Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival

Identification de mécanismes de régulation des fonctions des interférons: Rôle de la palmitoylation du récepteur de l'interféron de type I

Type I interferons (IFNs) play key roles in mediating innate and acquired immune responses, in host defense against viral infections, and exhibit antiproliferative and tumoricidal activity. Through the binding to their shared cell surface receptor, composed of the two subunits IFNAR1 and IFNAR2, they induce the JAK/STAT signaling cascade, thus leading to their biological effects. The goal of my thesis was to identify regulatory mechanisms of IFNs signaling. In this context, we analyzed the role of protein palmitoylation, a lipid modification which is known to be involved in trafficking and signaling of proteins. We found that pharmacological inhibition of palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of IFNAR1 where each or both of the two cysteines present in the cytoplasmic domain were replaced by alanines. We found that only Cys463, the most proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. Nevertheless, the lack of IFNAR1 palmitoylation results in major defects in JAK/STAT signaling and gene transcription activated by IFN-a. Suprisingly, it had no effect on IFN-a antiproliferative activity, in spite of the importance of palmitoylation for signaling.Les interférons (IFNs) de type I sont des cytokines qui jouent un rôle capital dans les défenses immunes, antivirales et antiprolifératives de l'organisme. En se liant à leur récepteur de surface, composé des deux sous-unités IFNAR1 et IFNAR2, ils induisent la cascade de signalisation JAK/STAT qui aboutit à leur effets biologiques. L'objectif de ma thèse était d'identifier des mécanismes de régulation de la signalisation des IFNs. Dans ce contexte, nous nous sommes intéressés à la palmitoylation du récepteur de l'IFN de type I, une modification lipidique souvent impliquée dans le trafic et la signalisation des protéines. Par marquage métabolique au palmitate tritié, nous avons montré qu'IFNAR1 et IFNAR2 sont palmitoylées. Le domaine cytoplasmique d'IFNAR1 contient deux cystéines, Cys463 et Cys502, qui sont des sites potentiels de palmitoylation. A l'aide de mutants sur chacune de ces cystéines, nous avons montré qu'IFNAR1 est palmitoylée uniquement sur sa cystéine la plus proche de la membrane plasmique, la Cys463. Un mutant non palmitoylé dans lequel cette cystéine a été remplacée par une alanine nous a permis de constater que la palmitoylation d'IFNAR1 n'est pas impliquée dans son trafic intracellulaire, dans son endocytose ni dans sa stabilité, mais qu'en revanche elle joue un rôle crucial dans l'activation de la voie de signalisation JAK/STAT. De façon concordante, la palmitoylation d'IFNAR1 est requise pour l'activation transcriptionnelle des gènes induits spécifiquement par l'IFN-a. Par contre, un défaut de palmitoylation n'influence nullement l'activité antiproliférative de l'IFN-a, en dépit du rôle de cette modification dans la signalisation

Identification de mécanismes de régulation des fonctions de interférons (Rôle de la palmitoylation du récepteur de l'interféron de type I)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

A Label-Free Optical Detection of Pathogens in Isopropanol as a First Step towards Real-Time Infection Prevention

The detection of pathogens is a major public health issue. Every year, thousands of people die because of nosocomial infections. It is therefore important to be able to detect possible outbreaks as early as possible, especially in the hospital environment. Various pathogen detection techniques have already been demonstrated. However, most of them require expensive and specific equipment, and/or complex protocols, which, most of the time, involve biochemical reaction and labelling steps. In this paper, a new method that combines microscopic imaging and machine learning is described. The main benefits of this approach are to be low-cost, label-free and easy to integrate in any suitable medical device, such as hand hygiene dispensers. The suitability of this pathogen detection method is validated using four bacteria, both in PBS (Phosphate Buffered Saline) and in isopropanol. In particular, we demonstrated an efficient pathogenic detection that is sensible to changes in the composition of a mixture of pathogens, even in alcohol-based solutions

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

International audienc

Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion

Background Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family. Methods Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line. Results In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of β1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments. Conclusion Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability. General significance After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity.ISSN:0304-4165ISSN:1872-800

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics

Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics