2015-‐2016

PROPOSITION DE SUJET DE STAGE MASTER-­‐2 RECHERCHE INFECTIOLOGIE FONDAMENTALE UNIVERSITÉ LYON-­‐1 2015-­‐2016 Titre du sujet de stage: Identification of host cell pathways controlled by Legionella protein kinases Nom, adresse de l’Unité d’accueil / Nom du responsable de l’Unité: Centre International de Recherche en Infectiologie (CIRI), INSERM U1111, CNRS UMR5308 46 Allée d’Italie 69364 Lyon Cedex 07/ François-­‐Loïc Cosset Nom, adresse de l’Equipe d’accueil/ Nom et adresse e-­‐mail du responsable d’Equipe à contacter: Pathogénèse des Légionelles Bat A. Lwoff 10 rue Dubois, Campus de la Doua 69622 Villeurbanne / Patricia Doublet, patricia.doublet@univ-­‐lyon1.fr Résumé du sujet de stage Background -­‐ Legionella pneumophila is the most common causative agent of the severe pneumonia legionellosis. Legionella pathogenic strains emerge from the environment after intracellular multiplication in amoeba, are disseminated by water aerosols technologies, and infect alveolar macrophages of human lungs. A Type 4 Secretion System (T4SS), which translocates approximately 300 effector proteins into the host cell cytoplasm, is absolutely required for immune defences evasion and successful replication of Legionella in phagocytic cells. Deciphering the individual contribution of each effector protein to the intracellular lifestyle of L. pneumophila remains a major, even the principal, challenge to understand the molecular basis of Legionella virulence. Our work aims at contributing to this question by characterizing the role of one family of these effectors, namely the protein kinases family, in L. pneumophila virulence. Results -­‐ In silico analysis and in vitro phosphorylation assays identified 5 functional protein kinases LegK1-­‐LegK5 encoded by the epidemic L. pneumophila Lens strain. Translocation assays showed that except LegK5, the Legionella protein kinases are T4SS effectors. We established that LegK2 plays a key role in bacterial virulence (Hervet et al., IAI, 2011). More precisely, we recently demonstrated that LegK2 interferes with host actin cytoskeleton and contributes to Legionella evasion from the endocytic pathway (Michard et al., Mbio, sous presse). Project -­‐ The internship aims at characterizing the other Legionella protein kinases by using the same experimental approach that we develop for LegK2. Briefly, the host cell proteins that interact with bacterial protein kinases will be indentified in a high throughput yeast two-­‐hybrid screen. Protein interactions will be then confirmed by pull-­‐down biochemical approaches in transfected mammalian cells and upon physiological conditions in Legionella infected cells. The human proteins that can interact with bacterial protein kinases will be assayed for in vitro and in cellulo phosphorylation to establish if they are substrates. According to the nature of the identified cellular targets, adapted approaches of biochemistry, cellular biology and genetics will be then developed to decipher in detail each host-­‐cell signalling pathway controlled by Legionella kinases.