CARACTERISATION DES PROTEINES DE GRANULES DENSES DE TOXOPLASMA GONDII :Etude des interactions protéiques et lipidiques et du rôle des hélices alpha-amphipatiques de GRA2

At the interface with the host cell compartment, the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii comprises several membranous systems, including the membranous nanotubular network formed upon secretion of the dense granule protein GRA2. Using biochemical approaches, I have shown that the GRA proteins form oligomeric complexes, which could explain the solubility of these membrane proteins within the dense granules and after secretion into the vacuolar soluble fraction. Using molecular and cellular approaches, I have investigated the involvement of each of the three GRA2 amphipathic alpha-helices in post-secretory membrane association of the protein, likely via association to phosphatidylinositol phosphates, and in the GRA2 induced-formation of the membranous nanotubular network. These studies led us to propose models for the interaction of GRA2 with membranes and for the tubulation of the membranous nanotubular network.A l'interface avec la cellule hôte, la vacuole parasitophore du parasite intracellulaire Toxoplasma gondii comprend plusieurs systèmes membranaires, dont un réseau de nanotubes formé suite à la sécrétion de la protéine de granules denses GRA2. Mes travaux ont montré, par des approches biochimiques, que les protéines GRA membranaires forment des complexes oligomériques qui pourraient expliquer leur solubilité dans les granules denses et lors de leur sécrétion dans la vacuole. Par des approches moléculaires et cellulaires, j'ai analysé l'importance relative des trois hélices alpha-amphipatiques de GRA2 dans son association post-sécrétoire au réseau de nanotubes, via la liaison potentielle de la protéine à des phosphoinositides, et dans la tubulation du réseau induite par GRA2. Ces travaux ont permis de proposer un modèle d'interaction de GRA2 avec les membranes et de formation des tubules membranaires du réseau de nanotubes

Rôles de l’immaturité des barrières épithéliales et du microbiote intestinal dans la susceptibilité néonatale à la méningite

International audienceLes méningites bactériennes sont la conséquence de l’accès au système nerveux central de bactéries pathogènes qui ont traversé la barrière hémato-encéphalique au niveau des micro-vaisseaux cérébraux, de la barrière hémato-méningée et/ou des plexus choroïdes (barrière sang-liquide cérébro-spinal) [...

CARACTERISATION DES PROTEINES DE GRANULES DENSES DE TOXOPLASMA GONDII (Etude des interactions protéiques et lipidiques et du rôle des hélices alpha-amphipatiques de GRA2 )

A l interface avec la cellule hôte, la vacuole parasitophore du parasite intracellulaire Toxoplasma gondii comprend plusieurs systèmes membranaires, dont un réseau de nanotubes formé suite à la sécrétion de la protéine de granules denses GRA2. Mes travaux ont montré, par des approches biochimiques, que les protéines GRA membranaires forment des complexes oligomériques qui pourraient expliquer leur solubilité dans les granules denses et lors de leur sécrétion dans la vacuole. Par des approches moléculaires et cellulaires, j ai analysé l importance relative des trois hélices alpha-amphipatiques de GRA2 dans son association post-sécrétoire au réseau de nanotubes, via la liaison potentielle de la protéine à des phosphoinositides, et dans la tubulation du réseau induite par GRA2. Ces travaux ont permis de proposer un modèle d interaction de GRA2 avec les membranes et de formation des tubules membranaires du réseau de nanotubes.At the interface with the host cell compartment, the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii comprises several membranous systems, including the membranous nanotubular network formed upon secretion of the dense granule protein GRA2. Using biochemical approaches, I have shown that the GRA proteins form oligomeric complexes, which could explain the solubility of these membrane proteins within the dense granules and after secretion into the vacuolar soluble fraction. Using molecular and cellular approaches, I have investigated the involvement of each of the three GRA2 amphipathic alpha-helices in post-secretory membrane association of the protein, likely via association to phosphatidylinositol phosphates, and in the GRA2 induced-formation of the membranous nanotubular network. These studies led us to propose models for the interaction of GRA2 with membranes and for the tubulation of the membranous nanotubular network.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Listeria monocytogenes ActA: a new function for a ‘classic’ virulence factor

International audienceListeria monocytogenes (Lm) is ubiquitous and widespread in the environment. It is responsible for one of the most severe human foodborne infection. Lm is a facultative intracellular bacterium that can cross the intestinal barrier, disseminate via the bloodstream and reach the liver, spleen, central nervous system and fetus. The bacterial surface protein ActA is one of the most critical and best characterized virulence factors of Lm. It fulfills many essential functions within host cells, allowing Lm escape from autophagy and recruiting an actin polymerization complex that promotes Lm actin-based motility, cell-to-cell spread and dissemination within host tissues. We have recently shown that ActA also acts extracellularly. It mediates Lm aggregation and biofilm formation in vitro and in vivo, and long-term colonization of the gut lumen. This new property of ActA favors Lm transmission and may participate in the selective pressure on Lm to maintain ActA

Microbial and immune factors regulate brain maintenance and aging

International audienceTissue aging can be viewed as a loss of normal maintenance; in advanced age, the mechanisms which keep the tissue healthy on daily bases fail to manage the accumulating "wear and tear", leading to gradual loss of function. In the brain, maintenance is provided primarily by three components: the blood-brain barrier, which allows the influx of certain molecules into the brain while excluding others, the circulation of the cerebrospinal fluid, and the phagocytic function of microglia. Indeed, failure of these systems is associated with cognitive loss and other hallmarks of brain aging. Interestingly, all three mechanisms are regulated not only by internal conditions within the aging brain, but remain highly sensitive to the peripheral signals, such as cytokines or microbiome-derived molecules, present in the systemic circulation. In this article, we discuss the contribution of such peripheral factors to brain maintenance and its loss in aging

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

International audienceThe brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions

Escherichia coli Resistance to Nonbiocidal Antibiofilm Polysaccharides Is Rare and Mediated by Multiple Mutations Leading to Surface Physicochemical Modifications

Antivirulence strategies targeting bacterial behavior, such as adhesion and biofilm formation, are expected to exert low selective pressure and have been proposed as alternatives to biocidal antibiotic treatments to avoid the rapid occurrence of bacterial resistance. Here, we tested this hypothesis using group 2 capsule polysaccharide (G2cps), a polysaccharidic molecule previously shown to impair bacterium-surface interactions, and we investigated the nature of bacterial resistance to a nonbiocidal antibiofilm strategy. We screened an Escherichia coli mutant library for an increased ability to form biofilm in the presence of G2cps, and we identified several mutants displaying partial but not total resistance to this antibiofilm polysaccharide. Our genetic analysis showed that partial resistance to G2cps results from multiple unrelated mutations leading to modifications in surface physicochemical properties that counteract the changes in ionic charge and Lewis base properties induced by G2cps. Moreover, some of the identified mutants harboring improved biofilm formation in the presence of G2cps were also partially resistant to other antibiofilm molecules. This study therefore shows that alterations of bacterial surface properties mediate only partial resistance to G2cps. It also experimentally validates the potential value of nonbiocidal antibiofilm strategies, since full resistance to antibiofilm compounds is rare and potentially unlikely to arise in clinical settings

GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network.

International audienceThe intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction