Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response

TANK-binding kinase 1 (TBK1) is of central importance for the induction of type-I interferon (IFN) in response to pathogens. We identified the DEAD-box helicase DDX3X as an interaction partner of TBK1. TBK1 and DDX3X acted synergistically in their ability to stimulate the IFN promoter, whereas RNAi-mediated reduction of DDX3X expression led to an impairment of IFN production. Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction

Function of Macrophage and Parasite Phosphatases in Leishmaniasis

The kinetoplastid protozoan parasites belonging to the genus Leishmania are the causative agents of different clinical forms of leishmaniasis, a vector-borne infectious disease with worldwide prevalence. The protective host immune response against Leishmania parasites relies on myeloid cells such as dendritic cells and macrophages in which upon stimulation by cytokines (e.g., interferon-γ) a complex network of signaling pathways is switched on leading to strong antimicrobial activities directed against the intracellular parasite stage. The regulation of these pathways classically depends on post-translational modifications of proteins, with phosphorylation events playing a cardinal role. Leishmania parasites deactivate their phagocytic host cells by inducing specific mammalian phosphatases that are capable to impede signaling. On the other hand, there is now also evidence that Leishmania spp. themselves express phosphatases that might target host cell molecules and thereby facilitate the intracellular survival of the parasite. This review will present an overview on the modulation of host phosphatases by Leishmania parasites as well as on the known families of Leishmania phosphatases and their possible function as virulence factors. A more detailed understanding of the role of phosphatases in Leishmania–host cell interactions might open new avenues for the treatment of non-healing, progressive forms of leishmaniasis

Function of Macrophage and Parasite Phosphatases in Leishmaniasis

The kinetoplastid protozoan parasites belonging to the genus Leishmania are the causative agents of different clinical forms of leishmaniasis, a vector-borne infectious disease with worldwide prevalence. The protective host immune response against Leishmania parasites relies on myeloid cells such as dendritic cells and macrophages in which upon stimulation by cytokines (e.g., interferon-γ) a complex network of signaling pathways is switched on leading to strong antimicrobial activities directed against the intracellular parasite stage. The regulation of these pathways classically depends on post-translational modifications of proteins, with phosphorylation events playing a cardinal role. Leishmania parasites deactivate their phagocytic host cells by inducing specific mammalian phosphatases that are capable to impede signaling. On the other hand, there is now also evidence that Leishmania spp. themselves express phosphatases that might target host cell molecules and thereby facilitate the intracellular survival of the parasite. This review will present an overview on the modulation of host phosphatases by Leishmania parasites as well as on the known families of Leishmania phosphatases and their possible function as virulence factors. A more detailed understanding of the role of phosphatases in Leishmania–host cell interactions might open new avenues for the treatment of non-healing, progressive forms of leishmaniasis

Phosphorylation des protéines au niveau de la tyrosine chez Staphylococcus Aureus (mécanisme moléculaire et rôle biologique)

Ce travail s'inscrit dans le cadre de l'étude de la phosphorylation réversible des protéines au niveau de la tyrosine chez Staphylococcus aureus. Nous avons tout d'abord identifié, chez cette bactérie, deux activités déphosphorylantes, les tyrosine-phosphatases de bas poids moléculaire Ptp5A et Ptp5B, et une activité phosphorylante, la tyrosine-kinase Cap5B2. Nous avons ensuite réalisé la caractérisation biochimique des protéines Ptp5A et Ptp5B. Nous avons également déterminé le mécanisme moléculaire d'activation de l'activité kinase de la protéine Cap5B2. Une étude comparable réalisée sur la tyrosine-kinase Wzc d'Escherichia coli a permis de mettre en évidence deux modes d'activation différents entre les tyrosine-kinases de ces deux espèces bactériennes. Enfin, nous avons identifié un substrat endogène de la protéine Cap5B2 : la protéine Cap5O codée par un gène appartenant à l'opéron de biosynthèse de la capsule. Cette protéine possède une activité UDP-N-acétylmannosamine déshydrogénase qui augmente lorsqu'elle est phosphorylée sur des résidus tyrosyles. L'étude au niveau moléculaire de ces tyrosine-kinases et tyrosine-phosphatases vise à mieux appréhender le rôle de ces enzymes dans le métabolisme des bactéries et, plus particulièrement, dans la régulation de leur virulenceLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Etapes du procédé RTM pour pièces de protections thermiques

Dans le cadre d'une application de tubes de protections thermiques, ce travail décrira les différentes étapes du procédé de fabrication, de type RTM. L'importance des étapes de dépose et de compaction sèche seront mise en évidence, par une approche expérimentale. Le choix des renforts en adéquation avec le procédé sera justifié. L’influence des paramètres, orientation et densité des mèches sur l'étape d'injection sera analysée. La qualité de la pièce finale sera étudiée par des tomographies

INJECTION OF A COMPLEX PREFORM BY RTM. OPTIMISATION OF A MOULD DESIGN AND PROCESS PARAMETERS

International audienceThis work concerns the manufacturing of a composite tube using RTM process. During the preforming stage, a woven braid is laid down and stacked on a mandrel so that reinforcement plies form conical shapes with a defined angle. An innovative experimental preforming procedure has been developed to respect the specific angle. The resin injection step has been studied both experimentally and numerically. In this work a focus is applied on the optimization of the mould geometry and on the way the resin is injected within the preform to prevent the appearance of defects such as displacement of plies and porosity. Different optimization steps are described in this work and a mould geometry is proposed for this specific application and the quality of the part obtained is discussed and analysed

Staphylococcus aureus Contains Two Low-Molecular-Mass Phosphotyrosine Protein Phosphatases

The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacterium Staphylococcus aureus suggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in an Escherichia coli system. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40°C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors, N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases

INJECTION OF A COMPLEX PREFORM BY RTM PROCESS.

International audienceThis work concerns the manufacturing of a composite tube using RTM process. During the preforming stage, a woven braid is laid down and stacked on a mandrel so that reinforcement plies form conical shapes with a defined angle. An innovative experimental preforming procedure has been developed to respect the specific angle. The resin injection step has been studied both experimentally and numerically. The influence of processing parameters such as the injection pressure or the imposed flow rate on the position and the size of possible defects has been evaluated. A numerical study has been conducted to model the flow of resin within the different areas of the complex preform. Planar and through-the-thickness permeability studies were conducted experimentally as a function of the fibre volume fraction and the shear angle. Within the preform and especially along a radius of the part, variation of shear, fiber volume fraction, orientation of yarns and consequently variation of the permeability tensor must be taken into account for the simulation of the resin injection. Simulation results presenting the evolution of the flow front within the preform for different injection cases are discussed and correlated to the experimental study results previously mentioned