrin alpha M, biology.protein, Integrin, beta 6, Female, Fibroblast Growth Factor 2, Laminin, Integrin alpha2beta1, Integrin alpha5beta1, Signal Transduction
Abstract: Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.
Published: 2010

31. Ubiquitin‐Mediated Proteasomal Degradation of Rho Proteins by the CNF1 Toxin

Author: Emmanuel Lemichez, Laurent Boyer, Anne Doye, Amel Mettouchi, Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Biologie et physiopathologie cutanées : expression génique, signalisation et thérapie, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), 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)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), and Lemichez, Emmanuel
Subjects: rho GTP-Binding Proteins, Proteasome Endopeptidase Complex, RHOA, Bacterial Toxins, MESH: Escherichia coli Proteins, RAC1, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, CDC42, Biology, 03 medical and health sciences, chemistry.chemical_compound, Ubiquitin, MG132, Escherichia coli, Humans, cdc42 GTP-Binding Protein, Deamidation, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Cells, Cultured, 030304 developmental biology, 0303 health sciences, MESH: Humans, MESH: cdc42 GTP-Binding Protein, MESH: Escherichia coli, Escherichia coli Proteins, MESH: Proteasome Endopeptidase Complex, Cell Membrane, 030302 biochemistry & molecular biology, Ubiquitin-Protein Ligase Complexes, MESH: Ubiquitin-Protein Ligase Complexes, MESH: rho GTP-Binding Proteins, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Bacterial Toxins, chemistry, Proteasome, biology.protein, Endothelium, Vascular, MESH: Endothelium, Vascular, MESH: Bacter, MESH: Cell Membrane, MESH: Cells, Cultured, Cysteine
Abstract: The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.
Published: 2006

32. Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes

Author: V. Kaddai, Nils C. Gauthier, Pascale Monzo, Anne Doye, Patrice Boquet, Vittorio Ricci, Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Signalisation moléculaire et obésité, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Human Physiology Section, Department of Experimental Medicine, Università degli Studi di Pavia, Lemichez, Emmanuel, 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)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), and Università degli Studi di Pavia = University of Pavia (UNIPV)
Subjects: rac1 GTP-Binding Protein, MESH: ADP-Ribosylation Factors, Endocytic cycle, Caveolin 1, Vesicular Transport Proteins, MESH: Membrane Microdomains, Dynamin II, MESH: Caveolin 1, MESH: Clathrin, cdc42 GTP-Binding Protein, Lipid raft, MESH: Bacterial Proteins, 0303 health sciences, biology, ADP-Ribosylation Factors, Pinocytosis, 030302 biochemistry & molecular biology, Transferrin, Articles, Cell biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Cdc42 GTP-Binding Protein, MESH: Cell Adhesion Molecules, MESH: Membrane Proteins, Endosome, Virulence Factors, Bacterial Toxins, Endosomes, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Actins, Clathrin, Cell Line, EEA1, 03 medical and health sciences, Membrane Microdomains, Bacterial Proteins, Humans, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Molecular Biology, 030304 developmental biology, Dynamin, rab5 GTP-Binding Proteins, MESH: Humans, Helicobacter pylori, MESH: Research Support, Membrane Proteins, Cell Biology, bacterial infections and mycoses, MESH: Pinocytosis, Actins, digestive system diseases, MESH: Cell Line, MESH: Bacterial Toxins, ADP-Ribosylation Factor 6, MESH: Endosomes, MESH: Dynamin II, biology.protein, bacteria, MESH: Helicobacter pylori, rhoA GTP-Binding Protein, Cell Adhesion Molecules
Abstract: The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.
Published: 2005

33. Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

Author: Philippe Poujeol, Pierre Gounon, Vittorio Ricci, Anne Doye, Nils C. Gauthier, Patrice Boquet, Michel Tauc, Toxines bactériennes dans la relation hôtes-pathogènes, 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)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Institut des Sciences Chimiques de Rennes (ISCR), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Ecole Nationale Supérieure de Chimie de Rennes (ENSCR)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Dipartimento di Metodi e Modelli Matematici, Università degli studi di Palermo - University of Palermo, Human Physiology Section, Department of Experimental Medicine, Università degli Studi di Pavia = University of Pavia (UNIPV), Services et Réseaux Communication - Institut Universitaire de Technologies Laval - Université du Maine (DÉPARTEMENT SERVICES ET RÉSEAUX DE COMMUNICATION - IUT. DE LAVAL), IUT de Laval, Le Mans Université (UM)-Le Mans Université (UM), Laboratoire d'Informatique de l'Université du Maine (LIUM), Le Mans Université (UM)-Centre National de la Recherche Scientifique (CNRS), Département Services et Réseaux de Communication (IUT DE LAVAL), Centre Commun de Microscopie Appliquée (CCMA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Laboratoire des images et des signaux (LIS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS), Centre commun de microscopie appliquée, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), Physiologie cellulaire et moléculaire des systèmes intégrés (PCMSI), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Bactériologie [CHU Nice], Centre Hospitalier Universitaire de Nice (CHU Nice)-Hôpital Archet 2 [Nice] (CHU), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Ecole Nationale Supérieure de Chimie de Rennes (ENSCR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA), Università degli Studi di Pavia, Université Nice Sophia Antipolis (... - 2019) (UNS), and Institut National Polytechnique de Grenoble (INPG)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Patch-Clamp Techniques, Glycosylphosphatidylinositols, Endosome, Cell, Biology, Endocytosis, MESH: Actins, Biochemistry, Ion Channels, MESH: Vacuoles, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Chlorides, MESH: Patch-Clamp Techniques, medicine, MESH: Cytoskeleton, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Humans, Channel blocker, MESH: Chlorides, Molecular Biology, Lipid raft, MESH: Bacterial Proteins, Cytoskeleton, 030304 developmental biology, Cytochalasin D, 0303 health sciences, Ion Transport, MESH: Humans, 030302 biochemistry & molecular biology, Membrane Proteins, Cell Biology, Actin cytoskeleton, bacterial infections and mycoses, MESH: Glycosylphosphatidylinositols, Actins, digestive system diseases, Cell biology, MESH: Hela Cells, MESH: Ion Transport, Cytosol, medicine.anatomical_structure, chemistry, Vacuoles, MESH: Ion Channels, bacteria, MESH: Membrane Proteins, HeLa Cells
Abstract: The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells (Ricci V., Galmiche, A., Doye, A., Necchi, V., Solcia, E., and Boquet, P. (2000) Mol. Biol. Cell 11, 3897-3909). Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity (Tombola, F., Oregna, F., Brutsche, S., Szabo, I., Del Giudice, G., Rappuoli, R., Montecucco, C., Papini, E., and Zoratti, M. (1999) FEBS Lett. 460, 221-225). We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.
Published: 2004

34. The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release

Author: Virginie De Thillot, Sébastien Cagnol, Jean-Claude Chambard, Vittorio Ricci, Antoine Galmiche, Stephanette Contamin, Patrice Boquet, Enrico Solcia, Anne Doye, Joachim Rassow, Ingo Just, and Ellen Van Obberghen
Subjects: Programmed cell death, Reticulocytes, Poly ADP ribose polymerase, Green Fluorescent Proteins, Stomach Diseases, Fluorescent Antibody Technique, Caspase 3, Apoptosis, Cytochrome c Group, Digitonin, Mitochondrion, Transfection, General Biochemistry, Genetics and Molecular Biology, Cell Line, Cytosol, Bacterial Proteins, Animals, Humans, Molecular Biology, Cell Nucleus, General Immunology and Microbiology, biology, General Neuroscience, Cytochrome c, fungi, Articles, bacterial infections and mycoses, Molecular biology, Immunohistochemistry, digestive system diseases, Cell biology, Mitochondria, Luminescent Proteins, Microscopy, Electron, Proto-Oncogene Proteins c-bcl-2, Caspases, biology.protein, bacteria, Electrophoresis, Polyacrylamide Gel, Rabbits, Poly(ADP-ribose) Polymerases, HeLa Cells
Abstract: The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.
Published: 2000

35. High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis

Author: Anne Doye, Antoine Galmiche, Vittorio Ricci, Patrice Boquet, Vittorio Necchi, and Enrico Solcia
Subjects: Nystatin, Carcinoma, Hepatocellular, Cytochalasin D, Endocytic cycle, Cell, Bacterial Toxins, Vacuole, CHO Cells, Biology, Endocytosis, Phosphatidylinositols, Clathrin, Article, Cell Line, Iodine Radioisotopes, Dogs, Bacterial Proteins, Cricetinae, medicine, Animals, Humans, Molecular Biology, Lipid raft, Dose-Response Relationship, Drug, Proteins, Cell Biology, Actin cytoskeleton, bacterial infections and mycoses, digestive system diseases, Actins, Cell biology, Actin Cytoskeleton, medicine.anatomical_structure, Cell culture, Type C Phospholipases, Vacuoles, biology.protein, lipids (amino acids, peptides, and proteins)
Abstract: Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains (“lipid rafts”), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.
Published: 2000

36. The p21 Rho-activating Toxin Cytotoxic Necrotizing Factor 1 Is Endocytosed by a Clathrin-independent Mechanism and Enters the Cytosol by an Acidic-dependent Membrane Translocation Step

Author: Patrice Boquet, Stephanette Contamin, Anne Doye, Alexandre Benmerah, Gilles Flatau, and Antoine Galmiche
Subjects: Bacterial Toxins, Caveolin 1, Coated Vesicles, Coated vesicle, Golgi Apparatus, Receptors, Cell Surface, Biology, Endocytosis, Clathrin, Caveolins, Microtubules, Article, Cell membrane, Cytosol, Dogs, Immunotoxin, Catalytic Domain, medicine, Tumor Cells, Cultured, Animals, Humans, Molecular Biology, Diphtheria toxin, Binding Sites, Cytotoxins, Escherichia coli Proteins, Cell Membrane, Membrane Proteins, Biological Transport, Cell Biology, Cell biology, Cell Compartmentation, medicine.anatomical_structure, ADP-ribosylation, biology.protein
Abstract: Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (Kd= 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of ≤5.2. CNF1 is the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.
Published: 2000

37. Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection

Author: P Lagadec, Jacques Robert Nicoli, Flaviano S. Martins, Dorota Czerucka, Véronique Imbert, Jean-François Peyron, Guillaume Dalmasso, Anne Doye, Rosa Maria Esteves Arantes, Patrick Rampal, and Emmanuel Lemichez
Subjects: Infectious Diseases/Gastrointestinal Infections, Salmonella, Phagocytosis, lcsh:Medicine, Inflammation, medicine.disease_cause, Saccharomyces, Cell Biology/Cell Signaling, Bacterial Adhesion, Microbiology, Infectious Diseases/Bacterial Infections, Microbiology/Applied Microbiology, Mice, Immune system, Microscopy, Electron, Transmission, medicine, Animals, lcsh:Science, Multidisciplinary, biology, Probiotics, lcsh:R, NF-kappa B, Microbiology/Medical Microbiology, Salmonella enterica, biology.organism_classification, Gastroenterology and Hepatology/Gastrointestinal Infections, Freeze Drying, Liver, Salmonella Infections, lcsh:Q, Mitogen-Activated Protein Kinases, medicine.symptom, Bacteria, Research Article, Signal Transduction, Saccharomyces boulardii
Abstract: Background Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-κB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea. Methodology/Principal Findings In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-κB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects. Conclusions Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.
Published: 2010

38. Nonsteroidal anti-inflammatory drugs counteract Helicobacter pylori-induced vacuolation of gastric epithelial cells in vitro

Author: Concetta Tuccillo, Patrizia Sommi, Anne Doye, Patrice Boquet, Marco Romano, Vittorio Ricci, Barbara A. Manzo, Raffaele Zarrilli, and Mariella Santangelo
Subjects: Nonsteroidal, Hepatology, biology, medicine.drug_class, business.industry, Gastroenterology, Pharmacology, Helicobacter pylori, biology.organism_classification, Anti-inflammatory, In vitro, chemistry.chemical_compound, chemistry, medicine, business
Published: 2000

39. The E3 Ubiquitin-Ligase HACE1 Catalyzes the Ubiquitylation of Active Rac1

Author: Anne Doye, Amel Mettouchi, Stéphanie Torrino, Caroline Stefani, Emmanuel Lemichez, Orane Visvikis, Patrick Munro, Gérard Gacon, Jacques Bertoglio, and Laurent Boyer
Subjects: rac1 GTP-Binding Protein, Proteasome Endopeptidase Complex, Ubiquitin-Protein Ligases, Cell, RAC1, CHO Cells, GTPase, General Biochemistry, Genetics and Molecular Biology, law.invention, Ubiquitin, law, Cricetinae, medicine, Animals, Humans, Small GTPase, Molecular Biology, Cells, Cultured, biology, Point mutation, Ubiquitination, Cell Biology, Ubiquitin ligase, Cell biology, HEK293 Cells, medicine.anatomical_structure, Biocatalysis, biology.protein, Suppressor, Developmental Biology
Abstract: SummaryRac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.
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40. ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments

Author: Didier Sauvaire, Philippe M. Loiseau, Sabrina Kali, Rahima Yousfi, Pascal Clayette, Daniel Gillet, Céline Bouclier, Noël Tordo, Jérôme Laporte, Amélie Goudet, Goulven Merer, Roman Lopez, Michel R. Popoff, Sandrine Cojean, Valérie Pons, Céline Aubenque, Claire Boulogne, Emmanuel Lemichez, Annette Fischer, Jean-Christophe Cintrat, Laetitia Panigai, Yu Wu, Cynthia Gillet, Roger Le Grand, Thomas Rudel, Robert A. Davey, Julien Barbier, Jo-Ana Herweg, Emilie Gobbo, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), 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 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), Service de Chimie Bio-Organique et de Marquage (SCBM), Agence nationale de sécurité du médicament et des produits de santé [Saint-Denis] (ANSM), Julius-Maximilians-Universität Würzburg [Wurtzbourg, Allemagne] (JMU), Stratégies antivirales, Institut Pasteur [Paris], Texas Biomedical Research Institute, CEA [Fontenay-aux-Roses] (UGRA / SETA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), 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), Molécules bioactives, conception, isolement et synthèse (MBCIS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Bactéries anaérobies et Toxines, Pharmacologie des rétrovirus, Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), 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), This work has been funded by the joint ministerial program of R&D against CBRNE risks and CEA, and benefited from the core facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by 'France-BioImaging' (ANR-10-INBS-04–01) and the Labex 'Saclay Plant Science' (ANR-11-IDEX-0003–02). This work was also supported by U.S. National Institutes of Health and National Institute of Allergy and Infectious Diseases grant R01AI063513, Defense Threat Reduction Agency grant HDTRA1–12–1–0002, and the Douglass and Ewing Halsell Foundations. Work on Simkania and Chlamydia was supported by the Deutsche Forschungsgemeinschaft (DFG) Priority programme 1580 to T.R. SIMOPRO and SCBM are members of the Laboratory of Excellence LERMIT supported by a grant from the Agence Nationale de la Recherche (ANR-10-LABX-33) and RetroLeishma project R3, We greatfully thank Anne Doye (Université de Nice Sophia-Antipolis) for technical assistance and Bruno Beaumelle for providing ricin, ANR-11-IDEX-0002,UNITI,Université Fédérale de Toulouse(2011), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-IDEX-0003,IPS,Idex Paris-Saclay(2011), Julius-Maximilians-Universität Würzburg (JMU), Institut Pasteur [Paris] (IP), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Popoff, Michel, Initiative d'excellence - Université Fédérale de Toulouse - - UNITI2011 - ANR-11-IDEX-0002 - IDEX - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, and Idex Paris-Saclay - - IPS2011 - ANR-11-IDEX-0003 - IDEX - VALID
Subjects: 0301 basic medicine, Benzylamines, Endosome, 030106 microbiology, Cell, Golgi Apparatus, lcsh:Medicine, Adamantane, Endosomes, Ricin, Endoplasmic Reticulum, Article, Mice, 03 medical and health sciences, symbols.namesake, ddc:570, Benzyl Compounds, Organelle, medicine, Animals, Humans, lcsh:Science, Pathogen, Toxins, Biological, Multidisciplinary, Chemistry, Antimicrobials, Endoplasmic reticulum, Intracellular parasite, lcsh:R, High-throughput screening, Golgi apparatus, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell Compartmentation, 3. Good health, Cell biology, 030104 developmental biology, medicine.anatomical_structure, symbols, Infectious diseases, lcsh:Q, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Lysosomes, Intracellular, HeLa Cells
Abstract: Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
Published: 2017

41. The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

Author: Patrick Rampal, Patrick Munro, Rodolphe Pontier-Bres, Emmanuel Lemichez, Dorota Czerucka, Jean-François Peyron, Centre Scientifique de Monaco (CSM), Inflammation, Cancer, Cancer Stem Cells (equipe 4 - U1065), 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)-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), Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), and The authors greatly acknowledge the C3M Imaging Core Facility (Microscopy and Imaging Platform Côte d’Azur, MICA). We also thank Monica Rolando and Anne Doye for comments during the manuscript redaction and technical help.
Subjects: Cell Membrane Permeability, Gastrointestinal Diseases, Health, Toxicology and Mutagenesis, [SDV]Life Sciences [q-bio], lcsh:Medicine, MESH: Bacterial Toxins/toxicity, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], MESH: Cell Membrane Permeability/drug effects, Toxicology, medicine.disease_cause, Saccharomyces, law.invention, Probiotic, 0302 clinical medicine, MESH: MAP Kinase Kinase Kinase 2/chemistry, law, MESH: Human Umbilical Vein Endothelial Cells/drug effects, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], 0303 health sciences, Tight junction, 3. Good health, Bacillus anthracis, Saccharomyces boulardii, MESH: Anthrax/prevention & control, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, 030211 gastroenterology & hepatology, MESH: Actins/chemistry, Bacterial Toxins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, MESH: Bacillus anthracis/chemistry, MAP Kinase Kinase Kinase 2, Article, Microbiology, Cell Line, Tight Junctions, Anthrax, 03 medical and health sciences, medicine, Human Umbilical Vein Endothelial Cells, Humans, 030304 developmental biology, MESH: Humans, Toxin, MESH: Tight Junctions/drug effects, MESH: Bacterial Toxins/antagonists & inhibitors, lcsh:R, Actin remodeling, MESH: Probiotics/pharmacology, biology.organism_classification, MESH: Saccharomyces, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Actins, MESH: Cell Line, probiotics, Cell culture, MESH: Gastrointestinal Diseases/prevention & control, anthrax toxins
Abstract: International audience; The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.
Published: 2015
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42. Inhibition of the GTPase Rac1 Mediates the Antimigratory Effects of Metformin in Prostate Cancer Cells

Author: Frédéric Larbret, Muriel Golzio, Olivier Cuvillier, Bernard Malavaud, Kathiane Laurent, Emmanuel Lemichez, Frédéric Bost, Fabienne Massa, Isabelle Ader, Amel Mettouchi, Mireille Cormont, Jean-François Tanti, Béatrice Dirat, 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), Physiopathologie Cellulaire et Moléculaire de l'Obésité et du Diabète (Equipe 7), 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)-Université Nice Sophia Antipolis (... - 2019) (UNS), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Laboratoire Tolérance Immunitaire (TIM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Toxines bactériennes dans la relation hôtes-pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR50-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), This study was supported by The European Foundation for the Study of Diabetes (EFSD), INCA (grants 2010-219 and 2010-214) and the 'Fondation ARC.' B. Dirat was supported by INCA grant 2010-219, the Cancerople PACA, and the Région PACA. F. Bost, J.F. Tanti, and A. Mettouchi are investigators of the Centre National de la Recherche Scientifique (CNRS). F. Massa is supported by ITMO-Cancer. This work was supported by the French Government (National Research Agency, ANR) through the 'Investments for the Future' LABEX SIGNALIFE (grant ANR-11-0028-01), The authors thank Anne Doye and Rachel Paul-Bellon for technical assistance, Pr. Mitchell and Dr. Becanovic for The HA–PREX plasmid, Issam Ben-Sahra, Stéphane Ricoult, Jérôme Gilleron, Sophie Giorgetti-Peraldi, and Yannick Le Marchand Brustel for their help and the critical reading of the article. The authors greatly acknowledge Damien Alcor of the C3M (or MicorBio) Cell Imaging Facility., and ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011)
Subjects: Male, rac1 GTP-Binding Protein, Cancer Research, Cytoskeleton organization, [SDV]Life Sciences [q-bio], Cell, MESH: Actin Cytoskeleton/drug effects, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], MESH: rac1 GTP-Binding Protein/metabolism, Prostate cancer, 0302 clinical medicine, MESH: Cell Proliferation/drug effects, Cell Movement, MESH: Actin Cytoskeleton/metabolism, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Cyclic AMP, Guanine Nucleotide Exchange Factors, MESH: Animals, Neoplasm Metastasis, 0303 health sciences, MESH: Chemokine CXCL12/pharmacology, Chemotaxis, Cell migration, MESH: Metformin/pharmacology, Metformin, 3. Good health, Actin Cytoskeleton, medicine.anatomical_structure, MESH: Chemotaxis/drug effects, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Oncology, 030220 oncology & carcinogenesis, MESH: Cell Movement/drug effects, MESH: rac1 GTP-Binding Protein/antagonists & inhibitors, medicine.drug, MESH: Xenograft Model Antitumor Assays, MESH: Cell Line, Tumor, MESH: Prostatic Neoplasms/enzymology, Mice, Nude, RAC1, MESH: Prostatic Neoplasms/pathology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, 03 medical and health sciences, DU145, Cell Line, Tumor, medicine, MESH: Cyclic AMP/metabolism, MESH: Mice, Nude, Animals, Humans, Neoplasm Invasiveness, 030304 developmental biology, Cell Proliferation, MESH: Humans, Cancer, Prostatic Neoplasms, MESH: Guanine Nucleotide Exchange Factors/metabolism, MESH: Neoplasm Invasiveness, medicine.disease, Xenograft Model Antitumor Assays, MESH: Neoplasm Metastasis, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Chemokine CXCL12, MESH: Male, Cancer research
Abstract: Cell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer. Mol Cancer Ther; 14(2); 586–96. ©2014 AACR.
Published: 2015

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