Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Department: Cell Biology of Infection
Title of the PhD project: Functional contribution of novel molecular effectors on cell infection by the bacterial pathogen Listeria
Name of the lab: Unité Interactions Bactéries-Cellules
Head of the lab: Pascale Cossart
PhD advisor: Javier Pizarro-Cerda
Email address: firstname.lastname@example.org
Web site address of the lab: http://www.pasteur.fr/ip/easysite/pasteur/fr/recherche/departements-scie...
Doctoral school affiliation and University: B3MI
Presentation of the laboratory and its research topics:
Our laboratory investigates the molecular and cellular basis of the infection by the Gram-positive bacterium Listeria monocytogenes, a model pathogen for the study of the intracellular parasitism. L. monocytogenes is responsible for severe food-borne infections leading to gastroenteritis, meningitis and abortion in pregnant women. This bacterium is characterized by its ability to cross three host barriers -the intestinal, blood-brain or feto-placental barriers- and to invade several cell types in which it multiplies. L. monocytogenes moves in the cytosol of infected cells and spreads from cell to cell using an original propelling process; i.e. cell actin polymerization at one pole of the bacteria.
Our present activities focuses on: 1) the identification and characterization of new non-coding RNAs involved in virulence, as well as new RNA-mediated regulations; 2) the investigation of new molecular pathways involved in bacterial entry into host cells and cell-to-cell spread; 3) organelle dynamics during infection; 4) the systematic analysis of post-translational modifications of host molecules during infection, in particular SUMOylation; 5) chromatin remodeling upon infection; 6) the characterization of new bacterial virulence factors identified by post-genomic approaches or by their effect on cellular responses; and 7) in-depth understanding of the intestinal phase of the L. monocytogenes infection by analyzing the impact of commensals on L. monocytogenes growth and the bacterial effect on the intestinal tissue.
Description of the project:
(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))
A human genome-wide siRNA screen, as well as a complete kinome, miRNA and drug screens have been performed in the framework of the Swiss systems biology consortium 'InfectX' (http://www.infectx.ch) to identify cellular factors required for Listeria monocytogenes infection of host cells. Using an image-based approach on 384-well plates, our readout for infection was based on monitoring the accumulation of the Listeria secreted protein InlC in the cytoplasm of infected HeLa cells after 5 hours of bacterial internalization. We have identified in this way a wide panel of effectors involved in many different cellular functions (cell differentiation, cytokinesis, apoptosis, fatty acid metabolism, membrane trafficking, vacuolar acidification, RNA splicing, transcription, molecules of unknown function, etc.) which either up-regulate or down-regulate the detected levels of InlC within host cells. Since the detection of InlC is a late readout for infection, it is possible to identify a posteriori molecular hits that affect specific stages of the Listeria cellular infection cycle including adhesion to host cells, bacterial survival during the vacuolar stage, escape to the cytoplasm, proliferation in the cytoplasmic space, actin-based bacterial motility, and cell-to-cell spread.
While a large number of potential hits are being validated systematically by re-screening with a different complementary siRNA libraries in the framework of the InfectX consortium, this Ph.D. project is particularly oriented towards the in-depth investigation of the specific contribution to the Listeria infection process of several of the most promising identified molecular hits. These candidates molecules will be initially inactivated by siRNA and their potential role on each step of the Listeria intracellular cycle using appropriate techniques: a) Bacterial adhesion: gentamicin invasion assays performed at 4° together with image analysis of bacterial numbers associated to host cells after differential fluorescent intra- versus extracellular bacterial staining will allow to determine whether the molecular candidates play a role on the most early interaction of Listeria with host cells; b) vacuolar stage: in collaboration with colleagues of the Pasteur Institute, we will monitor by FRET-based assays the cleavage of cytoplasmic fluorescent probes by bacterial-encoded enzymes
allowing to determine whether the molecular candidates affect the stability of the bacterial-containing compartment, favoring or inhibiting the translocation of Listeria to the cytosol; c) bacterial proliferation: gentamicin invasion assays at 37°C together with image analysis of bacterial numbers associated to host cells will determine whether bacterial multiplication is affect by the molecules under study; d) actin-based bacterial motility: using a mathematical algorithm developed by our colleagues at the Basel University specifically for this project, we will determine in an image-based analysis whether the presence of polymerized actin at the tail of Listeria is inhibited or increased by the deletion of the candidate effectors; e) cell-to-cell spread: co-culture of siRNA-treated cells infected with Listeria with cells transfected with fluorescent probes the detection of bacterial infection will determine wether infection of secondary infected cells is affected in the study conditions.
We will additionally implement bio-informatic tools to study the global distribution of bacteria in cells infected with Listeria and treated with our candidate siRNAs to have an additional readout of their possible contribution to the Listeria infection process. Once the specific function of the candidates has been identified, the precise characterization of their molecular mode-of-action will be investigated using the different cell biology, molecular biology, biochemistry and imaging approaches that we master in our laboratory. Finally, using public bioinformatic databases we will reconstruct the topology of the detected signaling pathway to integrate them in the framework of our precedent studies.
Pizarro-Cerdá, J. & P. Cossart. 2009. The Listeria monocytogenes membrane traffic and intracellular life-style: an exception or the rule?. Annual Review of Cell and Developmental Biology 25: 649-670.
Pizarro-Cerdá, J., M. Bonazzi & P. Cossart. 2010. Clathrin-mediated endocytosis: what works for small, also works for big. Bio-Essays 32: 496-504.
Pizarro-Cerdá, J., A. Kühbacher & P. Cossart. 2012. Mechanisms of entry of Listeria monocytogenes in mammalian cells. Cold Spring Harbor Laboratory Perspectives 2: doi a0100009.
Kühbacher, A., D. Rambouillet, E. Arnaud, P. Cossart & J. Pizarro-Cerdá. 2012. OCRL-mediated actin rearrangements are required for efficient Listeria entry within target cells. Journal of Biological Chemistry 287:13128-13136.
Kühbacher, A., Cossart P & J. Pizarro-Cerdá. 2013. Imaging late infection steps by the bacterial pathogen Listeria monocytoenes. Journal of Visual Experiments 73: In press.
Kühbacher, A., Cossart P & J. Pizarro-Cerdá. 2013. Internalization assays for Listeria monocytogenes. Methods in Molecular Biology 1000: In press.
Listeria monocytogenes, phagocytosis, siRNA, imaging, cytoskeleton
Expected profile of the candidate (optional):
Unité Interactions Bactéries-Cellules
INSERM U604-INRA USC2020
28 rue du Docteur Roux
75724 Paris Cedex 15 FRANCE
tel: +33(0)1 4061 3779
fax: +33(0)1 4568 8706