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1. Oxygen concentration modulates colibactin production

Author

Nadège Bossuet, Cécile Guyonnet, Camille V Chagneau, Min Tang-Fichaux, Marie Penary, Dorian Loubet, Priscilla Branchu, Eric Oswald, and Jean-Philippe Nougayrede

Subjects
Escherichia coli, colibactin, DNA damage, oxygen, hypoxia, Diseases of the digestive system. Gastroenterology, RC799-869
Abstract

ABSTRACTUp to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue.

Published
2023
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2. Interplay between siderophores and colibactin genotoxin biosynthetic pathways in Escherichia coli.

Author

Patricia Martin, Ingrid Marcq, Giuseppe Magistro, Marie Penary, Christophe Garcie, Delphine Payros, Michèle Boury, Maïwenn Olier, Jean-Philippe Nougayrède, Marc Audebert, Christian Chalut, Sören Schubert, and Eric Oswald

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4'-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.

Published
2013
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3. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways.

Author

Grégory Jubelin, Frédéric Taieb, David M Duda, Yun Hsu, Ascel Samba-Louaka, Rika Nobe, Marie Penary, Claude Watrin, Jean-Philippe Nougayrède, Brenda A Schulman, C Erec Stebbins, and Eric Oswald

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G₁/S and G₂/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.

Published
2010
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4. A novel toxic effect of foodborne trichothecenes: The exacerbation of genotoxicity

Author

Marion Garofalo, Delphine Payros, Marie Penary, Eric Oswald, Jean-Philippe Nougayrède, Isabelle P. Oswald, ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Biosynthèse & Toxicité des Mycotoxines (ToxAlim-BioToMyc), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), This work was supported by grants from the French Agence Nationale de la Recherche (Genofood ANR-19-CE34 and GenoMyc ANR-22-CE34). MG was supported by a fellowship from the French ministry for Higher Education and Research., ANR-19-CE34-0014,Genofood,Exacerbation de la génotoxicité du microbiote intestinal par un contaminant alimentaire(2019), LESUR, Hélène, and Exacerbation de la génotoxicité du microbiote intestinal par un contaminant alimentaire - - Genofood2019 - ANR-19-CE34-0014 - AAPG2019 - VALID

Subjects
[SDV] Life Sciences [q-bio], Carcinogenic potential, Microbiota, [SDV]Life Sciences [q-bio], Health, Toxicology and Mutagenesis, Colibactin, General Medicine, Mycotoxins, Pesticides, Toxicology, Pollution
Abstract

International audience; Trichothecenes (TCT) are very common mycotoxins. While the effects of DON, the most prevalent TCT, have been extensively studied, less is known about the effect of other trichothecenes. DON has ribotoxic, pro-inflammatory, and cytotoxic potential and induces multiple toxic effects in humans and animals. Although DON is not genotoxic by itself, it has recently been shown that this toxin exacerbates the genotoxicity induced by model or bacterial genotoxins. Here, we show that five TCT, namely T-2 toxin (T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FX), and the newly discovered NX toxin, also exacerbate the DNA damage inflicted by various genotoxins. The exacerbation was dose dependent and observed with phleomycin, a model genotoxin, captan, a pesticide with genotoxic potential, and colibactin, a bacterial genotoxin produced by the intestinal microbiota. For this newly described effect, the trichothecenes ranked in the following order: T-2>DAS > FX > NIV ≥ DON ≥ NX. The genotoxic exacerbating effect of TCT correlated with their ribotoxic potential, as measured by the inhibition of protein synthesis. In conclusion, our data demonstrate that TCT, which are not genotoxic by themselves, exacerbate DNA damage induced by various genotoxins. Therefore, foodborne TCT could enhance the carcinogenic potential of genotoxins present in the diet or produced by intestinal bacteria.

Published
2023

5. The bacterial stress-responsive Hsp90 chaperone is required for the production of the genotoxin colibactin and the siderophore yersiniabactin by Escherichia coli

Author

Alexander O. Brachmann, Jörn Piel, Alex J. McCarthy, Sébastien Houle, Eric Oswald, Christophe Garcie, Jean-Philippe Nougayrède, Pierre Genevaux, Peter W. Taylor, Amélie Garénaux, Patricia Martin, Sophie Tronnet, Marie Penary, Charles M. Dozois, Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Institut de Recherche en Santé Digestive (IRSD ), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Fédérale Toulouse Midi-Pyrénées, Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), University College of London [London] (UCL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Service de Bactériologie-Hygiène [Toulouse], This work was supported by the Agence Nationale de la Recherche (France) [grants ANR-13-BSV3-0015-02 and ANR-13-BSV1-0028-01], by the Medical Research Council (UK) [grant MR/K018396/1 to A.J.M.], and by the Natural Sciences and Engineering Research Council (Canada) [Discovery grant to C.M.D.], ANR-13-BSV3-0015,RESTRICTIRON,Limitation du fer disponible pour les pathogènes : mécanismes et contribution à la défense antimicrobienne de l'hôte(2013), ANR-13-BSV1-0028,Coliforlife,Répercussions de la colonisation néonatale par Escherichia coli au sein du microbiote intestinal sur la susceptibilité à développer des pathologies immunes.(2013), Courcelles, Michel, Blanc 2013 - Limitation du fer disponible pour les pathogènes : mécanismes et contribution à la défense antimicrobienne de l'hôte - - RESTRICTIRON2013 - ANR-13-BSV3-0015 - Blanc 2013 - VALID, Blanc 2013 - Répercussions de la colonisation néonatale par Escherichia coli au sein du microbiote intestinal sur la susceptibilité à développer des pathologies immunes. - - Coliforlife2013 - ANR-13-BSV1-0028 - Blanc 2013 - VALID, CHU Toulouse [Toulouse], Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Laboratoire de microbiologie et génétique moléculaires (LMGM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], and Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], medicine.disease_cause, Yersiniabactin, chemistry.chemical_compound, Protein Interaction Mapping, Immunology and Allergy, Escherichia coli Infections, chemistry.chemical_classification, biology, Escherichia coli Proteins, yersiniabactin, meningitis, Endopeptidase Clp, 3. Good health, [SDV] Life Sciences [q-bio], Infectious Diseases, Biochemistry, Female, colibactin, Virulence, Hsp90, Microbiology, 03 medical and health sciences, Polyketide, Phenols, Nonribosomal peptide, Heat shock protein, medicine, Escherichia coli, Animals, HSP90 Heat-Shock Proteins, Rats, Wistar, siderophores, stress response, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Pathogenicity island, Mice, Inbred C57BL, virulence, Disease Models, Animal, Thiazoles, 030104 developmental biology, chemistry, Polyketides, Chaperone (protein), heat shock proteins, biology.protein, HtpG, Peptides, Gene Deletion, Mutagens
Abstract

International audience; The genotoxin colibactin synthesized by Escherichia coli is a secondary metabolite belonging to the chemical family of hybrid polyketide/non-ribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the High-Pathogenicity Island, encoding the siderophore yersiniabactin that belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90 involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to the eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence

Published
2016

6. HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis

Author

Emmanuelle Helloin, Sébastien Houle, Tetsuya Hayashi, Marie Penary, Patricia Martin, Gaëlle Porcheron, Kazunori Murase, Charles M. Dozois, Eric Oswald, Jean-Philippe Nougayrède, University of Miyazaki, Université Fédérale Toulouse Midi-Pyrénées, Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Laboratoire de Comportement, INRA-CNRS, 37380 Nouzilly, France, Institut National de la Recherche Agronomique (INRA), Department of Applied Chemistry, Osaka University [Osaka], CHU Toulouse [Toulouse], This work was supported by the French government, through the Investments for the Future program (ANR-11-EQPX-0003), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants-in-Aid for Young [B] 26860293 to K. M.), the National Science and Engineering Research Council of Canada (Discovery grant to C. M. D.), and a Canada Research Chair (to C. M. D.)., ANR-11-EQPX-0003/11-EQPX-0003,ANINFIMIP,Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées(2011), ANR-11-EQPX-0003,ANINFIMIP,Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées(2011), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Courcelles, Michel, Equipements plateforme animalerie infectieuse de haute-sécurité de Midi Pyrénées - - ANINFIMIP2011 - ANR-11-EQPX-0003 - EQPX - VALID, Institut de Recherche en Santé Digestive (IRSD ), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], MESH: Escherichia coli Proteins, Vacuole, MESH: Virulence, medicine.disease_cause, Virulence factor, Immunology and Allergy, MESH: Animals, MESH: Phylogeny, Escherichia coli Infections, Phylogeny, Extraintestinal Pathogenic Escherichia coli, MESH: Gene Expression Regulation, Bacterial, biology, Virulence, MESH: Escherichia coli, Escherichia coli Proteins, pathogenesis, MESH: Chickens, OMV, 3. Good health, [SDV] Life Sciences [q-bio], MESH: Mutagenesis, Site-Directed, Infectious Diseases, Bacterial outer membrane, Hemolysin Factors, Virulence Factors, MESH: Poultry Diseases, MESH: Vacuoles, Microbiology, 03 medical and health sciences, medicine, Autophagy, Escherichia coli, Animals, Humans, MESH: Autophagy, Poultry Diseases, MESH: Virulence Factors, MESH: Escherichia coli Infections, MESH: Humans, Cell Membrane, Pathogenic bacteria, Gene Expression Regulation, Bacterial, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, HlyF, 030104 developmental biology, Vacuoles, MESH: Hemolysin Factors, Mutagenesis, Site-Directed, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Chickens, Bacteria, MESH: Cell Membrane
Abstract

International audience; Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

Published
2016

7. Distinct Chk2 Activation Pathways Are Triggered by Genistein and DNA-damaging Agents in Human Melanoma Cells

Author

Caroline Baudouin, Bernard Ducommun, Nathalie Escalas, Francoise Goubin-Gramatica, Fabrice Casagrande, Jean-Marie Darbon, and Marie Penary

Subjects
G2 Phase, Cell cycle checkpoint, DNA damage, Genistein, Cell Cycle Proteins, Protein Serine-Threonine Kinases, Biology, Biochemistry, Wortmannin, chemistry.chemical_compound, Caffeine, Tumor Cells, Cultured, Humans, cdc25 Phosphatases, Melanoma, Molecular Biology, Checkpoint Kinase 2, Etoposide, Cyclin-dependent kinase 1, Kinase, Cell growth, Choroid Neoplasms, Cell Cycle, Cell Biology, Cell biology, Enzyme Activation, chemistry, Doxorubicin, Protein Kinases, Cell Division, DNA Damage
Abstract

Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.

Published
2000

8. Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

Author

Eric Oswald, Christian Chalut, Marie Penary, Patricia Martin, Maïwenn Olier, Jean-Philippe Nougayrède, Delphine Payros, Michèle Boury, Marc Audebert, Christophe Garcie, Ingrid Marcq, Sören Schubert, Giuseppe Magistro, Centre de Physiopathologie Toulouse Purpan (CPTP - U1043 INSERM - UMR5282 CNRS - UT3), 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), Service de Bactériologie-Hygiène [CHU Toulouse] (Hôpital Purpan), Centre Hospitalier Universitaire de Toulouse, Neuro-Gastroentérologie et Nutrition (NGN), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole supérieure d'agriculture de Purpan (ESAP), ToxAlim (ToxAlim), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut de pharmacologie et de biologie structurale (IPBS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre de Physiopathologie Toulouse Purpan (CPTP), CHU Toulouse [Toulouse], Ecole supérieure d'agriculture de Purpan (ESAP)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Métabolisme et Xénobiotiques (ToxAlim-MeX), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), FEDER (MYCA), Centre de Physiopathologie de Toulouse Purpan (CPTP, Sepsis: the critical role of iron), French National Research Agency (ANR), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chalut, Christian, Service Bactériologie et hygiène [CHU Toulouse], Institut Fédératif de Biologie (IFB), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Pôle Biologie [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), and Institut National de la Recherche Agronomique (INRA)-Ecole supérieure d'agriculture de Purpan (ESAP)

Subjects
Siderophore, GENE-CLUSTER, Siderophores, Transferases (Other Substituted Phosphate Groups), HIGH-PATHOGENICITY ISLAND, COMPLETE GENOME SEQUENCE, PHOSPHOPANTETHEINYL TRANSFERASE, PHYLOGENETIC DISTRIBUTION, 4'-PHOSPHOPANTETHEINYL TRANSFERASE, IRON ACQUISITION, MAMMALIAN-CELLS, IN-VIVO, VIRULENCE, medicine.disease_cause, Yersiniabactin, chemistry.chemical_compound, Mice, Gene cluster, Biology (General), Escherichia coli Infections, ComputingMilieux_MISCELLANEOUS, Genetics, 0303 health sciences, Escherichia coli Proteins, Glycopeptides, Isoenzymes, Female, Research Article, Genomic Islands, QH301-705.5, Immunology, Virulence, Biology, Microbiology, Enterobactin, 03 medical and health sciences, Bacterial Proteins, Phenols, Virology, Sepsis, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Escherichia coli, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, 030304 developmental biology, 030306 microbiology, RC581-607, Pathogenicity island, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Mice, Inbred C57BL, Thiazoles, chemistry, Polyketides, Mutation, Parasitology, Immunologic diseases. Allergy, Peptides, Gene Deletion, Mutagens
Abstract

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies., Author Summary The synthesis of numerous molecules involved in the virulence potential and fitness of pathogenic bacteria requires a particular enzyme family, i.e. phosphopantetheinyl transferases (PPTases). To date, the synthesis of a given bioactive metabolite was thought to require a specific PPTase. As PPTases are being investigated as promising targets for antibacterial development, we addressed the question of a possible functional interchangeability between PPTases in Escherichia coli. PPTases are known to be involved in the synthesis of low-molecular weight iron chelators (siderophores), and of a genotoxin named colibactin. Here we demonstrated interplay between the synthesis pathways leading to the production of siderophores and of colibactin. We showed that inactivation of both PPTases abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model. To our knowledge, this is the first demonstration of interplay between multiple PPTases-requiring pathways leading to the biosynthesis of functionally distinctive virulence factors, in a given microorganism. The extensive substrate specificity of PPTase ClbA could account for the co-selection and co-evolution of genomic islands encoding colibactin and yersiniabactin siderophore.

Published
2013

9. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity

Author

Maïwenn Olier, Christel Salvador-Cartier, Michèle Boury, Thomas Secher, Ulrich Dobrindt, Jean Fioramonti, Ingrid Marcq, Eric Gaultier, Marie Penary, Jean-Philippe Nougayrède, Valerie Bacquie, Eric Oswald, ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Centre de Physiopathologie Toulouse Purpan (CPTP), 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), Neuro-Gastroentérologie et Nutrition (NGN), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole supérieure d'agriculture de Purpan (ESAP), Centre de Physiopathologie de Toulouse Purpan, Institut National de la Recherche Agronomique (INRA), University of Münster, CHU Toulouse [Toulouse], non-profit Association F. Aupetit (AFA), ANR-09-MIEN-0005,COLIBACTIN,Conséquences sur la santé de la colonisation néonatale du tractus digestif par des entérobactéries produisant la Colibactine(2009), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Centre de Physiopathologie de Toulouse-Purpan (INSERM U563 - CNRS UMR1037), CHU Toulouse [Toulouse]-Institut Claudius Regaud-Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de lutte contre le cancer (CLCC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Physiopathologie Toulouse Purpan ex IFR 30 et IFR 150 (CPTP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire de Purpan (CHU Purpan), ANR-09-MIEN-005-01, AFA, Institut National de la Recherche Agronomique (INRA)-Ecole supérieure d'agriculture de Purpan (ESAP), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Jean-Philippe, Nougayrede, and MIE : Maladies infectieuses, immunité et environnement - Conséquences sur la santé de la colonisation néonatale du tractus digestif par des entérobactéries produisant la Colibactine - - COLIBACTIN2009 - ANR-09-MIEN-0005 - MIE - VALID

Subjects
Male, colitis, Mutant, medicine.disease_cause, law.invention, Probiotic, Gene Knockout Techniques, law, genotoxin, DSS, 0303 health sciences, Gastroenterology, Colibactin, 3. Good health, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, CD4(+) CD45RB(high) T-cell transfer model, probiotic, Research Paper, Microbiology (medical), DNA damage, Biology, Microbiology, Cell Line, 03 medical and health sciences, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Escherichia coli, Animals, Colitis, Rats, Wistar, Gene, 030304 developmental biology, 030306 microbiology, Probiotics, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Epithelial Cells, medicine.disease, biology.organism_classification, Inflammatory Bowel Diseases, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Rats, Disease Models, Animal, inflammation, Polyketides, CD4+ CD45RBhigh T-cell transfer model, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Genotoxicity, Bacteria, Mutagens
Abstract

International audience; Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Published
2012

10. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways

Author

Jean-Philippe Nougayrède, C. Erec Stebbins, Ascel Samba-Louaka, Eric Oswald, Brenda A. Schulman, Claude Watrin, David M. Duda, Grégory Jubelin, Yun Hsu, Marie Penary, Rika Nobe, Frédéric Taieb, Unité de Microbiologie (MIC), Institut National de la Recherche Agronomique (INRA), Interactions hôtes-agents pathogènes [Toulouse] (IHAP), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Department of Structural Biology, Howard Hughes Medical Institute (HHMI)-St Jude Children's Research Hospital, Rockefeller University [New York], Institut de Recherche en Santé Digestive (IRSD ), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), St Jude Children's Research Hospital, Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), taieb, frederic, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and CHU Toulouse [Toulouse]

Subjects
Infectious Diseases/Gastrointestinal Infections, Cell, Cell Biology/Cell Growth and Division, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], NEDD8, Cell Biology/Cell Signaling, Infectious Diseases/Bacterial Infections, sécrétion, Ubiquitin, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], invertébré, Microbiology/Parasitology, Biology (General), Host cell nucleus, Cells, Cultured, Escherichia coli Infections, 0303 health sciences, ubiquitine, biology, Escherichia coli Proteins, 030302 biochemistry & molecular biology, Cell Cycle, Microbiology and Parasitology, Intracellular Signaling Peptides and Proteins, in vitro, Cell cycle, Microbiologie et Parasitologie, 3. Good health, Cell biology, Ubiquitin ligase, Protein Transport, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Signal transduction, escherichia coli, bactérie pathogène, Cyclin-Dependent Kinase Inhibitor p27, Signal Transduction, Research Article, Infectious Diseases/Tropical and Travel-Associated Diseases, Cyclin-Dependent Kinase Inhibitor p21, NEDD8 Protein, QH301-705.5, Immunology, Blotting, Western, Microbiology, 03 medical and health sciences, Virology, Two-Hybrid System Techniques, Genetics, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, Immunoprecipitation, [SDV.EE.SANT] Life Sciences [q-bio]/Ecology, environment/Health, Ubiquitins, Molecular Biology, Actin, 030304 developmental biology, Cell Nucleus, [SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health, SKP Cullin F-Box Protein Ligases, Ubiquitination, [SDV.EE.IEO] Life Sciences [q-bio]/Ecology, environment/Symbiosis, RC581-607, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, vertébré, Actins, Rats, biology.protein, Parasitology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immunologic diseases. Allergy, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.

Published
2010

11. In vivo restoration of RhoB expression leads to ovarian tumor regression

Author

Muriel Golzio, Cuider Allal, Bettina Couderc, Marie Penary, Anne Pradines, Arash Rafii, Gilles Favre, D Berg, A. Deviers, and Justin Teissié

Subjects
Cancer Research, Tumor suppressor gene, RHOB, Genetic Vectors, Immunoblotting, Biology, Adenocarcinoma, Metastasis, Adenoviridae, Ovarian tumor, Peritoneal cavity, Mice, Cell Line, Tumor, RhoB GTP-Binding Protein, medicine, Animals, Humans, rhoB GTP-Binding Protein, Molecular Biology, Ovarian Neoplasms, Genetic Therapy, medicine.disease, Gene Expression Regulation, Neoplastic, medicine.anatomical_structure, Microscopy, Fluorescence, Cancer research, Molecular Medicine, Ectopic expression, Female, Ovarian cancer
Abstract

Ovarian cancers are very aggressive cancers most often diagnosed when metastasis has already occurred in the entire peritoneal cavity. Ovarian adenocarcinoma cells present an undetectable level of RhoB GTPase. Using preclinical ovarian cancer models, we aimed to evaluate the potential use of RhoB cDNA as a tumor suppressor gene in gene therapy. RhoB restoration in vitro, through recombinant adenovirus transduction, resulted in the apoptosis of endogenous RhoB protein low-expressing cell lines (OVCAR-3 and IGROV-1) through the activation of the intrinsic apoptotic caspase cascade. We showed that a single injection of 10(8) p.f.u. of adenoviral vector encoding a reporter gene into the peritoneal cavity of ovarian tumor bearing mice can induce the gene modification of a large quantity of cells throughout the cavity. We thereby tested the effect of AdRhoB injections to treat ovarian cancer-bearing mice. The ectopic expression of RhoB, following its introduction via viral transduction into nude mice in vivo, was highly effective in suppressing tumor growth of ovarian cancer xenografts. Therapeutic agents designed to correct defects of RhoB at the molecular level may thereby provide innovative treatment options for patients not responding to standard therapies.

Published
2008

12. MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling

Author

Cyril Favard, Daniel Tovar, Barbara Lortal, Isabelle Lajoie-Mazenc, Marie Penary, Anne Pradines, Sophie Allart, Gilles Favre, Meryem Brihoum, Departement /u563 : Oncogenèse, Signalisation et Innovation thérapeutique, Centre de Physiopathologie Toulouse Purpan (CPTP), 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)-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 Claudius Regaud, Institut Claude de Préval (ICP), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut FRESNEL (FRESNEL), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Claudius Regaud, Université de Toulouse (UT)-Université de Toulouse (UT)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Signal Transduction, MESH: Epidermal Growth Factor, RHOA, Receptor expression, RHOB, Endocytic cycle, Fluorescent Antibody Technique, MESH: Amino Acid Sequence, MESH: Base Sequence, Biochemistry, Epidermal growth factor, MESH: RNA, Small Interfering, MESH: Microscopy, Confocal, RNA, Small Interfering, rhoB GTP-Binding Protein, MESH: Fluorescent Antibody Technique, 0303 health sciences, MESH: Guanosine Triphosphate, Microscopy, Confocal, biology, 030302 biochemistry & molecular biology, Cell biology, ErbB Receptors, Endocytic vesicle, Guanosine Triphosphate, Microtubule-Associated Proteins, Protein Binding, Signal Transduction, MESH: Cell Line, Tumor, Endosome, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Molecular Sequence Data, MESH: Receptor, Epidermal Growth Factor, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 03 medical and health sciences, Cell Line, Tumor, MESH: Protein Binding, Humans, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, MESH: rhoB GTP-Binding Protein, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, Epidermal Growth Factor, Cell Biology, Actin cytoskeleton, MESH: Microtubule-Associated Proteins, biology.protein
Abstract

International audience; Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.

Published
2007

13. Reversible inactivation of the transcriptional function of P53 protein by farnesylation

Author

Marie Penary, Anne Pradines, Danièle Berg, Gilles Favre, Antoine Casteignau, Mustapha Tohfe, Bettina Couderc, Departement /u563 : Oncogenèse, Signalisation et Innovation thérapeutique, Centre de Physiopathologie Toulouse Purpan (CPTP), 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)-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 Claudius Regaud, FTI-277 was generously provided by S. Sebti, Moffitt Cancer Center, USF, Tampa, FL with the permission of the University of Pittsburgh. This work was supported by INSERM (F), a grant from the REGION MIDI-PYRENNES (F) and a grant from the University Paul Sabatier (F). We are grateful to the GVPN Network (Genethon, Evry) which provides us with the AdeGFP., and Maylin, Françoise

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Apoptosis, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, MESH: Genetic Vectors, Enzyme Inhibitors, Cellular localization, 0303 health sciences, Farnesyl Transferase Inhibitor, MESH: Adenoviridae, Biochemistry, MESH: Recombinan, MESH: Enzyme Inhibitors, 030220 oncology & carcinogenesis, Biotechnology, Research Article, Transcriptional Activation, MESH: Cell Line, Tumor, MESH: Mutation, lcsh:Biotechnology, Recombinant Fusion Proteins, Genetic Vectors, MESH: Protein Isoprenylation, Protein Prenylation, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Adenoviridae, Proto-Oncogene Proteins p21(ras), 03 medical and health sciences, MESH: Proto-Oncogene Proteins p21(ras), Prenylation, [SDV.CAN] Life Sciences [q-bio]/Cancer, lcsh:TP248.13-248.65, MESH: Cell Proliferation, Cell Line, Tumor, Farnesyltranstransferase, Humans, MESH: Farnesyltranstransferase, 030304 developmental biology, Cell Proliferation, MESH: Humans, C-terminus, MESH: Apoptosis, Cell Membrane, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Protein Structure, Tertiary, Mutation, Protein prenylation, Lipid modification, Tumor Suppressor Protein p53, MESH: Cell Membrane
Abstract

Background The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function. Results We constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21waf1/CIP1 and Bax transcription, cell growth arrest, caspase activation and apoptosis. Conclusion Artificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor.

Published
2005

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