Titre de la thèse : Caractérisation d’un ARN non-codant atypique et ses mécanismes d’action chez le pathogène humain Staphylococcus aureus
Mots-clés : Staphylococcus aureus, ARN régulateurs bactériens, régulation transcriptionelle et post-transcriptionelle
Unité/équipe encadrante : INSERM U1230 BRM
2, avenue du Professeur Léon Bernard
35043 RENNES CEDEX France
https://biochpharma.univ-rennes1.fr/
Directeur de thèse : Yoann AUGAGNEUR
yoann.augagneur@univ-rennes1.fr
https://biochpharma.univ-rennes1.fr/interlocuteurs/yoann-augagneur
Contexte socioéconomique et scientifique :
L’émergence de pathogènes bactériens multi-résistants aux antibiotiques est un problème majeur de santé publique. En Europe, 33000 personnes décèdent annuellement des suites d’une bactérie résistante aux antibiotiques, dont 7000 dues à Staphylococcus aureus. S. aureus est un commensal de l’Homme et également un agent pathogène redoutable. Pendant la transition entre colonisation et infection, la bactérie induit ou réprime l’expression de nombreux gènes via des facteurs de transcription et des ARN régulateurs. Ces derniers sont impliqués dans divers processus dont la virulence et la résistance aux antibiotiques. Alors qu’ils étaient considérés comme exprimés à partir des régions intergéniques ou en antisens de leurs cibles, plusieurs exemples montrent que certains échappent à cette règle (Wang et al, 2019). Nos travaux sur S. aureus ont permis l’établissement d’une base de données sur les ARN régulateurs (Sassi et al, 2015 ; srd.genouest.org) puis l’identification de nouveaux ARN (Bronsard et al, 2017). L’un d’eux, Srn_9342, présente un profil atypique. Il s’agit du premier exemple d’ARN exprimé, chez S. aureus, sous deux formes distinctes à partir de la région 3’ d’un messager.
Hypothèses et questions posées :
Le projet de thèse s’articulera principalement autour de la caractérisation de Srn_9342 et la détermination de ses fonctions dans la bactérie. L’identification des cibles ARN de Srn_9342 par affinité couplée au séquençage haut-débit (MAPS) a révélé comme partenaires de Srn_9342 de nombreux ARN régulateurs et des messagers codant des acteurs de la virulence, de l’adaptation au stress et la résistance antibactérienne. Nous émettons l’hypothèse d’un rôle d’éponge à ARN régulateurs comme décrit chez Salmonella (Miyakoshi et al, 2015).
Grandes étapes de la thèse :
Dans ce projet, il s’agira de : (i) caractériser l’expression des deux formes de Srn_9342, (ii) identifier les mécanismes de régulation et, (iii) explorer l’impact de la délétion et surexpression de Srn_9342 sur la physiologie bactérienne. Dans la première partie du projet, nous développerons un système simple-hybride chez S. aureus pour identifier le(s) facteur(s) de transcription régulant l’expression de Srn_9342. Nous rechercherons également l’acteur protéique ou ribonucléique responsable au cours de la phase de croissance de la transition entre l’expression de la forme courte à longue de Srn_9342. Dans ce cadre, l’expression de Srn_9342 sera étudiée dans des souches délétées pour les endoribonucléases connues et les partenaires protéiques de Srn_9342 identifiés par purification d’affinité. Ensuite, nous utiliserons différentes techniques biochimiques pour caractériser les mécanismes de régulation de Srn_9342 sur ses cibles (retards sur gel, toeprints et systèmes double-plasmide). Finalement, nous évaluerons le rôle fonctionnel de Srn_9342 par l’utilisation de souches de délétion ou surexpression. Nous insisterons particulièrement sur des conditions pouvant être reliées aux cibles identifiées de Srn_9342, c’est-à-dire la réponse au stress oxydant (en culture et au cours de la phagocytose), la carence nutritionnelle, la résistance aux antibiotiques et la virulence (sur modèle murin). A l’instar des quelques exemples relatés chez les bactéries à Gram négatif, ce projet pourrait conduire à l’émergence d’une nouvelle classe d’ARN régulateurs multifonctionnels chez S. aureus : éponge à ARN régulateurs et régulateur de l’expression des gènes. La caractérisation de Srn_9342 pourrait apporter de nouvelles connaissances dans la fonction des ARN régulateurs et participer à l’émergence de nouvelles stratégies thérapeutiques ciblant les ARN régulateurs dans un contexte de régulation global.
Compétences scientifiques et techniques requises par le candidat :
L’étudiant(e) devra avoir des solides connaissances en biologie moléculaire (clonage, expression des gènes par Northern blot et RT-qPCR), en biochimie (retard sur gel etc.) et porter un intérêt pour la microbiologie des pathogènes. L’étudiant(e) devra avoir un goût prononcé pour la curiosité et faire preuve de rigueur scientifique.
Sujet déposé et validé dans le cadre du concours de l’école doctorale Biologie-Santé.
Date limite des candidatures : 6 juin 2020
Envoyer lettre de motivation, CV, notes de licences et de Master 1 & 2 avec classement à l’adresse mail suivante : yoann.augagneur@univ-rennes1.fr
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