11 research outputs found

    A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response

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    International audienceIntracellular pathogens such as Listeria monocytogenesListeria\ monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon IFN-stimulated genes (ISGs). IFN-λ\lambda expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntAlntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/^{+/-} mice or when lntAlntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ\lambda-mediated immune response to control bacterial colonization of the host

    Etude structurale et fonctionnelle de protéines impliquées dans la pathogénie bactérienne

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    La première partie de cette thèse s'intéresse au complexe PBP2 :MreC impliqué dans l'élongation d' Helicobacter pylori, pathogène responsable de pathologies gastriques. Ce complexe a pu être mis en évidence in vitro dont nous avons déterminé la stoechiométrie par différentes méthodes. Ce complexe 1 : 1 a été analysé par diffusion des rayons X aux petits angles permettant de modéliser une enveloppe du complexe à basse résolution. Des cristaux du complexe ont été obtenus ainsi qu'un jeu de données à 2.7 A. Par remplacement moléculaire, un domaine très conservé des PBP a pu être placé mais la présence de MreC est encore discutable. La seconde partie s'intéresse au système de sécrétion de type III, facteur de virulence majeur chez Pseudomonas a eruginosa , bactérie causant des maladies nosocomiales. PopB et PopD, deux protéines membranaires, forment un pore au niveau de la membrane d'une cellule-cible, nécessaire à la translocation de toxines. PopB a été purifiée sous forme soluble en présence de sa chaperonne, PcrH, complexe dissociable en présence de détergents et lipides. PopB seule, serait capable d'oligomériser en hexamère en formant des structures annulaires. La topologie du pore a également été déterminée dans des liposomes montrant ainsi que Po pB est insérée dans la membrane alors que PopD n'est associée que superficiellement. Pour finir, la structure de PcrH, a été résolue avec un peptide de PopD. Les résidus de Pc rH intervenant dans ce complexe sont essentiels à la sécrétion de PopD et à la formation du pore de translocation. Nous cherchons actuellement de petites molécules qui seraient capables d'interagir avec Pc rH et empêcheraient son interaction avec PopB et PopD.The first part of this thesis focuses on the PBP2 :MreC complex which is implicated in the elogation steps of the cell wall of Helicobacter pylori, a pathogen involved in gastric adenocarcinoma. The existence of such a complex was demonstrated in vitro which we were able to determine its stoichiometry by different methods. This 1: 1 complex was also analyzed by Small Angle X-ray Scattering with which we could model an envelope at low resolution. Crystals were obtained which diffracted up to 2.7 A. By molecular replacement we were able to place a highly conserved domain of PBP but the presence of MreC is not yet certain. The second part focuses on the type III secretion system of Pseudomonas aeruginosa, major virulence factor of this bacterium. PopB and PopD, two membrane proteins, form a pore in the membrane of a host-cell, and are thus able to translocate toxins directly into the eukaryotic cytoplasm. We purified PopB in soluble form associated to its chaperone then dissociated it in the 'presence of detergents and lipids. PopB alone is then able to oligomerize into hexamers and form ring-like structures which are visible by electron microscopy. We then determined the topology of thE pore in liposomes. We have shown that PopB is embedded in the membrane whereas PopD is superficially associated to it. We have finally solved the structure of the chaperone of Po pB and PopD, PcrH, with a PopD synthetic peptide. The PcrH residues involved in this complex are essential for the secretion of PopD and thus formation of the translocation pore formation. We are currently searching for molecules that are able to interact with PcrH and thus prevent the interaction with PopB and PopD.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

    Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins*

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    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development

    Membrane targeting and pore formation by the type III secretion system translocon.

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    International audienceThe type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed

    Molecular architecture of the PBP2–MreC core bacterial cell wall synthesis complex

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    Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth

    Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

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    International audienceThe definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology

    Places, rôles et vécus des acteurs et des actrices dans les recherches en éducation et formation

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    Ce dossier a pour objectif de réunir des contributions qui interrogent les places, rôles et vécus des acteurs et actricesdans les recherches en éducation et formation. Les huit articles se répartissent en deux groupes distincts mais complémentaires. Le premier groupe permet de mieux réfléchir certaines formes des dispositifs mis en place dans des recherches de type collaboratif, en soulignant que, néanmoins, leur déploiement n’est pas dénué de tensions, de difficultés quand il est question des places et rôles de chacun et chacune. Le second groupe propose des analyses développant plus particulièrement les vécus des acteurs et actriceset leurs effets, en termes de développement professionnel, de transformations et d’apprentissages. Dans un groupe comme dans l’autre, on perçoit certaines des vertus de la collaboration entre les acteurs et actricesdes recherches en éducation et formation, sans pour autant verser dans une forme d’angélisme. The aim of this issue is to bring together contributions that question the places, roles and experiences of the actors in education. The eight articles are divided into two distinct but complementary groups. The first group invites to reflect on certain working modalities in collaborative research, by underlining that, nevertheless, their deployment is not without tensions and difficulties when it comes to the places and roles of each person. The second group proposes analyses that focus on the experiences of the actors and their effects in terms of professional development, transformation and learning. In both groups some of the virtues of collaboration between researchers in education have been showed, without falling into a form of angelism

    Sociétés, environnements, santé

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    Les domaines de la santé et de l’environnement ont longtemps été considérés, tant par les décideurs que par les scientifiques, comme des réalités disjointes. Il a fallu attendre les dernières décennies pour que soit reconnu et que se concrétise le lien entre environnements et santé avec la création d’agences spécialisées. Qu’il s’agisse de foyers de maladies parasitaires ou de pollution atmosphérique, les situations d’exposition à un risque sanitaire lié à l’environnement relèvent de facteurs particuliers : épidémiologiques, géographiques, politiques, et bien évidemment sociaux. À partir de zones géographiques situées tant au Sud qu’au Nord, les auteurs de cet ouvrage présentent un large éventail de configurations dans lesquelles s’exprime la complexité des relations entre santé et environnements dans leur rapport avec les groupes humains. Ils mettent l’accent sur l’importance de la perception et de la pratique des acteurs (des décideurs aux bénéficiaires des mesures, en passant par les médecins) pour penser le risque sanitaire selon les milieux. Ils soulignent tout l’intérêt de l’apport des sciences sociales et des pratiques interdisciplinaires dès lors qu’il s’agit d’envisager les relations entre un pathogène et l’homme. Chacune des approches s’attache ainsi à éclairer les différentes facettes du risque environnemental, depuis le comportement des individus jusqu’aux politiques nationales, en invitant le lecteur à se départir de toute vision déterministe et simplificatrice

    Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan

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    International audienceThe maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance to date no structure of a protein in complex with an intact bacterial peptidoglycan has been re-solved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spec-troscopy to derive for the first time an atomic model of an L,D-transpeptidase from Bacillussubtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains – the catalytic domain as well as the proposed peptidoglycan recognition domain – are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the impli-cation of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidogly -can complex paves the way for the design of new antibiotic drugs targeting L,D-transpeptidases. The strategy devel-oped here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis
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