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La Déramification Des Polysaccharides Définit Un Mécanisme Universel Et Polyphylétique Pour La Synthèse D’Amidon Chez Les Bactéries Et Les Eucaryotes

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La Déramification Des Polysaccharides Définit Un Mécanisme Universel Et Polyphylétique Pour La Synthèse D’Amidon Chez Les Bactéries Et Les Eucaryotes Thèse de Ugo Pierre Cenci, Lille 1, 2013 UNIVERSITE DES SCIENCES ET TECHNOLOGIES DE LILLE ECOLE DOCTORALE BIOLOGIE SANTE LILLE NORD DE FRANCE Thèse de Doctorat / Doctorate Thesis Ugo Pierre CENCI En vue de l’obtention du titre de : Docteur de l’Université des Sciences et Technologies Lille 1 Spécialité : Génétique Microbienne / Microbial Genetics La déramification des polysaccharides définit un mécanisme universel et polyphylétique pour la synthèse d’amidon chez les bactéries et les eucaryotes Polysaccharide debranching defines a universal and polyphyletic mechanism for starch accumulation in bacteria and eukaryotes 18th October 2013 Jury: Président : Pr. C. D’Hulst Université Lille 1 Rapporteurs : Pr. W. Löffelhardt Universität Wien Dr. B. Henrissat DR CNRS Université d’Aix-Marseille Examinateurs : Pr. Y. Nakamura Akita Prefectural University Pr. D. Bhattacharya Rutgers University Dr. E. Suzuki Akita Prefectural University Dr. J.-L Putaux DR CNRS Université Joseph Fourrier Dr. C. Colleoni Université Lille 1 Directeur de thèse : Pr. S.G. Ball Université Lille 1 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 UNIVERSITE DES SCIENCES ET TECHNOLOGIES DE LILLE ECOLE DOCTORALE BIOLOGIE SANTE LILLE NORD DE FRANCE Thèse de Doctorat / Doctorate Thesis Ugo Pierre CENCI En vue de l’obtention du titre de : Docteur de l’Université des Sciences et Technologies Lille 1 Spécialité : Génétique Microbienne / Microbial Genetics La déramification des polysaccharides définit un mécanisme universel et polyphylétique pour la synthèse d’amidon chez les bactéries et les eucaryotes Polysaccharide debranching defines a universal and polyphyletic mechanism for starch accumulation in bacteria and eukaryotes 18th October 2013 Jury: Président : Pr. C. D’Hulst Université Lille 1 Rapporteurs : Pr. W. Löffelhardt Universität Wien Dr. B. Henrissat DR CNRS Université d’Aix-Marseille Examinateurs : Pr. Y. Nakamura Akita Prefectural University Pr. D. Bhattacharya Rutgers University Dr. E. Suzuki Akita Prefectural University Dr. J.-L Putaux DR CNRS Université Joseph Fourrier Dr. C. Colleoni Université Lille 1 Directeur de thèse : Pr. S.G. Ball Université Lille 1 1 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 Résumé A l’état natif, l'amidon s’agrège sous forme de grains insolubles semicristallins, à la différence des particules de glycogène qui sont hydrosolubles. Chez les eucaryotes photosynthétiques, le passage à l'accumulation de l'amidon est survenu après l'endosymbiose du plaste, à partir d'un réseau métabolique synthétisant du glycogène cytosolique chez l'hôte. Ceci a impliqué le recrutement d'une enzyme de débranchement d'origine chlamydienne, un parasite intracellulaire obligatoire. Cette enzyme de débranchement est considérée comme responsable de l’épissage des branches mal placées au sein d’un polysaccharide précurseur qui, sans ce mécanisme, serait soluble dans l'eau. Nous présentons l'implication de l'enzyme de débranchement dans le processus d’édification de l’amidon dans des cyanobactéries unicellulaires qui accumulent à la fois du glycogène et des grains d'amidon semicristallins. Nous montrons que, dans ces organismes, une enzyme de nature analogue à l'enzyme de débranchement de la plante, mais d'origine bactérienne différente, a été recrutée pour la même fonction. Les enzymes de débranchement des cyanobactéries et des plantes ont évolué séparément à partir d’une enzyme impliquée dans le catabolisme du glycogène pour donner de l'amidon. La nature polyphylétique de la transition de l’accumulation de glycogène à celle d'amidon, chez les Archaeplastida et les cyanobactéries, ainsi que l'évolution convergente de mécanismes biochimiques communs, suggèrent que le débranchement des polysaccharides au cours de leur synthèse est une exigence universelle de l'évolution du métabolisme de l'amidon dans les cellules vivantes. Mots clefs : Amidon, Isoamylase, Enzyme de débranchement, Glycogène, Cyanobacterie, Evolution, Archaeplastida, Cyanobacterium sp. CLg, Polysaccharide cristallin. 2 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 Abstract Starch, unlike hydrosoluble glycogen particles aggregates into insoluble, semi-crystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a pre-existing cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semi-crystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably both the plant and cyanobacterial enzymes have evolved a novel yet identical substrate specificity from a preexisting enzyme which originally displayed the much narrower substrate preferences required for glycogen catabolism. The polyphyletic nature of the transition from glycogen to starch in Archaeplastida and cyanobacteria together with the convergent evolution of a common biochemical mechanism suggest that polysaccharide debranching defines a universal requirement for the evolution of starch metabolism in living cells. Key words: Starch, Isoamylase, Debranching enzyme, Glycogen, Cyanobacteria, Evolution, Archaeplastida, Cyanobacterium sp. CLg1, Cristalline polysaccharide. 3 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 Remerciements Ce qui me plaît dans les remerciements ; c’est qu’ils peuvent être complètement décousus, comme mon esprit ! Alors à ceux qui liront ces lignes si vous ne comprenez pas tout, tant pis ! Pourvu que vous compreniez ce qui est pour vous. Tout d’abord je remercie tous les membres de mon jury d’avoir bien voulu prendre du temps pour juger mon travail. I would first like thank my jury members to have accepted this work. Je remercie particulièrement, les Drs J.-L Putaux et B. Henrissat, pour les échanges très intéressants autour de mon manuscrit. Ensuite, je dois remercier ceux grâce à qui j’ai attrapé une crampe au poignet, un problème aux yeux… Pour rédiger ma thèse bien évidemment ! « C'est le rôle essentiel du professeur d'éveiller la joie de travailler et de connaître. » Albert Einstein Je dédie cette phrase à ceux qui ont su éveiller cette joie, aussi bien : Her Docteur Professeur Steven G. Ball que Her Docteur Christophe Colleoni. Mes remerciements vont aussi aux différentes personnes que j’ai croisées pendant cette thèse et qui m’ont accueilli, et aidé à avancer dans ma thèse : Dr JL Putaux et Amandine Durand Terrasson, Pr D. Bhattacharya et toute son équipe, en particulier Dr Huan Qiu pour l’aide en phylogénie, et Dr M.-M. Perrineau pour les pauses midi et les discussions scientifiques. Toutefois, je n’oublie pas mes professeurs qui m’ont éveillé -voire réveillé !- durant ma scolarité. Je remercie aussi Maria Cecilia Arias pour son aide en bioinfo et dans la rédaction de ma thèse, ainsi que Catherine Tirtiaux et Dr Jenifer Nirmal Raj qui ont abattu un énorme travail, et permis cette thèse. Enfin une pensée pour ceux avec qui j’ai travaillé à l’UGSF. « Un enfant pourra difficilement transmettre plus tard ce qu'il ne reçoit pas de ses parents. » P.D James Donc, avant tout cela, il y a eu ceux qui m’ont préparé lorsque je n’étais que petit garçon, et qui m’ont armé pour cette expédition. Ceux qui m’ont appris à marcher droit devant (Mes 4 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 parents et ma famille proche : Pépé, Grand Papa, Mémé, Mamé, Papa, Maman, Gilles, Marion, Fabien, Léo). Sans oublier ceux qui m’ont aidé à corriger mon bout de thèse en français. Je n’oublie évidemment pas toutes les personnes que j’ai croisées plus récemment et qui font maintenant partie de ma famille (ta famille Gillou, et les pièces rapportées en Jul… qui prennent soin des yeux verts). Je vous remercie, vous, ma famille algérienne d’ici (y en a pas tant que ça : Sara et Fatiha), et d’au-delà des océans et des mers : celle des Etats Unis - sans laquelle mon passage aux USA aurait été sérieusement compromis : Nasser, Aziza et leurs enfants, et, celle d’Algérie - là, ça fait beaucoup de monde… je n’en nommerai que deux : les parents de Malika : Rabah et Megdouda. « Aux vrais amis tout est commun » Euripide Merci à vous mes amis des différents coins du monde et à ceux ici ; aussi bien les sportifs, que les rigolos de l’UGSF, et avec qui j’ai pu partager de la bonne humeur, et passé du bon temps. Je voudrais aussi faire une spéciale dédicace à ceux avec qui j’ai partagé la pipette ici (Mathieu Ducatez, Laura Boyer, Maud Facon, Justin Findinier, Hande Tuncay, Derifa Kadouche, Jenifer Nirmal Raj). « A deux vous êtes une équipe qui gagne » Gilles et Marie Françoise Arcis Et surtout Malika que j’aime. Je te remercie, ma femme, toi, qui as permis que j’arrive jusqu’ici : tu fais tant de choses si bien pour moi, veillant au moindre détail... y compris de la coupe de cheveux à l’étincelant de ma bague ! Toi, avec qui je discute science, ce qui nous permet de progresser mutuellement. 5 © 2014 Tous droits réservés. http://doc.univ-lille1.fr Thèse de Ugo Pierre Cenci, Lille 1, 2013 Table of Contents Résumé 2 Abstract 3 Remerciements/Acknowledgments 4 Table of Contents 6 Abréviations en français 9 Abbreviations 12 Foreword : Description of my works 15 Aim of this study 18 Résumé en français 19 1. Introduction générale 20 2. Chapitre évolution 22 2.1. Les endosymbioses et leurs rôles dans l’évolution du vivant 22 2.2. L’endosymbiose primaire du plaste ayant donné naissance aux Archaeplastida 23 2.3.
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