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Etude De La Méthylation De L'adn Chez La Bactérie Pathogène D'insectes

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Etude De La Méthylation De L'adn Chez La Bactérie Pathogène D'insectes Etude de la méthylation de l’ADN chez la bactérie pathogène d’insectes Photorhabdus luminescens Amaury Payelleville To cite this version: Amaury Payelleville. Etude de la méthylation de l’ADN chez la bactérie pathogène d’insectes Photorhabdus luminescens. Bactériologie. Université Montpellier, 2018. Français. NNT : 2018MONTG063. tel-02045899 HAL Id: tel-02045899 https://tel.archives-ouvertes.fr/tel-02045899 Submitted on 22 Feb 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE M ONTPELLIER En Biologie des Interactions - Mécanismes des Interactions Parasitaires Symbiotiques et Pathogènes École doctorale GAIA Unité de recherche 1333, DGIMI Titre de la thèse Étude de la méthylation de l’ADN chez Photorhabdus luminescens TT01 Présentée par Amaury PAYELLEVILLE Le 16 Novembre 2018 Sous la direction de Julien BRILLARD et Alain GIVAUDAN Devant le jury composé de Didier LERECLUS, DR, INRA Rapporteur Eric CASCALES, DR, CNRS Rapporteur Alice GUIDOT, CR, INRA Examinatrice Mathieu SICARD, PR, UM Examinateur Julien BRILLARD, CR, INRA Directeur de thèse Alain GIVAUDAN, DR, INRA Co-Directeur de thèse À mes chers parents, Monique VINOT et Alain PAYELLEVILLE Résumé : Photorhabdus luminescens est une entérobactérie retrouvée en symbiose avec les nématodes du genre Heterorhabditis . Dans les sols, ce complexe némato-bactérien est pathogène d’insectes ravageurs et est utilisé en contrôle biologique. Le nématode pénètre dans l’insecte et libère la bactérie dans l’hémolymphe. Photorhabdus va ensuite se multiplier et secréter divers facteurs de virulence comme des toxines. L’insecte meurt de septicémie puis le nématode et la bactérie vont se nourrir du cadavre. Une fois les ressources épuisées, le complexe némato-bactérien va se reformer et sortir du cadavre à la recherche d’une nouvelle cible. Plusieurs exemples d’hétérogénéité phénotypique ont été décrits chez cette bactérie amenant chacun à la présence de sous-populations dans une culture bactérienne. Cette hétérogénéité phénotypique peut-être causée par des mécanismes épigénétiques et plus précisément par la méthylation de l’ADN. Chez les entérobactéries, la méthyltransférase Dam est très conservée. Elle méthyle les adénines des sites GATC et est impliquée dans la réparation des erreurs lors de la réplication de l’ADN, la régulation du cycle cellulaire mais aussi la régulation de divers gènes. Cette méthyltransférase est en compétition avec certain régulateurs transcriptionnel. Selon qui de la méthyltransférase ou du régulateur se fixera en premier, le gène sera ou non exprimé donnant naissance à deux sous-populations. Cette thèse a pour objectif de mettre en évidence les rôles de la méthyltransférase Dam chez Photorhabdus luminescens . Dans un premier temps, j’ai montré que la surexpression de Dam (Dam+) amène une diminution de la mobil ité et du pouvoir pathogène de la bactérie mais à l’inverse augmente sa capacité à former des biofilms. Une analyse transcriptomique (RNAseq) a montré des différentiels d’expression de certains gènes impliqués dans les phénotypes observés. En recombinant l a souche de Photorhabdus Dam+ avec les nématodes hôtes, l’effet sur la pathogénicité a été augmenté en comparaison des résultats après injection de la bactérie seule. L’établissement de la symbiose némato-bactérienne avec cette souche Dam+ n’est pas signif icativement impacté par rapport à la souche sauvage. Enfin l’analyse du méthylome (détection de tous les sites méthylés sur le génome grâce à la technique SMRT) de Photorhabdus dans diverses phases de croissance nous a permis de déterminer que la méthylation par Dam semble être stable aux différents temps de la croissance bactérienne testés chez Photorhabdus . Le méthylome de la souche Dam+ a confirmé l’hypothèse que cette surexpression augmentait le taux de méthylation des sites GATC sur le génome. La comparaison combinée entre le RNAseq et les sites GATC différentiellement méthylés entre la souche contrôle et Dam+ a mis en évidence certains gènes candidats ressortant de ces deux analyses. En effet, certains gènes sont différentiellement exprimés dans les deux souches et ont un différentiel de méthylation au niveau de sites GATC dans leur région promotrice, l’étude détaillée de leur régulation par la méthylation fait maintenant partie des perspectives et permettront peut-être d’expliquer une partie des phénotypes observés chez Photorhabdus luminescens . Mots-clefs : Méthyltransférase, Virulence, Régulation de la transcription, Méthylome, RNAseq Abstract : Photorhabdus luminescens is an Enterobacteriaceae found in soils in symbiosis with a nematode from the genus Heterorhabditis . This nemato-bacterial complex is highly pathogenic against insect pest crops and so used in biocontrol. The nematode enters into the insect and releases Photorhabdus in the hemolymph of the insect. Photorhabdus multiplies and produces diverse virulence factors as toxins. Insect die from septicemia and both nematodes and bacteria feed on the nutrients in the cadaver. Once nutrients are lacking, the nematodes and the bacteria reassociate and exit from the cadaver to find new insects to infect. Photorhabdus is switching between pathogenic and symbiotic state. This bacterium displays phenotypic heterogeneity as we observe subpopulations coexisting in a same bacterial culture. Phenotypic heterogeneity can be explained by epigenetic mechanisms such as DNA methylation. In Enterobacteriaceae , Dam methyltransferase is broadly distributed. It methylates the adenine of GATC sites. Dam is involved in post-replicative mismatch repair, cell-cycle regulation and also gene transcription regulation. This methyltransferase can be in competition with some transcriptional regulators. Depending on which will bind first on the promoter region, gene will be expressed or not, leading to the rise of two subpopulations. This thesis aims to understand roles of Dam in Photorhabdus luminescens . Overexpression of the methyltransferase leads to a decrease in motility and pathogenicity of Photorhabdus Dam+ strain whereas it increases biofilms formation. A transcriptomic analysis (RNAseq) revealed differential expression of genes involved in the observed phenotypes. Symbiosis establishment does not seem to be strongly impacted in Dam+ strain as the only difference observed when compared to the nematode associated with the control strain is the same as with bacteria alone (a delayed virulence). A methylome analysis was also done (screening of all methylated sites in the genome using SMRT sequencing) in several growth conditions which revealed that DNA methylation is stable over growth kinetics. Dam+ strain methylome analysis confirmed the hypothesis that Dam overexpression increases GATC methylation over the genome. Comparative analysis of methylome and RNAseq experiments between control and Dam+ strains highlighted several common genes. In fact, some genes are differentially expressed between both strains and also have GATC sites differentially methylated in their promoter region. Their transcription regulation by methylation is a future aim and may give some explanation for a part of the phenotypes observed in Photorhabdus luminescens . Keywords: Methyltransferase, Virulence, Gene transcription regulation, Methylome, RNAseq Remerciements Je tiens tout d’abord à remercier Didier LERECLUS et Eric CASCALES pour avoir accepté d’être membres rapporteurs du jury de cette thèse. Je tiens tout autant à remercier les deux membres examinateurs de ce jury : Alice GUIDOT et Mathieu SICARD. Je tiens a ussi à remercier l’école doctorale GAIA pour m’avoir financé et permis de réaliser ces trois ans de thèse ainsi que l’Université de Montpellier pour la formation m’ayant été fournie. J’aimerais aussi remercier l’INRA et plus particulièrement le département SPE m’ayant permis l’aboutissement de cette thèse. Merci aux membres de mon comité de thèse, Josep CASADES US, Anne-Blanc POTARD et Guillaume CHARRIERE pour leur aide lors des choix difficile et leur point de vue sur l’avancée de mes travaux. J’ai forcément envie d’adresser un énorme remerciement à mes encadrants, Julien BRILLARD et Alain GIVAUDAN. Tout d’abord Julien po ur le temps que tu as pris pour me former et la patience dont tu as fait preuve. Tu m’as permis de m’épanouir dans une thèse ou tout n’a pas toujours été facile. Ta confiance en moi et les libertés que tu m’as laissé prendre m’ont poussé à avancer et donner le meilleur au travers de cette épopée. Alain pour ta disponibilité malgré tes responsabilités et la qualité de tes réflexions sur mon travail. Vous m’avez tout deux permis d’arriver au bout de cette thèse et à me donner une envie de continuer dans la recherche encore plus grande qu’à mon arrivée dans ce laboratoire. Je n’oublierais pas non plus le s nombreuses discussions œnologiques nous ayant rassemblé et permis de s’éclipser du cadre du travail. Encore un immense merci à vous deux ! J’ai une grande pensée pour Anne LANOIS qui m’a accompagné et guidé dans mes diverses péripéties de biologie moléculaire sans perdre espoir et en m’en redonnant quand je n’en avais plus ! Cette thèse n’aurait pas été la même sans ton expérience et ton investissement. Je tiens aussi grandement à remercier Sylvie PAGES pour ton aide durant les suivis de pathogénèse et ta gentillesse. Jean -Claude OGIER pour ta bonne humeur et ton aide précieuse sur la phylogénie. Bernard DUVIC pour ta précieuse aide en informatique quand rien ne marc hait ainsi que Sophie GAUDRIAULT pour ton point de vue et tes opinions originales si importantes en science. Comment ne pas remercier la « Dream team » du labo Magalie EYCHENNE, Marie FRAYSSINET et Pierre -Alain GIRARD vous avez apporté votre bonne humeur dans ce travail et en dehors.
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