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Ehrlichia Ruminantium : Rôle Du Système De Sécrétion De Type IV Et Des Protéines De La Membrane Externe

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Ehrlichia Ruminantium : Rôle Du Système De Sécrétion De Type IV Et Des Protéines De La Membrane Externe UNIVERSITE DES ANTILLES ET DE LA GUYANE U.F.R. Sciences de la Vie et de la Terre THESE pour l’obtention du grade de DOCTEUR DE L’UNIVERSITE DES ANTILLES ET DE LA GUYANE Discipline : Sciences de la vie - Microbiologie Ecole Doctorale Pluridisciplinaire de l'Université des Antilles Guyane Présentée et soutenue publiquement par Amal MOUMENE Le 16 janvier 2015 Caractérisation de déterminants moléculaires du pouvoir pathogène d’Ehrlichia ruminantium : rôle du système de sécrétion de type IV et des protéines de la membrane externe Co-Directeurs de thèse : Thierry LEFRANCOIS et Olivier GROS Encadrant de thèse : Damien MEYER Devant le jury composé de : Matthieu ARLAT, Professeur de l’Université Paul Sabatier, Toulouse ; Rapporteur Matteo BONAZZI, Chargé de Recherche, HDR, CNRS, Montpellier ; Rapporteur Murielle VAYSSIER-TAUSSAT, Directrice de Recherche, Inra, Maison-Alfort ; Examinatrice Thierry LEFRANCOIS, Directeur Unité CMAEE, CIRAD, Montpellier ; Examinateur Olivier GROS, Professeur de l’Université Antilles-Guyane, Guadeloupe, Examinateur Damien MEYER, Chargé de Recherche, CIRAD, Guadeloupe ; Examinateur Recherches effectuées au sein de l’UMR CIRAD-INRA CMAEE, «Contrôle des maladies animales exotiques et émergentes », Domaine de Duclos, 97170 Petit-Bourg, Guadeloupe Financement de thèse : Union européenne, FEDER 1/1.4-30305, « Risques en santé animale et végétale » et CIRAD AUTEUR : Amal Moumène TITRE : Caractérisation de déterminants moléculaires du pouvoir pathogène d'Ehrlichia ruminantium: rôle du système de sécrétion de type IV et des protéines de la membrane externe. DIRECTEURS DE THESE : Thierry Lefrançois / Olivier Gros ENCADRANT : Damien Meyer RESUME Identifier les déterminants moléculaires des bactéries pathogènes et comprendre comment ils sont régulés pour permettre l’adaptation à l’environnement et à l’hôte est crucial pour imaginer des méthodes de contrôle innovantes et proposer des alternatives thérapeutiques. Ehrlichia ruminantium est une bactérie intracellulaire obligatoire de la famille des Anaplasmataceae, vectorisée par les tiques du genre Amblyomma et causant la cowdriose, une maladie fatale des ruminants. Dans le cadre de cette thèse, nous avons caractérisé certains déterminants du pouvoir pathogène d’E. ruminantium selon trois niveaux de résolution différents. Une approche globale sans a priori, nous a tout d’abord permis de déterminer le protéome de la membrane externe de la forme infectieuse d’E. ruminantium. Cette étude nous a permis d’avoir une meilleure vision de l’architecture de la membrane externe qui constitue la première interface d’échanges entre la bactérie et sa cellule hôte. Ensuite, nous avons montré la fonction d’ErxR comme régulateur central du système de sécrétion de type IV (SST4) et de la famille multigénique map1 en permettant d’intégrer les signaux environnementaux que sont la carence en fer et l’acidité du milieu. En outre, ce travail a permis d’établir pour la première fois le lien entre les protéines Map1 de la membrane externe et le SST4 et suggère donc qu’elles puissent avoir un rôle direct dans la virulence. Enfin, une analyse in silico utilisant le logiciel S4TE a conduit à la caractérisation d’Erip1, le premier effecteur du SST4 d’E. ruminantium. Cet effecteur, phosphorylé sur les tyrosines et injecté dans le noyau de la cellule hôte, ne présente aucune homologie dans les bases de données et pourrait donc représenter une nouvelle famille de nucléomodulines. La recherche d’éventuels partenaires protéiques et l’identification des cibles intracellulaires de cet effecteur permettront de mieux comprendre comment E. ruminantium manipule la cellule hôte à son profit. Enfin, la caractérisation des voies de signalisation ciblées par Erip1 sera riche d’enseignements sur la réponse cellulaire à l’infection par E. ruminantium. MOTS-CLES : Ehrlichia ruminantium, protéome, système de sécrétion de type IV, protéines de la membrane externe, Map1, ErxR, tr1, effecteurs, Erip1 DISCIPLINE : Microbiologie SOMMAIRE AVANT-PROPOS ................................................................................. 4 INTRODUCTION GENERALE ............................................................................. 6 Manuscrit préliminaire : Ehrlichia and Anasplama’s molecular tricks to manipulate their host ............................................................................................ 7 RESULTATS .............................................................................................. 33 PARTIE 1 ...................................................................................... 34 IDENTIFICATION DES PROTEINES DE LA MEMBRANE EXTERNE D’E. RUMINANTIUM ............... 34 1. Préambule ..................................................................................... 34 2. Publication: Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium ................................ 36 PARTIE 2 ...................................................................................... 80 ETUDE DE LA REGULATION DU SST4 ET DES PROTEINES MAP1 DE LA MEMBRANE EXTERNE D’E. RUMINANTIUM ................................................................................. 80 1. Préambule ..................................................................................... 80 2. Manuscrit préliminaire: Acidic and iron starvation conditions upregulate Ehrlichia ruminantium Type IV secretion system and map1 genes through the master regulatory protein ErxR ........................................................................ 82 PARTIE 3 ..................................................................................... 118 CARACTERISATION D’ERIP1, UN NOUVEL EFFECTEUR DU SST4 D’E. RUMINANTIUM .......... 118 1. Préambule ................................................................................... 118 2. Manuscrit préliminaire: Erip1, a new substrate of the E. ruminantium type IV secretion system, is tyrosine phosphorylated and imported into host cell nucleus119 DISCUSSION GENERALE ............................................................................. 154 DISCUSSION GENERALE ET PERSPECTIVES ..................................................... 155 REFERENCES BIBLIOGRAPHIQUES ................................................................. 169 ANNEXES .............................................................................................. 176 - 1 -1 Avant-Propos Avant-propos Les maladies animales et zoonotiques émergentes provoquent chaque année de lourdes pertes parmi les élevages et présentent un risque croissant en termes de santé humaine pour les populations. Certaines bactéries pathogènes intracellulaires obligatoires de la famille des Anaplasmataceae sont les principaux agents des maladies émergentes transmises par les tiques (Rikihisa 2010). Comprendre les mécanismes moléculaires impliqués dans l’interaction hôte-pathogène, et plus spécifiquement l’adaptation à l’hôte et la mise en place du pouvoir pathogène chez des bactéries telles que Ehrlichia spp. ou Anaplasma spp. revêt un enjeu majeur pour la recherche de nouveaux traitements contre ces bactéries pathogènes. Au laboratoire, nous étudions la bactérie Ehrlichia ruminantium, responsable d’une maladie mortelle des ruminants, la cowdriose. Alors que les bactéries du genre Ehrlichia subvertissent certains mécanismes majeurs de défense de l’hôte, les facteurs de virulence bactériens responsables de l’échec de ces défenses sont encore peu connus. Un des facteurs de virulence important pour l’infection intracellulaire chez les Ehrlichia est le système de sécrétion de type IV (SST4), qui délivre directement des protéines effectrices (ET4s) à l’intérieur des cellules hôtes eucaryotes. Ces ET4s ont été identifiés chez de nombreuses bactéries pathogènes (Agrobacterium tumefaciens, Bartonella henselae, Brucella abortus, Anaplasma spp., Ehrlichia chaffeensis, Coxiella burnetii, Legionella pneumophila) (Chen et al. 2010; Marchesini et al. 2011; Rikihisa and Lin 2010; Schulein et al. 2005; Vergunst et al. 2005; Zhu et al. 2011). Ils jouent un rôle crucial lors de l’infection et dans le développement de la maladie comme par exemple en inhibant la réponse immunitaire innée de l’hôte, en détournant les voies de signalisation cellulaire et le trafic vésiculaire ou en encore en utilisant les voies de recyclage liées à l’autophagie (Ivanov and Roy 2009; Newton, McDonough, and Roy 2013; Niu et al. 2012). Plusieurs études ont révélé que le SST4 de nombreuses bactéries était sous le contrôle de mécanismes de régulation fins et variés (facteurs de transcription, signaux environnementaux) affectant l’activité des promoteurs des gènes codant le SST4 à différents degrés au cours du cycle de développement bactérien (Z. Cheng, Wang, and Rikihisa 2008; Wu et al. 2012). La compréhension globale et détaillée des voies de régulation du SST4 chez E. ruminantium en réponse à différents signaux environnementaux nous permettrait d’imaginer de nouvelles stratégies thérapeutiques en renforçant par exemple les voies perturbées par la bactérie par des approches pharmaceutiques. 4 4 Avant-Propos Ma thèse a été menée au sein de l’unité « Contrôle des maladies animales exotiques et émergentes » au CIRAD en Guadeloupe et s’inscrit dans la thématique globale du laboratoire visant à étudier les interactions étroites entre la bactérie, la tique et l'hôte. Des approches sans a priori de type « omics » (transcriptomique, protéomique et métabolomique) sont actuellement développées au laboratoire mais des approches
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