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Etude De Petits ARN Régulateurs Chez Helicobacter Pylori

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Etude De Petits ARN Régulateurs Chez Helicobacter Pylori UFR Sciences de la Vie, Laboratoire INSERM U869, 146, rue Léo Saignat. 146, rue Léo Saignat. 33076 Bordeaux Cedex 33076 Bordeaux Cedex Thèse de Doctorat de l’Université Bordeaux II – Victor Segalen Ecole doctorale des Sciences de la Vie et de la Santé Option : Microbiologie Etude de petits ARN régulateurs chez Helicobacter pylori Présentée et soutenue publiquement Le 14 Décembre 2010, Par Jérémy Reignier Né le 11 Février 1984, au Creusot Membres du Jury Mr. Christophe Cullin, Professeur des Universités (CNRS, Bordeaux) Président Mme. Hilde de Reuse, Directeur de Recherche (Institut Pasteur, Paris) Rapporteur Mr. Francis Repoila, Chargé de Recherche (INRA, Paris) Rapporteur Mr. Pablo Radicella, Directeur de Recherche (CEA, Paris) Examinateur Mr. Philippe Lehours, Maitre de conférences (INSERM, Bordeaux) Examinateur Mr. Fabien Darfeuille, Chargé de Recherche (INSERM Bordeaux) Directeur de Thèse 1 Remerciements Je tiens à remercier le Professeur Christophe Cullin pour avoir accepté de présider mon jury de thèse. J’exprime également mes sincères remerciements envers le Docteur Hilde de Reuse et le Docteur Francis Repoila, qui ont accepté de corriger mon travail de thèse. Je remercie aussi le Docteur Pablo Radicella et le le Docteur Philippe Lehours, qui ont aimablement accepté de participer à mon jury de thèse. Je remercie le Docteur Fabien Darfeuille, mon directeur de thèse, pour sa disponibilité, le soutien constant qu’il m’a apporté tout au long de ces trois années et auprès duquel j’ai beaucoup appris. Je le remercie aussi pour la patience dont il a parfois dû faire preuve (il faut bien l’avouer !). Sa passion pour les sciences, son honnêteté et son éloquence sur une large variété de sujets resteront pour moi d’excellents souvenirs. Je remercie également le Docteur Jean-Jacques Toulmé pour m’avoir accueilli et permis de réaliser ce travail au sein de son unité, le laboratoire INSERM U869. Je remercie les groupes du Professeur Jôrg Vogel et du Professeur Peter Stadler, pour la collaboration que nous avons entretenue tout au long de ces trois années et pour nous avoir accueilli plusieurs fois dans la belle cité berlinoise. Je tiens à adresser également un grand merci à toute l’équipe de l’ « Unité postulante de pathogénèse de Helicobacter » dirigée par le Docteur Hilde de Reuse, pour sa grande expertise d’Helicobacter pylori, qu’elle a aimablement et régulièrement partagée avec notre équipe. Je tiens à remercier toutes les personnes de l’unité INSERM U869 qui ont apporté leur aide précieuse à la réalisation de mon projet de thèse : Merci Sandrine pour ta bonne humeur et ton expertise en biologie moléculaire. Merci Hélène, pour ton soutien et ton implication dans le projet. Merci pour ton dynamisme et tes éclats de rire. Merci aux docteurs Cathy Staedel et Isabelle Iost pour leur gentillesse et leur regard critique sur le projet. Merci au « très bientôt Docteur» Cédric Belair, pour ses conseils techniques. Merci au Docteur Jaydip Gosh, pour les nombreuses discussions que nous avons échangées, sur tous les sujets, et qui ont permis de m’enrichir autant sur le plan scientifique que culturel Merci à toutes les stagiaires qui ont participé au projet : Merci à Claire et Jessica (désormais thésardes, bravo les filles), Merci à Valérie à l’accent québécois si charmant et merci à Madline, sérieuse et toujours motivée. Merci à tous les membres de l’équipe chimie de l’unité INSERM U869 : Bernard, Laurent, Arnaud, Alex, Isabelle et Phlippe, grâce à qui j’ai pu parfois détérerrer de lointains souvenirs de chimie. Je remercie tout particulièrement Nathalie, qui m’a apporté son aide à plusieurs reprises et Salim, pour sa jovialité toujours contagieuse, son humanité, et sa culture qu’il aime à partager. 2 Un grand merci aussi à mes collègues de bureau ou de paillasse, présents ou passés, Ahissan, Khalid, Amit, Guilhem, Emilie, Laurent, Christophe, pour les bons moments passés ensemble. Je remercie également Kati pour son soutien administratif. Et je n’oublie pas Jacques et Sonia, pour leur gentillesse et leur humour, qui ont maintes fois animés les couloirs du laboratoire. Merci aussi à Jacques pour avoir élargi mes connaissances sur les champignons, sur un plan toutefois plus gastronomique que microbiologique. Enfin, j’adresse les plus sincères remerciements à tous mes proches, qui m’ont aimé, aidé et soutenu tout au long de cette aventure. Je remercie tout particulièrement mon frère Valentin, mes grands parents, Laura, et bien entendu mes parents Maria et Jean-François, à qui je dédie cette thèse. 3 Table des matières Abréviations ................................................................................................................................................... 7 Organismes .................................................................................................................................................... 8 Liste des illustrations ..................................................................................................................................... 9 Liste des tableaux ........................................................................................................................................ 11 Chapitre 1 : Les ARN régulateurs bactériens ............................................................................................... 13 1. Introduction ..................................................................................................................................... 13 2. Caractéristiques et modes d’action ................................................................................................. 16 2.1. Mécanisme antisens ................................................................................................................ 17 2.2. Inhibition de la traduction ....................................................................................................... 19 2.3. Activation de la traduction ...................................................................................................... 21 2.4. Action sur la stabilité des ARNm ............................................................................................. 22 2.5. Les petits ARN ciblant les protéines régulatrices .................................................................... 23 2.6. Facteurs influençant la régulation par les petits ARN ............................................................. 26 2.7. Conclusion ............................................................................................................................... 30 3. Implication des petits ARN bactériens dans la virulence et l’adaptation à l’hôte .......................... 31 3.1. Introduction ............................................................................................................................. 31 3.2. Réponse aux stress .................................................................................................................. 32 3.3. Contrôle de l’expression des gènes de virulence .................................................................... 34 3.4. Quorum-sensing ...................................................................................................................... 37 4. Les sRNA codés en cis au sein des systèmes toxines/antitoxines de type I .................................... 38 4.1. Introduction ............................................................................................................................. 38 4.2. Découverte et distribution des cassettes TA de type I ............................................................ 40 4.3. Régulation ................................................................................................................................ 42 4.1. Propriétés des toxines ............................................................................................................. 45 4.2. Fonctions biologiques .............................................................................................................. 46 5. Méthodes d’identification des petits ARN régulateurs ................................................................... 50 5.1. Prédiction bio-informatique .................................................................................................... 51 5.2. Les analyses globales : Microarray et Hfq-coimmunoprécipitation ........................................ 53 5.3. La RNomique (clonage shotgun) ............................................................................................. 54 5.4. L’ère du séquençage haut-débit .............................................................................................. 55 Chapitre 2 : Helicobacter pylori ................................................................................................................... 57 1. Introduction ..................................................................................................................................... 57 4 2. Facteurs bactériens impliqués dans la pathogénèse de l’infection à H. pylori ............................... 58 2.1. Colonisation de la niche gastrique .......................................................................................... 60 2.2. Facteurs de pathogénicité ....................................................................................................... 62 3. Le génome de la bactérie ................................................................................................................ 65 3.1. Diversité
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