Tesi sul tema "Système de Sécrétion de Type IV Cag"
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Lopez-Lozano, Nina. "Caractérisation structurale de nanomachines bactériennes impliquées dans l'adaptabilité et la virulence". Electronic Thesis or Diss., Bordeaux, 2023. http://www.theses.fr/2023BORD0482.
Testo completoThis thesis is divided into two themes.The first theme focuses on the cag Type IV secretion system (cag-T4SS) of the bacterium Helicobacter pylori. This is a complex secretion machinery embedded in the bacterium's cellular envelope, enabling it to inject the CagA oncoprotein into human gastric epithelial cells. This toxin is considered a major virulence factor of H. pylori. It interacts with host proteins, disrupting cell signaling and leading to changes that can promote the development of gastrointestinal diseases, including gastric ulcers and cancers. The cag-T4SS is subdivided into three parts: (i) an inner membrane complex, composed essentially of ATPases providing the energy required for its assembly and/or its function; (ii) an outer membrane complex, or core complex, forming a channel that connects the inner and outer membranes; and (iii) an extracellular pilus, the existence of which is still controversial, and which would establish contact between the bacterium and its target, and possibly transfer substrates across the host membrane.The first project focuses on the extracellular pilus. The aim is to obtain data concerning a putative interaction between the CagI and CagL proteins, which are essential for secretion and are thought to be involved in the composition of the cag-T4SS pilus. We overexpressed recombinant versions of these proteins in Escherichia coli and co-purified them by affinity chromatography, demonstrating a direct interaction between them. The ability of DARPins and Nanobodies to bind this complex was tested. Analysis of these complexes was also undertaken by cryo-electron microscopy (cryoEM).The second project focuses on the core complex, with the aim of obtaining its structure at high resolution in order to shed light on the remaining grey areas concerning this imposing assembly. Various techniques have been used to solubilize this complex. Its purification remains to be optimized before it can be analyzed by cryoEM. Obtaining such structures could lead to a better understanding of how cag-T4SS functions, and to consider strategies to inhibit its assembly and/or function, thus depriving H. pylori of a major virulence factor.The second theme concerns bacterial spirosomes. The AdhE enzyme is highly conserved in the bacterial kingdom and in certain eukaryotic organisms. It is a bifunctional alcohol/aldehyde dehydrogenase enzyme, responsible for the conversion of acetyl-CoA to acetaldehyde and then to ethanol during anaerobic alcoholic fermentation. This enzyme is commonly found in its oligomeric form, known as spirosome. Depending on the ligands present in the medium, E. coli spirosomes can have a compact or extended conformation, the latter constituting the active form of the enzyme. Unlike E. coli spirosomes, Streptococcus pneumoniae ones are naturally stabilized in their extended conformation.The aim of this project is to understand the mechanisms behind this conformational difference. CryoEM enabled us to obtain a high-resolution structure of the S. pneumoniae spirosome and thus comparing it with the extended E. coli spirosome. Functional mutagenesis experiments with complementation enabled us to determine which residues are involved in the extension of these spirosomes. As they are involved in pathogenicity and have been shown to be essential to bacterial physiology in the absence of oxygen, in-depth study of their conformation could lead to the discovery of molecules capable of regulating their activity, which could be of major interest in the fields of biotechnology and healthcare
Pichon, S. "Système de sécrétion de type IV et protéines à domaines ankyrines dans les interactions Wolbachia-arthropodes". Phd thesis, Université de Poitiers, 2009. http://tel.archives-ouvertes.fr/tel-00551985.
Testo completoPichon, Samuel. "Système de sécrétion de type IV et protéines à domaines ankyrines dans les interactions Wolbachia-arthropodes". Poitiers, 2009. http://theses.edel.univ-poitiers.fr/theses/2009/Pichon-Samuel/2009-Pichon-Samuel-These.pdf.
Testo completoWolbachia are intracellular Gram(-) bacteria that are reproductive manipulators of many arthropods. In the isopod Armadillidium vulgare, the Wolbachia wVulC strain induces male feminization. Here, we characterized two vir operons which are expressed in all host tissues and which encode a type IV secretion system (T4SS) used to translocate bacterial effectors into host cytoplasm. Gene organization and sequence comparison in 37 Wolbachia strains highlighted the high conservation of both vir operons and their importance for the biology of the bacteria. We also identified in the on-going assembly of the wVulC genome, 66 ankyrin domain-encoding genes. Ankyrin motifs are known to mediate protein-protein interactions in eukaryotic organisms and thus are suggested to mediate in Wolbachia the interaction with host molecules. We showed that one of the three copies of the wVulC pk2 gene is only expressed in feminizing strains but not in the three strains inducing cytoplasmic incompatibility in terrestrial isopods. The associated Pk2 protein could be involved in male feminization. We thus tested the interaction between three T4SS proteins and five ankyrins (including Pk2) via the yeast twohybrid and CRAfT (Cre-recombinase Reporter Assay for Translocation) methods. None of the five ankyrin proteins were revealed to be secreted by the wVulC strain. Nevertheless, this promising approach may enable us to identify Wolbachia effectors
Botella, Eric. "Interactions entre le pilus du système de sécrétion de type IV de Brucella suis et la cellule hôte". Montpellier 2, 2006. http://www.theses.fr/2006MON20118.
Testo completoBacteria of the genus Brucella are facultative intracellular pathogens which have developed the ability to survive and replicate in professional and non-professsional phagocytes. The VirB type IV secretion system (T4SS) is a key virulence determinant used by Brucella, thought to deliver effector proteins directly into eukaryotic host cells, where these molecules subvert host cell biology. VirB mutants have lost the ability to perturb vesicular trafficking essential for the establishment of the bacterium's intracellular replication niche. Structural models suggest that the T4SS machinery is composed of inner membrane associated ATPases, a putative pore structure spanning the envelope, and a pilus like appendage exposed on the bacterial surface. It is not known how the T4SS interacts with the host cell, however the components of the pilus are prime candidates. Recently, plant proteins that interact with the Agrobacterium VirB pilus have been identified. We used the B. Suis VirB2 and VirB5 proteins as baits to screen a Hela cell cDNA library to identify « host interacting proteins » (HIPs). Three HIPs interacting with VirB2 and one interacting with VirB5 were identified. The three VirB2 HIPs are membrane proteins; CD98hc is a cell surface protein that acts as a receptor for certain viruses and modulates integrins functions, LZIP is a transcriptional activator involved in the response to viral infections and MCL-1 is an anti-apoptotic factor. The protein Galectin-1 that interacts with VirB5 is a lectin like. With confocal microscopy, we find that CD98 is recruited to wild type, but not virB- Brucella containing phagosomes from the early stages of infection. Furthermore, we showed using cd98hc-/- cells that CD98hc is involved in Brucella (in a VirB2 independent manner) and Listeria monocytogenes internalization into the host cells but, it has no effect on Salmonella enteritica serovar Typhimurium entry
Rancès, Edwige. "Étude des déterminants génétiques bactériens impliqués dans les interactions Wolbachia-hôtes : analyse fonctionnelle du système de sécrétion de type IV". Lyon 1, 2008. http://www.theses.fr/2008LYO10132.
Testo completoWolbachia is a strict intracellular bacterium which infects certain filarial nematodes and a great number of arthropods. For the marjority of insects, Wolbachia is considered a reproductive parasite. Nevertheless, in hymenoptera Asobara tabida, the bacterium became obligatory for insect oogenesis. Despite the scientific interest carried with Wolbachia, the molecular mechanisms implied in the interactions with its hosts are poorly knwn. The sequencing Wolbachia genomes highlighted the presence of genes encoding the type IV secretion system ( T4SS) often involved in the dialogue between bacteria and hosts. In the context, this study consisted in developing tools to handle the bacterium, then to decipher the functionality of the T4SS in Wolbachia. Polyclonal antibodies raised against the VirB6 protein was produced and confirmed the functionality of the T4SS translocon in cellulo and in insecta
Bergé, Célia. "Étude biochimique et structurale de composants essentiels à la biogenèse du pilus du système de sécrétion de type IV de la bactérie Helicobacter pylori". Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1336.
Testo completoHelicobacter pylori is a bacterium that colonizes the human stomach in half of the world population. It is estimated that 20% and 3% of patients develop peptic ulcer and gastric cancer, respectively. For these reasons, H. pylori was identified as a group 1 carcinogen by the World Health Organization (WHO) in 1994. The most virulent strains of H. pylori carry a type IV secretion system (Cag-T4SS) responsible for the injection of the oncoprotein CagA into gastric epithelial cells. One remarkable feature of the cagT4SS is its external pilus which composition is not clear. CagL, CagH and CagI proteins are critical components of the Cag-T4SS because these proteins are necessary for CagA translocation and are involved in pilus formation. Moreover CagL forms a sub-assembly with CagI and CagH but the molecular details of the complex are still to be discovered. Our objective is to better understand the molecular basis of CagLIH complex by interaction and structural study. CagL interacts with CagI and CagH with a with Kds of 5 µM. CagI does not interact with CagH. The structural study of CagL/CagI complex has been investigated by a two-pronged approach. First I have purified the CagL/CagI complex and collected cyo-EM micrographs. In parallel I have collected NMR spectra of CagL in the presence of CagI and identify the changes in the spectra to determine the residues involved in the interaction. In this study we have, for the first time, characterize the CagL-CagI-CagH interactions and obtained structural informations of the CagI-CagL complex
Moumene, Amal. "Caractérisation de déterminants moléculaires du pouvoir pathogène d'Ehrlichia ruminantium : rôle du système de sécrétion de type IV et des protéines de la membrane externe". Antilles-Guyane, 2015. http://www.theses.fr/2014AGUY0818.
Testo completoIdentify the molecular determinants of pathogenic bacteria and understand how they are controlled to allow adaptation to the environment and the host is crucial to develop innovative control methods and provide therapeutic alternatives. Ehrlichia ruminantium is an obligate intracellular bacterium of the family Anaplasmataceae, vectored by ticks of the genus Amblyomma and causing heartwater, a fatal disease of ruminants. As part of this thesis, we characterized some determinants of pathogenicity E. Ruminantium three levels of resolution different. A comprehensive approach without preconceptions, first of all we determined the proteome of the outer membrane of the infectious form of E. Ruminantium. This study will allowed to have a better vision of the architecture of the outer membrane, which is the first interface exchanges between the bacterium and its host cell. Then we showed the function of ErxR as a central regulator of the type IV secretion system (SST4) and map1 multigene family for integrating environmental signals that are iron deficiency and acidity of the medium. In addition, this work has established for the first link between Map1 proteins of the outer membrane and the SST4 and suggests that may have a direct role in virulence. Finally, an in silico analysis using the software S4TE led to the characterization of Erip1, the first effector SST4 E. Ruminantium. This effector phosphorylated tyrosines and injected into the nucleus of the host cell, removed no homology in the databases and may therefore represent a new family of nucleomodulines. Protein partners and identification of intracellular targets of the effector will better understand how E. Ruminantium manipulates the host cell to its advantage. Finally, channels of the characterization of signaling targeted Erip1 be instructive on the cellular response to infection E. Ruminantium
Jacobsen, Theis. "Structure and assembly of bacterial type IV filaments unravelled by an integrative approach". Electronic Thesis or Diss., Sorbonne université, 2022. http://www.theses.fr/2022SORUS146.
Testo completoThe type IV filament (TFF) superfamily is a group of molecular machineries located in the membrane of bacteria and archaea. These machineries assemble non-covalent protein polymers called pili extending away from the cell to perform multiple functions which have evolved specifically to adapt to different host organisms. The TFF superfamily includes the type II secretion system (T2SS) and the type IVa pili (T4aP). The T2SS promotes the secretion of substrates in Gram-negative bacteria. These substrates are in general enzymes degrading complex carbohydrates, peptidoglycan, and lipids, resulting in the release of nutrients. The T4aP are long flexible fibres anchored in the membrane and enable various functions such as twitching motility, DNA uptake and biofilm formation. The mechanism by which the T2SS and T4aP pilus fulfil their different functions is still not completely understood. To understand the mechanism of secretion by T2SS, we studied the structure of the pseudopilin OutG, the major component of the pseudopilus in Dickeya dadantii by Nuclear Magnetic Resonance (NMR). In a second part, we aimed to address the structure and the assembly of minor pilins, protein components of Enterohemorrhagic Escherichia coli T4aP. We optimised the overexpression, purification and labelling of the minor pilins for their structural study by NMR. Furthermore, molecular modelling of the minor pilins and crosslinking mass spectrometry were performed on whole T4aP and T2SS pseudopili purified samples as a methodology to determine the structure and the interactions of pilins and pseudopilins within the native pilus
Allombert, Julie. "Rôles des voies de signalisation à di-GMP cyclique chez Legionella pneumophila". Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10161/document.
Testo completoLegionella pneumophila is a bacterium that proliferates in fresh water environments through the replication within amoebas. These bacteria can persist in these environments through biofilm formation. The inhalation of aerosolized contaminated water through hot water systems or cooling towers can induce the infection of human lungs, leading to a severe pneumonia called legionellosis. Cyclic di‐GMP (c‐di‐GMP) in involved, in various bacterial species, in the motility‐to‐sessility transition, and in some pathogens, in virulence control. My work aims to demonstrate the involvement of signaling pathways that use c‐di‐GMP in virulence control and biofilm formation of L. pneumophila. This involvement was investigated by systematically inactivating each gene encoding a c‐di‐GMP‐metabolizing enzyme in L. pneumophila Lens strain. Our work revealed that 3 of these proteins, Lpl0780, Lpl0922 and Lpl1118 are specifically involved in virulence control and, particularly, in the early survival during host cell infection through the orchestration of virulence factors secretion within host cell. Lpl1118 is particularly required for replicative vacuole biogenesis. Five other proteins, participate in the formation and architecture of biofilms. One of them is more specifically involved in biofilm formation in the presence of nitric oxide. These results help to better understand the complexity and the specificity of c‐di‐GMP signaling pathways in L. pneumophila and should allow the exploration of more effective ways to fight this pathogen
Felix, Christine. "Etude moléculaire de la bactérie intracellulaire féminisante Wolbachia chez Armadillidium vulgare (crustacé isopode terrestre)". Phd thesis, Université de Poitiers, 2004. http://tel.archives-ouvertes.fr/tel-00011718.
Testo completoArantxa, Camus Etchecopar. "Mécanismes moléculaires impliqués dans la formation de biofilm à l’interface eau-composés organiques hydrophobes". Thesis, Pau, 2014. http://www.theses.fr/2014PAUU3032/document.
Testo completoHydrophobic organic compounds (HOC), a large family of naturally-produced or anthropogenic molecules including lipids and hydrocarbons, represent a significant part of organic matter in marine ecosystems. Because of their low solubility in water, bacteria that degrade those compounds require the establishment of specific cell functions to increase their biodisponibility. Biofilm formation in water-HOC interface is one of these adaptations. The model of bacteria used in our laboratory, Marinobacter hydrocarbonoclasticus SP17, is able to form a biofilm on a wide range of HOC, such as alkanes, fatty alcohols and triglycerides, in order to use them as a carbon and energy source. The main purpose of my work was to broaden the knowledge of how bacteria adhere to and from biofilms on HOC, through the functional characterization of 10 candidate genes highlighted during proteomic and transcriptomic studies. Genetic tools and a gene-specific functional characterization have been developed in order to carry out this project. Functional study conducted on MARHY2686 revealed its involvement in the formation of biofilm on alkanes. Co-expression of MARHY2686 and the adjacent genes MARHY2687 and MARHY2685 durnig transcriptomic analysis together with their phylogenetic distribution and synteny conservation suggest that these three genes are involved in the same biological process. According to the high peptide sequence identity between MARHY2686 and AdeT, a periplasmic protein of a tripartite efflux pump system of Acinetobacter baumanii, MARHY2686 in combination with MARHY2687 and MARHY2685 could be the components of such a system. Other phenotypic observations would consider the involvement of MARHY2686 either in the assimilation of HOC or in the accumulation of intracellular lipid reserves. M. hydrocarbonoclasticus SP17 uses type IV pili during biofilm formation on HOC. These appendages are involved in the adhesion of this strain to and in a detachment process from HOC. Type IV pili could either act directly to allow bacteria to detach from the surface to which it is adhered, or indirectly through the action of bacteriophages. The presence of twitching motility on HOC has also been suggested. Finally, the role of the type VI secretion system (T6SS), a well-known protein system which allows interactions between bacteria and host cells, during the formation of a mono-species biofilm on HOC where no other microorganism than M. hydrocarbonoclasticus SP17 is present, has been studied
Dangla, Pélissier Gauthier. "Identification et caractérisation des régulateurs du transfert horizontal de l’îlot de pathogénicité PAPI-1 chez Pseudomonas aeruginosa PA14". Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0136.
Testo completoHorizontal transfer of DNA is one of the major motors of evolutive forces. It allows bacteria to obtain extra biological functions such as new metabolic pathways, resistance factors against environmental stresses and adaption to therapeutic strategies. The opportunistic human pathogen P. aeruginosa PA14 is a gram-negative bacterium with a large genome plasticity partially due to genomic island acquisition. The major genomic acquisition is PAPI-1 which considerably increases the virulence potential of the PA14 strain. Indeed, a strain of P. aeruginosa without PAPI-1 is less infectious against various organisms. This genomic island, belonging to ICE family, is self-transmissible among P. aeruginosa species via a conjugative machinery (T4SS-GI) requiring at least 55 genes encoded within it. During my PhD, I proved that MvaT repress the conjugative pilus biosynthesis and that the anti-H-NS NdpA2 can release this repression to allow the transcriptional regulation factor (TRF) TprA to induce T4SS-GI synthesis. I also proved that TprA controls phenotype changes in P. aeruginosa PA14 through the regulation of the majority of PAPI-1 encoded genes. Through this work I also characterised the domain leading to the specificity of action of RHH family TRF. As a matter of fact, the N-terminal region of these TRF interacts directly with DNA leading their binding specificity. At last, by a random mutagenesis screening, I identified what seems to be a regulation cascade of horizontal transference in P. aeruginosa PA14
Casu, Bastien. "Caractérisation biochimique, structurale et inhibition du système de sécrétion de type IV par l’étude des protéines VirB8". Thèse, 2018. http://hdl.handle.net/1866/20745.
Testo completoMary, Charline. "Analyse du rôle de l’interaction de VirB6 avec VirB10 dans le système de sécrétion de type IV". Thèse, 2018. http://hdl.handle.net/1866/21188.
Testo completoSharifahmadian, Mahzad. "Structural and Biochemical Characterization of VirB8 Protein in Type IV Secretion Systems". Thèse, 2017. http://hdl.handle.net/1866/19323.
Testo completoLa sécrétion est le passage de macromolécules à travers les membranes cellulaires. Chez les bactéries, la sécrétion est essentielle pour la virulence et la survie. Les bactéries à Gramnégatif utilisent le système de sécrétion de type IV (SST4) pour la sécrétion de toxines et de nucléoprotéines. Les SST4 contribuent notamment à la propagation des gènes de résistance aux antibiotiques. Pour cette raison, les composants du SST4 sont des cibles potentielles pour le développement de médicaments antivirulence. Le SST4 est un complexe protéique qui s’étend entre la double membrane de la bactérie à Gram-négatif. Les protéines qui le composent sont insérées dans les membranes cellulaires ou solubles. Bien que la structure du pore central du SST4 ait été résolue récemment, les détails de l'assemblage et la structure de ce complexe ne sont pas connus. VirB8 est une protéine de la membrane interne qui interagit avec de nombreuses autres sous-unités du SST4. Il s’agit d’un acteur central de l'assemblage du SST4. Des études biophysiques, et notamment des expériences de RMN ont ainsi été réalisées pour caractériser les aspects structuraux des interactions avec VirB8. Des regions dynamiques dans la structure de VirB8 ont été identifiées par spectroscopie RMN lors de la transition entre la forme monomérique et dimérique. Les analyses de cristallographie et de RMN ont révélé des différences structurales dans les régions hélicoïdales (α1 et α4) de VirB8 wild-type et du variant monomérique VirB8M102R. Le criblage de fragments a permis d’identifier de petites molécules capables de se lier à VirB8 ainsi qu’au variant monomérique. Les analyses d’arrimage moléculaire in silico suggèrent que la rainure de surface dans la structure VirB8 est importante pour laliaison de ces petites molécules. Les expériences de RMN et les essais biochimiques révèlent que le feuillet β (β1 en particulier) constitue l'interface d’interaction entre VirB8 et VirB10. Cette interface d’interaction est d’ailleurs importante pour la conjugaison du SST4. De plus, j'ai identifié des changements dans la structure de VirB8 lors de l'interaction avec VirB5. Les études sur la protéine VirB8 nous ont permis de caractériser la séquence d'événements entre VirB8 et d'autres protéines VirB, régulant l'assemblage et la fonction du SST4.