Bacterial lipopolysaccharide (LPS) is a key virulence factor for both innate and acquired host responses to infection. Intriguingly, Legionella pneumophila (Lp) serogroup 1 (Sg1) strains, are responsible for over 80% of confirmed Legionnaires’ disease cases, a severe pneumonia that is often fatal when not treated promptly. The Sg of Lp is determined by the LPS. Here we propose to analyse the molecular mechanisms underlying the predominance of Lp Sg1 in human disease. We have recently developed a zebrafish model of Lp infection, recapitulating key features of human infection. We will exploit this powerful non-mammalian model to study immune cell behaviour and host-pathogen interactions to identify the role(s) of LPS in host-pathogen interaction and disease in vivo comparing 3 selcted strains. A Lp Sg1 and a Lp Sg 6 strain (Sg6 is the second cause of human disease) and a Sg9 strain that has the same genomic backbone as the Sg1 strain but differs only in its LPS cluster. This “isogenic pair” will allow to investigate differences in the host response that are only due to its specific LPS. In parallel we will analyse the impact of extracellular vesicles (EVs) shed by Lp. We have shown recently that Lp-EVs of Sg1 impact the host response, however it may differ depending on the strain the EVs are shed off. We will analyse virulence, trafficking and the immune response by analyzing phagocyte behavior and phagocyte Lp interaction in vivo in our zebrafish model, monitor bacterial dissemination and survival, analyze the host and bacterial response by dual RNAseq and decipher the molecular mechanisms by generating knock out zebrafish lines in key immune genes regulated differently by these strains or EVs. This knowledge will be the basis to better understand the impact of LPS and EVs on immune cells and to uncover virulence mechanisms of particularly successful bacterial clones.

Cell migration is a fundamental process during development and in the adult where is allows delivery of immune cells to infection sites, wound healing or else dissemination of tumour cells during metastasis. Establishment, orientation and regulation of cell polarity play a key role in the initiation and the regulation of migration. Cell polarity basically corresponds to the spatial organization of intracellular organites along a favoured cell axis defined by the centrosome and the nucleus position. Our research project aims at determining the molecular mechanisms controlling cell polarization and their role in cell migration. We are particularly interested in cell migration within the central nervous system (CNS). Astrocytes are the major glial cells of the CNS. They fulfill a wide range of functions contributing to neuronal development and functions. Following cerebral lesions, astrocytes polarize and migrate to participate to the glial scar which limits cerebral regions accessible to axonal regeneration. Astrocyte can also give rise to brain tumors exhibit a strong ability to invade the surrounding tissue. By developing new in vitro polarization models and by using a variety of cellular models, we aim at studying the regulation of astrocyte polarization and migration. We have previously deciphered a 'polarity pathway' that is controlled by cell adhesion to the extracellular matrix. In this project we plan: - to further characterize integrin-induced polarity pathway. We will focus our study on the regulation of the small GTPase Cdc42 which is a major regulator of cell polarity from yeast to mammals. - to determine how extracellular stimuli such as soluble chemoattractants or intercellular contacts can modulate this pathway to regulate cell polarity and cell migration. We will, in particular, investigate the role of astrocyte interactions with neighboring neuronal or non neuronal cells in the regulation of astrocyte polarity and migration - to study if and how polarization of the microtubule network, which plays a key role in astrocyte migration, can induce the polarization the other cytoskeletal networks (actin microfilaments and intermediate filaments) in order to promote cell migration. This project should help us define very fundamental molecular mechanisms controlling cell polarization. This project also aims at characterizing the molecular mechanisms specifically controlling astrocyte polarization and migration in order to better understand the migratory behaviors associated with cerebral responses to trauma and brain tumor dissemination and identify potential therapeutic targets that will help us control astrocyte and brain tumor dissemination. This should eventually lead to the identification of molecular therapeutic targets in order to modulate astrocyte or glioma migration.

Recent advances have shown that placing genes in a specific nuclear compartment plays a crucial role in the regulation of coordinated gene expression. Dynamic changes in gene activation and silencing are often accompanied by gene relocations in relation to other nuclear compartments. Unravelling of mechanisms and dynamics of chromatin positioning will lead to an understanding of gene regulation. This applies above all to protozoan pathogens causing human disease such as malaria. Plasmodium falciparum, the causal protozoan agent of the most severe form of human malaria (2-3 million deaths per year), has a complex life cycle with two different hosts, the Anopheles mosquito and humans. P. falciparum relies on its capacity of differential gene expression in different cellular environments and a sophisticated system of phenotypic variation to survive in the different host compartments. A large proportion of the estimated 5000 genes of P. falciparum encode virulence factors genes, which are organized in several multi-gene families located predominantly in subtelomeric regions and some few chromosome central regions. Gene expression studies revealed that immune evasion via antigenic variation is a main feature of these families. This is generally achieved by the expression of clonally variant molecules either at the erythrocyte membrane or merozoite surface. Switching of expression to another variant molecule avoids immune clearance and prolongs the period of infection. Very little is known about how one gene is brought to expression to the exclusion of all or most other members of the family although evidence is available to suggest that there is active repression of the silent members at the transcriptional level involving epigenetic mechanisms. The molecular mechanisms of 'gene counting' (resulting in monoallelic expression), 'epigenetic memory' (preserving a certain expression profile for several generations), 'switching expression' (changing the phenotype in isogenic parasites) remain elusive and can not be studied in unicellular model systems such as yeast. Pathogens are likely to have evolved unusual mechanisms of gene regulation to cope with harsh and varying environment in the host. Here we propose a number of novel approaches based on the nuclear organization to shed light on the mechanism of gene regulation in P. falciparum. The studies involve a genome wide analysis of factors that determine the spatial nuclear organization and the characterisation of the nuclear remodelling machinery. In particular we aim deciphering the parasite's histone 'writing' and 'reading' machinery and the histone 'code'. The activities will be expanded to look at chromosome dynamics and the relocation of genes loci during the activation/inactivation process. State of the art technologies such as Fluorescent in Situ Hybridisation (FISH), ChIP-on-chip or ChIP-sequencing, chromosome conformation capture (3C) will be applied. The aim of this project is to get a better understanding of gene regulation in a major human pathogen, which is able to establish chronic infection under strong immune pressure. The long-term goal is to use this knowledge to develop new intervention strategies that would target the parasite's escape mechanism.