Cell Biology and Infection Department Activity Report

Highlights

The group headed by Pascale Cossarthas reached her long lasting goal i.e. the understanding of the key molecular mechanisms underlying the targeting and crossing of the feto-placental barrier by Listeria monocytogenes. This long term project with Marc Lecuit, now head of the group « Microorganisms and crossing of host barriers » has led to the identification of the conjugated roles of two bacterial proteins internalin(InlA) and InlB. This result was obtained using two complementary animal models which overcome the stringent host specificities of InlA and InlB, the gerbil, a natural host for Listeria and a new knock-in mouse line which expresses a humanized E-cadherin in place of the mouse. This work demonstrates that internalin which also allows an efficient crossing of the intestinal barrier has a key role during infection. However, it is not sufficient to allow crossing of the placental barrier and requires InlB. This work provides the first function for InlB in vivo and highlights fundamental differences between two host barriers and consequently the different strategies used by a pathogen to cross these barriers. Work on the mechanisms used by InlA and InlB has highlighted functional similarities in bacterial entry, clathrin mediated endocytosis and cell-cell adhesion.

The group headed by Alice Dautry-Varsathas shown that one protein, translocated by Chlamydiainto the membrane of the compartment in which the bacteria develop, mimics a fundamental motif for membrane fusion in eukaryotic cells, the SNARE motif. This study provides the first example of SNARE mimicry by an intracellular bacterium. Regarding our work on clathrin-independent endocytosis, we have identified a cascade composed of Rac1, Pak and cortactin, which stimulates specifically the endocytosis of cytokine receptors. This internalization process does not involve clathrin nor caveolin. Our data indicate that proteins involved in several entry pathways, such as cortactin, could be regulated differently depending on the pathway.

Group headed by Anne Dejean.

The work in our lab is dedicated to the study of the molecular and cellular mechanisms involved in the development of human cancers with a particular emphasis on the role of the post-translational modification by SUMO. Our work combines fundamental projects, based on experimental systems, as well as more clinically-oriented projects devoted to the study of human liver cancer.

- We defined a signature of 14 miRNAs specifically associated with liver cancer progression and identified miR221 as a potent oncogene.

-We described a novel mechanism of regulation of PARP1 transcriptional activity through a sumoylation-coupled ubiquitination pathway.

-We identified the SUMO E3 ligase PIASy as an important co-regulator of FIP200 function TSC/mTOR signaling.

- We generated mouse models deficient/attenuated for sumoylation which should provide valuable tools for the elucidation of the general roles of this pathway in normal and disease-related fundamental processes.

- We investigated the role of sumoylation and related modifications in cellular senescence and identified the deubiquitinating USP1 enzyme as a key player in this process.

The group headed by Nancy Guillencharacterized the gene expression profile of Entamoeba histolytica during chemotaxis towards the human tumour necrosis factor. Their results suggest the repression of endocytosis and autophagy is closely related to cytoskeleton activation. In addition, they showed that the parasite’s lysine rich proteins, including KERP1 and KRiP3, which were previously identified as potential virulence factors, are effectors of the parasite’s stress responses to inflammation. Furthermore, in collaboration with clinicians, we developed an ex vivo human colon model to study of amoebiasis, highlighting the potential roles of the parasite’s cysteine proteinases in the onset of human inflammatory responses at the early stage of intestinal invasion.

The group headed by Alain Israël(Unité de Signalisation Moléculaire et Activation Cellulaire (SMAC)) is involved in the dissection of two signaling pathways, NF-kB and Notch. Regarding the former, we have completed (in collaboration with M Véron/ F Agou) a detailed structure-function analysis of NEMO, the core element of the NF-kB cascade. Regarding the Notch pathway, we have clarified the function of Itch, an ubiquitin-ligase known to negatively regulate Notch activation in Drosophila, and demonstrated that Itch is responsible for lysosomal degradation of non-activated Notch in mammalian cells, through the use of atypical K29-linked polyubiquitin chains. In parallel we have dissected the mechanisms responsible for generating active Notch ligands : genetic studies had suggested that ubiquitination and endocytosis of the ligands were required for activation of Notch signaling; our biochemical analysis has further demonstrated that the intracellular region of the ligands regulates their trafficking, and that an ubiquitination-dependent recycling step was required for their full signaling activity.

The group headed by Odile Kellerman (Stem cells, Signalisation and Prions laboratory (Pasteur-associated laboratory, INSERM UMR-S747, Université Paris Descartes, 45 rue des Saints-Pères, 75006) is focusing on the mechanisms that control stem cell differentiation and sustain the homeostasis of their differentiated progenies. We develop three major axes dealing with (i) the regulation of neuronal functions (ii) the role of the cellular prion protein and the impact of infection in a neuronal context (iii) the formation of mineralized tissus.

¯ We have demonstrated that pathogenic prions alter monoaminergic functions and trigger the production of oxidized derivatives of serotonin or catecholamines using a neuroectodermal precursor able to convert into fully-functional serotonergic or noradrenergic neuronal cells.

¯ We have identified a new PrP^C-dependent signal transduction effectors such as CREB transcription factor and its target genes (Egr1, c-fos, MMP9.. ) in neuronal cellswhich provides a foundation for elucidating how the deviation of PrP^Cnormal function may lead to prion diseases

¯We have developed of a 3D scanner imaging project allowing longitudinal studies to unravel phenotypic defects in genetically modified mice (monitoring of bone mineral density, dental and cranio-facial defects, adipose tissue distribution, lung morphology…).

¯We have set up of repair protocols using murine dental pulp progenitor cells.

Group headed by Philippe Sansonetti

- Several aspects related to entry of Shigellainto epithelial cells have been unravelled, such as capture of the bacteria by newly described retractile nanopodial extensions, and published, such as the polar secretion of Shigellatype III effectors, and the critical role of IpaC-activated c-srcin the spread of cytoskeletal rearrangements achieving efficient bacterial internalization.

- In the emerging field of manipulation of the innate immune response by Shigella, we have confirmed, including by structural analysis, that IpaH proteins formed a completely new family of E3 ubiquitine ligases. Following demonstration of the major role of gut antimicrobial peptides in the protection against Shigella, we have shown that this pathogen, via intra-epithelial injection of effectors controled by the MxiE regulon (i.e. Osp and IpaH proteins), suppressed expression of the beta-defensins, and infiltration of the lamina propria by dendritic cells. This novel strategy of subversion of innate defenses has also an impact on the profile of the adaptive response.

- We have published the first phase I trial of a live Shigella dysenteriae1vaccine candidate (SC599) and completed a phase II trial of this vaccine with promising results.

- Regarding the pathogenesis of lung infection, we have been able to reproduce and characterize for the first time Mickulick cells, the spumous macrophages characteristic of infection by Klebsiella rhinoscleromatis.

G5 headed by Christophe Zimmer

High resolution mapping of gene territories in living yeast cells

The position of genes inside nuclei of eukaryotic cells is not random, and plays a role in gene expression, DNA repair and replication. However, the spatial organization of the genome remains largely uncharted, owing to the limited resolution of light microscopy, moderate cell sampling and lack of nuclear landmarks. We have developed and validated a computational technique, which automatically analyses images of thousands of nuclei and generates high resolution maps of gene territories in the yeast Saccharomyces cerevisiae. Applying the method, we found that several genomic loci are confined to ’gene territories’ much smaller than the nucleus, which can be remodeled during transcriptional activation. We revealed a territorial organization of genes significantly more pronounced than observed before. These results and the technique are likely to have important implications for understanding gene expression and DNA repair. This work also shows how mere computational means can overcome standard limitations of light microscopy.

BEST PUBLICATIONS in 2008

Berger, A.B., Cabal, G.G., Fabre, E., Duong, T., Buc, H., Nehrbass, U., Olivo-Marin, J.C., Gadal, O., and Zimmer, C. (2008). High-resolution statistical mapping reveals gene territories in live yeast. Nature Methods, 5: 1031-1037.

Delevoye, C., Nilges, M., Dehoux, P., Paumet, F., Perrinet, S., Dautry-Varsat, A. and Subtil, A. (2008). SNARE Protein Mimicry by an Intracellular Bacterium. PLoS Pathogens, 4: e1000022.

Disson, O., Grayo, S., Huillet, E., Nikitas, G., Langa-Vives, F., Dussurget, O., Ragon, M., Le Monnier, A., Babinet, C., Cossart, P. and Lecuit, M.(2008). Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis. Nature, 455: 1114-1118.

Heuss, S., Ndiaye-Lobry, D., Six, E., Israël, A. and Logeat, F. (2008). The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.

J. Biol. Chem.283: 23782-23790.

Martin, N*., Schwamborn, K*., Urlaub, H., Gan, B., Guan, J.L. and Dejean, A. (2008) (* co-first authors) Spatial interplay between PIASy and FIP200 in the regulation of signal transduction and transcriptional activity. Mol Cell Biol. 28: 2771-2781.

Mouillet-Richard, S., Nishida, N., Pradines, E., Laude, H., Schneider, B., Feraudet, C., Grassi, J., Launay, JM., Lehmann, S. and Kellermann, O. (2008). Prions impair bioaminergic functions through serotonergic- or catecholaminergic-derived neurotoxins in neuronal cells.

Proc. Natl. Acad. Sci. USA. 105: 11212-11217.

Santi-Rocca, J., Weber, C., Guigon, G., Sismeiro, O., Coppée, J.-Y., and Guillen, N.(2008). The lysine- and glutamic acid-rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis. Cell Microb. 10:202-217.

Sperandio, B., Regnault, B., Guo, J., Zhang, Z., Stanley, S.L. Jr., Sansonetti, P.J. and Pedron, T. (2008).Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression. J. Exp. Med.205: 1121-1132.

HONORS AND AWARDS in 2008

Pascale Cossart

Descartes Prize for excellence in transnational research (Brussels, 2008)

Louis Jeantet Prize for medicine (Geneva, 2008)

Awarded European Research Council (ERC) Advanced Research Grant (2008)

Philippe Sansonetti :

Nomination to the rank of Professeur au Collège de France, Titulaire de la Chaire de

Microbiologie et Maladies Infectieuses (2008)

Awarded European Research Council (ERC) Advanced Research Grant (2008)

Awarded the « GlaxoSmithKline International Member of the Year Award », American Society for Microbiology (2009).

Guy Tran Van Nhieu :

Awarded a new INSERM Unit, ranked A+ by INSERM scientific instances. Will move as « Equipe accueillie » to the Collège de France.

Stéphane Roméro :

Awarded the "Prix Delheim 2008" from the Collège de France