1,721,002 research outputs found
Assessment of Research and Innovation on Food Systems by European member States: Policy and Funding Analysis
No full textFood not only supports human life; food production side-streams are also used for bio-fuel or bio-based products. Food has important historical, social, cultural, environmental and economic dimensions. Currently the EU and global food systems are affected by major challenges such as climate change, migration, a growing world population, urbanisation and resource scarcity, in addition to the “triple burden” of malnutrition (undernutrition, obesity, and hidden hunger), ageing and food poverty. Research and innovation (R&I) is key to developing high-impact solutions to future-proof our food systems. There is a pressing need to avoid fragmentation, ensure policy coherence, and align programmes in order to adopt a food system approach that can effectively address multiple objectives
La machinerie de sécrétion de type II Xcp de Pseudomonas aeruginosa : relations structure-fonction et interactome
No full textLes bactéries à Gram négatif sont entourées par une enveloppe cellulaire qui, contrairement aux bactéries à Gram positif, possèdent une organisation membranaire complexe composée d’une membrane interne appelée généralement membrane cytoplasmique, un espace périplasmique contenant une matrice de peptidoglycane et une membrane externe asymétrique constituée d’une monocouche de phospholipides surmontée d’une assise de lipopolysaccharide (LPS). Afin de franchir cette barrière, les bactéries à Gram négatif ont développé différentes voies de sécrétions spécifiques dédiées à l’export des protéines (effecteurs) du milieu intracellulaire vers le milieu extracellulaire. Jusqu'à présent, six systèmes de sécrétion ont été identifiés chez ces bactéries. Chez Pseudomonas aeruginosa, une bactérie pathogène opportuniste, le système de sécrétion de type II appelé aussi sécréton Xcp constitue l’un des facteurs principales de sa virulence. Le sécréton Xcp est un complexe macromoléculaire formé par 12 protéines, nommées XcpAO et XcpPC-XcpZM. Ce complexe macromoléculaire est organisé en trois sous-complexes : i) une plateforme d’assemblage ancrée dans la membrane interne formé par les protéines XcpRESFYLZM ii) un pore de sécrétion localisé dans la membrane externe formé par l’oligomérisation d’une protéine appelé la sécrétine XcpQD. Le pore de sécrétion est connecté à la plateforme de la membrane interne par une protéine appelée XcpPC iii) un pseudopilus périplasmique sous forme de fibre hélicoïdale qui est formé par la multimérisation d’une protéine appelée la pseudopiline majeure XcpTG. D’autres protéines appelées les pseudopilines mineures XcpUH-VI-WJ-XK intègrent le pseudopilus. La première partie du travail effectué au cours de cette thèse a eu pour but d’étudier et de comprendre par des approches structurales, biochimiques et biophysiques le mécanisme d’assemblage des pseudopilines en pseudopilus. La deuxième partie de ce travail a porté sur l’étude des réseaux d’interactions entre les substrats sécrétés et les composants de la machinerie Xcp. Durant cette thèse, nous avons ainsi i) identifier grâce à l’étude des interactions protéine-protéine l’existence d’un complexe quaternaire entre les pseudopilines mineures XcpUH-VI-WJ-XK localisées au sommet du pseudopilus ii) déterminer les structures de la pseudopiline majeure XcpTG par RMN et de la pseudopiline mineure XcpWJ par cristallographie aux rayons X iii) déterminer les différents éléments du sécréton qui interagissent avec les exoprotéines du sécréton. Ce réseau d’interaction nous a permis de proposer un modèle de fonctionnement du sécréton qui élucide le cheminement des exoprotéines dans le sécréton afin qu’elles soient exportées vers le milieu extracellulaire.Gram-negative bacteria are characterized by a complex organization of their cell envelope composed by the inner membrane (IM) called cytoplasmic membrane, the periplasmic space containing a peptidoglycan layer and the outer membrane (OM) covered by the lipopolysaccharide matrix. Gram-negative bacteria have evolved several specialized machines called secretion systems to export their effectors from the intracellular medium to the extracellular milieu or to the host cells. Up to now, at least six secretion systems have been identified. In the opportunistic pathogen Pseudomonas aeruginosa, the type II secretion system called the Xcp secreton is the major pathway for the release of virulence factors. The Xcp secreton is a macromolecular complex composed by 12 proteins called XcpAO, XcpPC-XcpZM. This machinery is organized in 3 sub-complexes: i) the assembly platform localized in the IM implicating XcpRESFYLZM proteins ii) the OM pore composed by the oligomerization of the secretin XcpQD. The connection between the assembly platform and the secretin is performed by XcpPC anchored in the IM iii) a periplasmic pseudopilus consisting of the multimerization of the so-called major pseudopilin XcpTG. The pseudopilus is a helicoidally filament spanning the periplasmic area and pushing the substrate into the secretin pore. Four other proteins, the minor pseudopilins XcpUH-VI-WJ-XK, were found in the pseudopilus. In the present work we first focused on the study of the pseudopilus components by biochemical, biophysical and structural strategies to understand their assembly. Secondly, we investigate the protein interactome between periplasmic secreton component and secreted substrates. Thus, we revealed the presence of a quaternary complex composed by XcpUH-VI-WJ-XK located at the tip of the pseudopilus. To understand at atomic scale the regulation of the pseudopilus, we determined the structure of two components of the pseudopilus XcpTG by NMR and XcpWJ by X-ray crystallography. Using systematic protein-protein interaction studies between secreton components and purified exoproteins of Pseudomonas aeruginosa, we identified 5 proteins of the secreton able to interact with exoproteins. This interaction network allowed us to propose a model for the secretion process including the sequential steps followed by exoproteins inside the secreton to leave the cell envelop
Assessment of Research and Innovation on Food Systems by European member States: Policy and Funding Analysis
No full textFood not only supports human life; food production side-streams are also used for bio-fuel or bio-based products. Food has important historical, social, cultural, environmental and economic dimensions. Currently the EU and global food systems are affected by major challenges such as climate change, migration, a growing world population, urbanisation and resource scarcity, in addition to the “triple burden” of malnutrition (undernutrition, obesity, and hidden hunger), ageing and food poverty. Research and innovation (R&I) is key to developing high-impact solutions to future-proof our food systems. There is a pressing need to avoid fragmentation, ensure policy coherence, and align programmes in order to adopt a food system approach that can effectively address multiple objectives
Assessment of Research and Innovation on Food Systems by European member States: Policy and Funding Analysis
No full textFood not only supports human life; food production side-streams are also used for bio-fuel or bio-based products. Food has important historical, social, cultural, environmental and economic dimensions. Currently the EU and global food systems are affected by major challenges such as climate change, migration, a growing world population, urbanisation and resource scarcity, in addition to the “triple burden” of malnutrition (undernutrition, obesity, and hidden hunger), ageing and food poverty. Research and innovation (R&I) is key to developing high-impact solutions to future-proof our food systems. There is a pressing need to avoid fragmentation, ensure policy coherence, and align programmes in order to adopt a food system approach that can effectively address multiple objectives
Deciphering the structural organization of OrfG, the VirB8-like of ICESt3 Type IV secretion system (T4SS)
No full textInternational audienceAlthough predominantly monomeric in solution, the data presented above show that OrfG forms functional trimers in vivo with the same structural organization observed in its crystal structure. Since TraM and TcpC show also the same packing in their crystal structures, this suggests that VirB8-like in Gram+ operate as trimers in their corresponding T4SS. This family of proteins in Gram- bacteria has been a target of choice for the design of conjugation inhibitors. Our study of OrfG structural organization and cellular localization which indicates that the soluble domain of OrfG is located in the cell wall and accessible on the surface of S. thermophilus (data not shown), makes this protein a very interesting target for the design of conjugation inhibitors targeting the trimer contact interfaces
The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus
No full textAssessment of Research and Innovation on Food Systems by European member States: Policy and Funding Analysis
No full textFood not only supports human life; food production side-streams are also used for bio-fuel or bio-based products. Food has important historical, social, cultural, environmental and economic dimensions. Currently the EU and global food systems are affected by major challenges such as climate change, migration, a growing world population, urbanisation and resource scarcity, in addition to the “triple burden” of malnutrition (undernutrition, obesity, and hidden hunger), ageing and food poverty. Research and innovation (R&I) is key to developing high-impact solutions to future-proof our food systems. There is a pressing need to avoid fragmentation, ensure policy coherence, and align programmes in order to adopt a food system approach that can effectively address multiple objectives
Study of the molecular and cellular mechanism involved on the conjugative transfer of ICESt3 from Streptococcus thermophilus
No full textLa conjugaison est l'un des principaux mécanismes du transfert horizontal de gènes, notamment les gènes de résistances aux antibiotiques. Elle permet l'échange des éléments génétiques mobiles, incluant les plasmides conjugatifs et les éléments conjugatifs intégratifs (ICE), entre bactéries de manière contact dépendant. Le transfert de ces éléments est médié par un complexe multiprotéique localisé dans l'enveloppe cellulaire de la cellule donneuse et appelé système de sécrétion de type IV (T4SS). On sait peu de choses sur l'architecture et la fonction des T4SS chez les bactéries Gram-positives par rapport à leurs homologues chez les bactéries Gram-négatives. Le but de mon projet de thèse a été d'étudier l'assemblage et l'architecture du T4SS chez les bactéries Gram-positives. Notre modèle d'étude est le T4SS codé par ICESt3, un élément génétique mobile présent chez Streptococcus thermophilus, capable de se transférer par conjugaison entre des microorganismes de la même espèce ou d'espèces différentes. Afin de répondre aux questionnements scientifiques de mon projet doctoral, nous avons utilisé plusieurs approches complémentaires notamment la biochimie des protéines, la biologie structurale, l'immunologie, la génétique, la biologie moléculaire ainsi que la microscopie. Plusieurs de ces techniques ont nécessité la mise en place de nombreuses collaborations entre notre laboratoire et d'autres laboratoires au niveau nationale et international. Nous avons démontré que OrfG, la VirB8-like de ICESt3, fonctionne sous forme de sous-unité trimétrique. La détermination de sa structure nous a révélé que son assemblage est conservé chez la famille des protéines VirB8 de la classe β, très répondu chez les Firmicutes. Nous avons également identifié une interaction de OrfG avec l'ADN et d'autres composants du T4SS ; en particulier la prédite hydrolase du peptidoglycane OrfA. Grace à ces données, nous proposons un modèle d'assemblage du T4SS chez les bactéries Gram-positives qui serait composé de deux complexes : le complexe de la paroi cellulaire formé par les sous-unités VirB8-like et VirB1-like puis le translocon formé par les autres protéines du T4SS. Nous renforçons ce modèle par l'étude de OrfD, la VirB4-like du système où nous avons mis en évidence sa multimérisation et son interaction avec OrfG mais aussi avec l'ADN. Notre étude apporte ainsi des nouvelles perspectives sur le processus de conjugaison et plus spécifiquement dans l'assemblage du T4SS.Conjugation is one of the main mechanisms of horizontal gene transfer, and plays a major role in the spread of antibiotic resistance among bacteria. The transfer of conjugative plasmids and integrative and conjugative elements (ICEs) is mediated by a multiprotein complex localized in the cell envelope of the donor cell and is called the type IV secretion system (T4SS). Little is known about the architecture and the function of T4SSs in Gram-positive bacteria compared with their counterparts in Gram-negative bacteria. The aim of my project is to identify proteins involved in the assembly and function of T4SSs in Gram-positive bacteria. Our model is ICESt3, a mobile genetic element found in Streptococcus thermophilus shown to propagate efficiently by conjugation to various Gram-positive bacteria. ICESt3 comprises a conjugation module which encodes a set of proteins involved in T4SS assembly. The approach we used for this study involved techniques in protein biochemistry, structural biology, immunology, genetics, molecular biology and also fluorescence microscopy. Many of these techniques have required the establishment of several collaborations between our laboratory and several other laboratories at national and international level. In this project, we demonstrated that OrfG, the VirB8-like of ICESt3, functions as a trimeric subunit. Structure determination revealed that its assembly is shared among VirB8-like proteins within β-class, which is widespread in Firmicutes. We also identified the interaction of OrfG with DNA and other T4SS components, in particular the predicted peptidoglycan hydrolase OrfA, the VirB1-like of the system. On the basis of these data, we propose a model for T4SS assembly in Gram-positive bacteria, consisting of two complexes: the cell wall complex formed by the VirB8-like and VirB1-like subunits, and the translocon formed by the other T4SS proteins. We reinforced this model by studying OrfD, the VirB4-like of the system, where we demonstrated its multimerisation, its interaction with OrfG, as well as with DNA. Our study thus provides new insights into the conjugation process and more specifically into T4SS assembly
Protein–Protein Interactions: Surface Plasmon Resonance
No full textInternational audienceSurface plasmon resonance (SPR) is one of the most commonly used techniques to study protein-protein interactions. The main advantage of SPR is it gives on the ability to measure the binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the ligand, on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the analyte, over the surface containing the ligand. The binding is monitored by subsequent changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 10 years, SPR has been intensively used in the study of secretion systems because of its ability to detect highly dynamic complexes that are difficult to investigate using other techniques. This chapter will guide users in the setup of SPR experiments in order to identify protein complexes and to assess their binding affinity or kinetics. It will include detailed protocols for (i) the immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetic measurements, and (iv) data analysis.Secretion systems are multiprotein complexes allowing the transport of a large number of effectors from the inside to the outside of bacterial cells. The assembly of these supramolecular machineries is ensured by the formation of protein complexes with extremely different times of stability, from transitory to stable interactions. To understand the function of these machineries as well as their modes of association, it is important to study their building blocks by identifying the different interacting partners and assessing their relative affinities and association/dissociation kinetics. For that purpose, scientists combine genetic, biochemical, and biophysical tools. During the last decade, the use of surface plasmon resonance (SPR) in the study of secretion systems has increased spectacularly [1-12]. This in vitro approach is the method of choice to study such dynamic systems owing to its ability to detect both weak and strong interactions ranging from the millimolar to the nanomolar range [13, 14]. SPR can be used as a primary tool to screen interacting partners or as a validation tool for interactions previously identified by other methods (e.g., bacterial two-hybrid, co-immunoprecipitation, chemical crosslinking). The determination of the affinity or kinetics of an interaction, as can be done by SPR, is fundamental to understanding the nature of binding at the cellular level
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