Etude de l'interaction entre les lectines et les lipopolysaccharides par Résonance Magnétique Nucléaire.

For an equilibrated survival of humans against bacterial infections, immune cells have a well-defined function to distinguish between self and non-self structures. Escherichia coli is a Gram-negative bacterium that reside in the gut microbiota, either as a beneficial or harmful microorganism.The tolerance of pathogenic strains or their clearance is covered by many immune cell receptors such as Pathogen Recognition Receptors (PRRs) including C-type lectin receptors (CLRs) that specifically interact with carbohydrates moieties. Bacterial Lipopolysaccharide (LPS) is one of the bacterial signatures and it is the hallmark of Gram-negative bacteria. The molecular diversity of both LPS (i.e. various carbohydrates, lengths and heterogeneity levels) and lectins (variable binding sites architectures) would confer them the particularity of controlling variable situations and targeting specific interactions. The understanding of the mechanisms of LPS-Lectins interactions is challenging and requires an interdisciplinary approach.On the above basis, we sought to investigate the interaction between a C-type lectin i.e. human Macrophage Galactose-type Lectin MGL and LPSs (from E. coli R1, R3 mutants and O157:H7 strain). Bacterial LPSs were extracted, purified, and thereafter considered for the investigation of this large and complex interaction system. The difficult experimental handling of such native biomolecules directed the use of a divided set of approaches. . In fact, our scientific strategy includes the use of Nuclear Magnetic Resonance NMR spectroscopy, fluorescence microscopy, and molecular binding essays. By combining these methods, we studied two distinct Lectin-LPS interaction systems: i) the molecular interaction between the Carbohydrate Recognition Domain (CRD) of MGL and soluble LPS versions by using NMR titrations and computational methods; ii) the recognition of E. coli mutants and LPS glycoconjugates by the Extracellular Domain (ECD) of MGL at both molecular and cellular levels by STD-NMR combined with computational analyses, Biolayer Interferometry (BLI), Electron Microscopy (EM) and fluorescence microscopy coupled to flow Cytometry.When only the CRD of MGL is considered, all tested E. coli LPS were found to bind in the millimolar affinity range, to an extended interacting area that includes a putative secondary binding region, in addition to the canonical calcium binding site, common in C-type lectins.Spectroscopic and microscopic investigations provided promising results about the recognition between ECD MGL and E. coli LPS/LOS. Trimeric human MGL interacts specifically with E. coli R1 LOS mainly through binding to the terminal di-galactose moiety. In addition, the dissociation constant (Kd) was estimated to be in the nanomolar range thus indicating a strong molecular binding. Moreover, the specific interaction between fluorescently labelled human MGL and E. coli R1 bacteria was investigated by single-cell essays. MGL-bound E. coli R1 bacteria were fluorescent (up to 40% of the bacterial population), exclusively at stationary phase, whereas E. coli R3 were not. Here again, binding specificity and selectivity was confirmed for ECD MGL-R1 interaction system.We showed that MGL strongly and specifically interacts with E. coli R1 glycoconjugates at the surface of bacteria through its terminal di-galactose motif. The contribution of a putative secondary binding site on MGL in glycoconjugates recognition remains to be investigated. This PhD work showed that, despite the difficulties that such large system studies may encounter, many findings are attainable by using our scientific strategy. The possibility of investigating data from atomic to cellular scale on LPS-lectin interactions in either modified or native states, opens prominent horizons for the study of bacterial infections.Pour que les humains aient une survie stable contre les infections bactériennes, les cellules immunitaires ont une fonction bien définie qui leurs permet de distinguer les antigènes du soi et du non-soi. Escherichia coli est une bactérie à Gram-negative qui réside dans le microbiote humain en étant un microorganisme bénéfique mais qui peut être aussi pathogene. La tolérance des souches pathogène ou leur élimination est assuré par les récepteurs des cellules immunitaires tels que les Pathogen Recognition Receptors PRRs incluant les lectines du type-C CLRs qui interagissent d’une manière spécifique avec les carbohydrates. Le Lipopolysaccharide LPS est la signature des bactéries à Gram-negatives en étant à la fois un motif structural et de reconnaissance par la cellule hôte. La diversité structurale des LPS (i.e. différents niveaux d’hétérogénéités) et des lectines humaines (sites de liaison avec différentes architectures) leurs confère la particularité de contrôler des situations variables en ciblant des interactions spécifiques. Ainsi, comprendre les mécanismes des interactions LPS-Lectine est un défi et ceci exige une approche scientifique multidisciplinaire. Sur cette base, nous avons cherché à étudier l’interaction entre la lectine humaine Macrophage Galactose-type Lectine MGL, et, les LPSs des mutants E. coli R1, R3 et wild-type O157:H7. Les LPSs ont été extrait, purifiés et ensuite considérés pour les analyses de ce système d’interaction large et complexe. Nous avons étudié deux systèmes d’interaction LPS-Lectine entre: i) le domaine de reconnaissance des carbohydrates CRD de la MGL humaine et les versions solubles des LPSmoyennant des titrations RMN et études computationnels; ii) le domaine extracellulaire ECD de la MGL, et LPS, sous forme d’assemblages membranaire ou directement sur la surface des bactéries E. coli, à travers la STD-RMN combinée aux analyses computationnel,l’interférométrie BLI,la microscopie électronique EM et la microscopie à fluorescence couplée à la cyrtométrie en flux. Lorsque le CRD de la MGL est concerné, tous les LPSs exprimés par E. coli ont interagit avec une affinité de liaison faible, sur une surface élargie incluant un second site putatif, en plus du premier site calcique commun aux lectines type-C. Nos études ont fourni des résultats prometteurs sur la reconnaissance des LPS d’E. coli par le domaine ECD de la MGL humaine. La MGL trimérique interagit d’une manière spécifique avec le LOS, une structure courte du LPS exprimé chez la souche E. coli R1, principalement à travers la liaison au di-galactose l’épitope terminal du LOS. La constante de dissociation (Kd) a été estimée de l’ordre du nanomolaire indiquant une forte interaction. En outre, laspécificité de cette interaction a été confirméepar l’étude de la MGL marqué par un fluorophore où les bactéries E. coli R1 ont été visualisée, sous le microscope, en temps réel. Les bactéries E. coli R1 liées à la MGL étaient fluorescentes (jusqu’à 40% de la population bactérienne), exclusivement en phase stationnaire, tandis que les bactéries E. coli R3 ne manifestèrent aucune luminescence. Là encore, le choix de la MGL d’interagir d’une manière sélective et spécifique était orienté vers les glycoconjugués d’E. coli R1. Nous avons démontré que la MGL humaine ECD interagit fortement et spécifiquement avec les glycoconjugués d’E. coli R1 à la surface bactérienne à travers les motifs di-galactoses des LOSs. La contribution du second site de liaison sur la MGL CRD à la reconnaissance des glycoconjugués d’E. coli reste à analyser. Ce travail de doctorat a montré que, malgré la complexité du système d’interaction, il est possible d’étudier la reconnaissance des agents pathogènes, moyennant la combinaison d’approches. La possibilité d’analyser les données brutes obtenus sur les interactions LPS-Lectine (en état natif ou décomposé), de l’échelle atomique à la cellulaire, permettrait d’ouvrir de nouvel horizon pour l’étude des infections bactériennes

Etude de l'interaction entre les lectines et les lipopolysaccharides par Résonance Magnétique Nucléaire.

For an equilibrated survival of humans against bacterial infections, immune cells have a well-defined function to distinguish between self and non-self structures. Escherichia coli is a Gram-negative bacterium that reside in the gut microbiota, either as a beneficial or harmful microorganism.The tolerance of pathogenic strains or their clearance is covered by many immune cell receptors such as Pathogen Recognition Receptors (PRRs) including C-type lectin receptors (CLRs) that specifically interact with carbohydrates moieties. Bacterial Lipopolysaccharide (LPS) is one of the bacterial signatures and it is the hallmark of Gram-negative bacteria. The molecular diversity of both LPS (i.e. various carbohydrates, lengths and heterogeneity levels) and lectins (variable binding sites architectures) would confer them the particularity of controlling variable situations and targeting specific interactions. The understanding of the mechanisms of LPS-Lectins interactions is challenging and requires an interdisciplinary approach.On the above basis, we sought to investigate the interaction between a C-type lectin i.e. human Macrophage Galactose-type Lectin MGL and LPSs (from E. coli R1, R3 mutants and O157:H7 strain). Bacterial LPSs were extracted, purified, and thereafter considered for the investigation of this large and complex interaction system. The difficult experimental handling of such native biomolecules directed the use of a divided set of approaches. . In fact, our scientific strategy includes the use of Nuclear Magnetic Resonance NMR spectroscopy, fluorescence microscopy, and molecular binding essays. By combining these methods, we studied two distinct Lectin-LPS interaction systems: i) the molecular interaction between the Carbohydrate Recognition Domain (CRD) of MGL and soluble LPS versions by using NMR titrations and computational methods; ii) the recognition of E. coli mutants and LPS glycoconjugates by the Extracellular Domain (ECD) of MGL at both molecular and cellular levels by STD-NMR combined with computational analyses, Biolayer Interferometry (BLI), Electron Microscopy (EM) and fluorescence microscopy coupled to flow Cytometry.When only the CRD of MGL is considered, all tested E. coli LPS were found to bind in the millimolar affinity range, to an extended interacting area that includes a putative secondary binding region, in addition to the canonical calcium binding site, common in C-type lectins.Spectroscopic and microscopic investigations provided promising results about the recognition between ECD MGL and E. coli LPS/LOS. Trimeric human MGL interacts specifically with E. coli R1 LOS mainly through binding to the terminal di-galactose moiety. In addition, the dissociation constant (Kd) was estimated to be in the nanomolar range thus indicating a strong molecular binding. Moreover, the specific interaction between fluorescently labelled human MGL and E. coli R1 bacteria was investigated by single-cell essays. MGL-bound E. coli R1 bacteria were fluorescent (up to 40% of the bacterial population), exclusively at stationary phase, whereas E. coli R3 were not. Here again, binding specificity and selectivity was confirmed for ECD MGL-R1 interaction system.We showed that MGL strongly and specifically interacts with E. coli R1 glycoconjugates at the surface of bacteria through its terminal di-galactose motif. The contribution of a putative secondary binding site on MGL in glycoconjugates recognition remains to be investigated. This PhD work showed that, despite the difficulties that such large system studies may encounter, many findings are attainable by using our scientific strategy. The possibility of investigating data from atomic to cellular scale on LPS-lectin interactions in either modified or native states, opens prominent horizons for the study of bacterial infections.Pour que les humains aient une survie stable contre les infections bactériennes, les cellules immunitaires ont une fonction bien définie qui leurs permet de distinguer les antigènes du soi et du non-soi. Escherichia coli est une bactérie à Gram-negative qui réside dans le microbiote humain en étant un microorganisme bénéfique mais qui peut être aussi pathogene. La tolérance des souches pathogène ou leur élimination est assuré par les récepteurs des cellules immunitaires tels que les Pathogen Recognition Receptors PRRs incluant les lectines du type-C CLRs qui interagissent d’une manière spécifique avec les carbohydrates. Le Lipopolysaccharide LPS est la signature des bactéries à Gram-negatives en étant à la fois un motif structural et de reconnaissance par la cellule hôte. La diversité structurale des LPS (i.e. différents niveaux d’hétérogénéités) et des lectines humaines (sites de liaison avec différentes architectures) leurs confère la particularité de contrôler des situations variables en ciblant des interactions spécifiques. Ainsi, comprendre les mécanismes des interactions LPS-Lectine est un défi et ceci exige une approche scientifique multidisciplinaire. Sur cette base, nous avons cherché à étudier l’interaction entre la lectine humaine Macrophage Galactose-type Lectine MGL, et, les LPSs des mutants E. coli R1, R3 et wild-type O157:H7. Les LPSs ont été extrait, purifiés et ensuite considérés pour les analyses de ce système d’interaction large et complexe. Nous avons étudié deux systèmes d’interaction LPS-Lectine entre: i) le domaine de reconnaissance des carbohydrates CRD de la MGL humaine et les versions solubles des LPSmoyennant des titrations RMN et études computationnels; ii) le domaine extracellulaire ECD de la MGL, et LPS, sous forme d’assemblages membranaire ou directement sur la surface des bactéries E. coli, à travers la STD-RMN combinée aux analyses computationnel,l’interférométrie BLI,la microscopie électronique EM et la microscopie à fluorescence couplée à la cyrtométrie en flux. Lorsque le CRD de la MGL est concerné, tous les LPSs exprimés par E. coli ont interagit avec une affinité de liaison faible, sur une surface élargie incluant un second site putatif, en plus du premier site calcique commun aux lectines type-C. Nos études ont fourni des résultats prometteurs sur la reconnaissance des LPS d’E. coli par le domaine ECD de la MGL humaine. La MGL trimérique interagit d’une manière spécifique avec le LOS, une structure courte du LPS exprimé chez la souche E. coli R1, principalement à travers la liaison au di-galactose l’épitope terminal du LOS. La constante de dissociation (Kd) a été estimée de l’ordre du nanomolaire indiquant une forte interaction. En outre, laspécificité de cette interaction a été confirméepar l’étude de la MGL marqué par un fluorophore où les bactéries E. coli R1 ont été visualisée, sous le microscope, en temps réel. Les bactéries E. coli R1 liées à la MGL étaient fluorescentes (jusqu’à 40% de la population bactérienne), exclusivement en phase stationnaire, tandis que les bactéries E. coli R3 ne manifestèrent aucune luminescence. Là encore, le choix de la MGL d’interagir d’une manière sélective et spécifique était orienté vers les glycoconjugués d’E. coli R1. Nous avons démontré que la MGL humaine ECD interagit fortement et spécifiquement avec les glycoconjugués d’E. coli R1 à la surface bactérienne à travers les motifs di-galactoses des LOSs. La contribution du second site de liaison sur la MGL CRD à la reconnaissance des glycoconjugués d’E. coli reste à analyser. Ce travail de doctorat a montré que, malgré la complexité du système d’interaction, il est possible d’étudier la reconnaissance des agents pathogènes, moyennant la combinaison d’approches. La possibilité d’analyser les données brutes obtenus sur les interactions LPS-Lectine (en état natif ou décomposé), de l’échelle atomique à la cellulaire, permettrait d’ouvrir de nouvel horizon pour l’étude des infections bactériennes

Deciphering the recognition patterns between lipopolysaccharides and human lectins using nuclear magnetic resonance spectroscopy

Pour que les humains aient une survie stable contre les infections bactériennes, les cellules immunitaires ont une fonction bien définie qui leurs permet de distinguer les antigènes du soi et du non-soi. Escherichia coli est une bactérie à Gram-negative qui réside dans le microbiote humain en étant un microorganisme bénéfique mais qui peut être aussi pathogene. La tolérance des souches pathogène ou leur élimination est assuré par les récepteurs des cellules immunitaires tels que les Pathogen Recognition Receptors PRRs incluant les lectines du type-C CLRs qui interagissent d’une manière spécifique avec les carbohydrates. Le Lipopolysaccharide LPS est la signature des bactéries à Gram-negatives en étant à la fois un motif structural et de reconnaissance par la cellule hôte. La diversité structurale des LPS (i.e. différents niveaux d’hétérogénéités) et des lectines humaines (sites de liaison avec différentes architectures) leurs confère la particularité de contrôler des situations variables en ciblant des interactions spécifiques. Ainsi, comprendre les mécanismes des interactions LPS-Lectine est un défi et ceci exige une approche scientifique multidisciplinaire. Sur cette base, nous avons cherché à étudier l’interaction entre la lectine humaine Macrophage Galactose-type Lectine MGL, et, les LPSs des mutants E. coli R1, R3 et wild-type O157:H7. Les LPSs ont été extrait, purifiés et ensuite considérés pour les analyses de ce système d’interaction large et complexe. Nous avons étudié deux systèmes d’interaction LPS-Lectine entre: i) le domaine de reconnaissance des carbohydrates CRD de la MGL humaine et les versions solubles des LPSmoyennant des titrations RMN et études computationnels; ii) le domaine extracellulaire ECD de la MGL, et LPS, sous forme d’assemblages membranaire ou directement sur la surface des bactéries E. coli, à travers la STD-RMN combinée aux analyses computationnel,l’interférométrie BLI,la microscopie électronique EM et la microscopie à fluorescence couplée à la cyrtométrie en flux. Lorsque le CRD de la MGL est concerné, tous les LPSs exprimés par E. coli ont interagit avec une affinité de liaison faible, sur une surface élargie incluant un second site putatif, en plus du premier site calcique commun aux lectines type-C. Nos études ont fourni des résultats prometteurs sur la reconnaissance des LPS d’E. coli par le domaine ECD de la MGL humaine. La MGL trimérique interagit d’une manière spécifique avec le LOS, une structure courte du LPS exprimé chez la souche E. coli R1, principalement à travers la liaison au di-galactose l’épitope terminal du LOS. La constante de dissociation (Kd) a été estimée de l’ordre du nanomolaire indiquant une forte interaction. En outre, laspécificité de cette interaction a été confirméepar l’étude de la MGL marqué par un fluorophore où les bactéries E. coli R1 ont été visualisée, sous le microscope, en temps réel. Les bactéries E. coli R1 liées à la MGL étaient fluorescentes (jusqu’à 40% de la population bactérienne), exclusivement en phase stationnaire, tandis que les bactéries E. coli R3 ne manifestèrent aucune luminescence. Là encore, le choix de la MGL d’interagir d’une manière sélective et spécifique était orienté vers les glycoconjugués d’E. coli R1. Nous avons démontré que la MGL humaine ECD interagit fortement et spécifiquement avec les glycoconjugués d’E. coli R1 à la surface bactérienne à travers les motifs di-galactoses des LOSs. La contribution du second site de liaison sur la MGL CRD à la reconnaissance des glycoconjugués d’E. coli reste à analyser. Ce travail de doctorat a montré que, malgré la complexité du système d’interaction, il est possible d’étudier la reconnaissance des agents pathogènes, moyennant la combinaison d’approches. La possibilité d’analyser les données brutes obtenus sur les interactions LPS-Lectine (en état natif ou décomposé), de l’échelle atomique à la cellulaire, permettrait d’ouvrir de nouvel horizon pour l’étude des infections bactériennes.For an equilibrated survival of humans against bacterial infections, immune cells have a well-defined function to distinguish between self and non-self structures. Escherichia coli is a Gram-negative bacterium that reside in the gut microbiota, either as a beneficial or harmful microorganism.The tolerance of pathogenic strains or their clearance is covered by many immune cell receptors such as Pathogen Recognition Receptors (PRRs) including C-type lectin receptors (CLRs) that specifically interact with carbohydrates moieties. Bacterial Lipopolysaccharide (LPS) is one of the bacterial signatures and it is the hallmark of Gram-negative bacteria. The molecular diversity of both LPS (i.e. various carbohydrates, lengths and heterogeneity levels) and lectins (variable binding sites architectures) would confer them the particularity of controlling variable situations and targeting specific interactions. The understanding of the mechanisms of LPS-Lectins interactions is challenging and requires an interdisciplinary approach.On the above basis, we sought to investigate the interaction between a C-type lectin i.e. human Macrophage Galactose-type Lectin MGL and LPSs (from E. coli R1, R3 mutants and O157:H7 strain). Bacterial LPSs were extracted, purified, and thereafter considered for the investigation of this large and complex interaction system. The difficult experimental handling of such native biomolecules directed the use of a divided set of approaches. . In fact, our scientific strategy includes the use of Nuclear Magnetic Resonance NMR spectroscopy, fluorescence microscopy, and molecular binding essays. By combining these methods, we studied two distinct Lectin-LPS interaction systems: i) the molecular interaction between the Carbohydrate Recognition Domain (CRD) of MGL and soluble LPS versions by using NMR titrations and computational methods; ii) the recognition of E. coli mutants and LPS glycoconjugates by the Extracellular Domain (ECD) of MGL at both molecular and cellular levels by STD-NMR combined with computational analyses, Biolayer Interferometry (BLI), Electron Microscopy (EM) and fluorescence microscopy coupled to flow Cytometry.When only the CRD of MGL is considered, all tested E. coli LPS were found to bind in the millimolar affinity range, to an extended interacting area that includes a putative secondary binding region, in addition to the canonical calcium binding site, common in C-type lectins.Spectroscopic and microscopic investigations provided promising results about the recognition between ECD MGL and E. coli LPS/LOS. Trimeric human MGL interacts specifically with E. coli R1 LOS mainly through binding to the terminal di-galactose moiety. In addition, the dissociation constant (Kd) was estimated to be in the nanomolar range thus indicating a strong molecular binding. Moreover, the specific interaction between fluorescently labelled human MGL and E. coli R1 bacteria was investigated by single-cell essays. MGL-bound E. coli R1 bacteria were fluorescent (up to 40% of the bacterial population), exclusively at stationary phase, whereas E. coli R3 were not. Here again, binding specificity and selectivity was confirmed for ECD MGL-R1 interaction system.We showed that MGL strongly and specifically interacts with E. coli R1 glycoconjugates at the surface of bacteria through its terminal di-galactose motif. The contribution of a putative secondary binding site on MGL in glycoconjugates recognition remains to be investigated. This PhD work showed that, despite the difficulties that such large system studies may encounter, many findings are attainable by using our scientific strategy. The possibility of investigating data from atomic to cellular scale on LPS-lectin interactions in either modified or native states, opens prominent horizons for the study of bacterial infections

The human macrophage galactose‐type lectin, MGL, recognizes the outer core of E. coli lipooligosaccharide

International audienceCarbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite the growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Here we describe a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of E. coli strain R1. Saturation Transfer Difference (STD) NMR analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through the recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognize glycan moieties exposed on Gram-negative bacterial surfaces

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces

The unique 3D arrangement of macrophage galactose lectin enables Escherichia coli lipopolysaccharide recognition through two distinct interfaces

International audienceAbstract Lipopolysaccharides are a hallmark of gram-negative bacteria, and their presence at the cell surface is key for bacterial integrity. As surface-exposed components, they are recognized by immunity C-type lectin receptors present on antigen-presenting cells. Human macrophage galactose lectin binds Escherichia coli surface that presents a specific glycan motif. Nevertheless, this high-affinity interaction occurs regardless of the integrity of its canonical calcium-dependent glycan-binding site. NMR of macrophage galactose-type lectin (MGL) carbohydrate recognition domain and complete extracellular domain revealed a glycan-binding site opposite to the canonical site. A model of trimeric macrophage galactose lectin was determined based on a combination of small-angle X-ray scattering and AlphaFold. A disulfide bond positions the carbohydrate recognition domain perpendicular to the coiled-coil domain. This unique configuration for a C-type lectin orients the six glycan sites of MGL in an ideal position to bind lipopolysaccharides at the bacterial surface with high avidity

Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN

Summary: Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy

Diagnosis of Mediterranean Visceral Leishmaniasis by Detection of Leishmania Antibodies and Leishmania DNA in Oral Fluid Samples Collected Using an Oracol Device ▿

Current methods for diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures such as visceral aspiration and/or blood drawing. The use of diagnostic tests using oral fluid, which is easier to collect, would be more simple and practical for VL diagnosis, especially under field conditions. Oral fluids from 37 VL patients and 40 healthy controls were collected using Oracol devices. Blood samples and oral fluid specimens from both groups were analyzed by recombinant protein K39 (rK39) enzyme-linked immunosorbent assay and quantitative real-time PCR. Detection of antibodies in the oral fluid had a sensitivity of 100% and a specificity of 97.5%. Antibody levels measured in serum and oral fluid showed a significant positive correlation (ρ = 0.655 and P = 0.01). Detection of Leishmania DNA in oral fluid had a sensitivity of 94.6% and a specificity of 90%. The median parasite load estimated in blood was 133 parasites/ml (interquartile range [IR], 10 to 1,048), whereas that in oral fluid specimens was 3 parasites/ml (IR, 0.41 to 92). However, there was no significant linear relationship between parasite loads assessed in the two biological samples (ρ = 0.31 and P = 0.06). VL diagnosis based on specific antibody detection and Leishmania DNA identification using oral fluid samples was equivalent in accuracy to that using blood and therefore is promising for clinical use

Alpha-mannosidosis in Tunisian consanguineous families: Potential involvement of variants in GHR and SLC19A3 genes in the variable expressivity of cognitive impairment

International audienceAlpha-Mannosidosis (AM) is an ultra-rare storage disorder caused by a deficiency of lysosomal alpha-mannosidase encoded by the MAN2B1 gene. Clinical presentation of AM includes mental retardation, recurrent infections, hearing loss, dysmorphic features, and motor dysfunctions. AM has never been reported in Tunisia. We report here the clinical and genetic study of six patients from two Tunisian families with AM. The AM diagnosis was confirmed by an enzymatic activity assay. Genetic investigation was conducted by Sanger sequencing of the mutational hotspots for the first family and by ES analysis for the second one. In the first family, a frameshift duplication p.(Ser802GlnfsTer129) was identified in the MAN2B1 gene. For the second family, ES analysis led to the identification of a missense mutation p.(Arg229Trp) in the MAN2B1 gene in four affected family members. The p.(Ser802GlnfsTer129) mutation induces a premature termination codon which may trigger RNA degradation by the NMD system. The decrease in the levels of MAN2B1 synthesis could explain the severe phenotype observed in the index case. According to the literature, the p.(Arg229Trp) missense variant does not have an impact on MAN2B1 maturation and transportation, which correlates with a moderate clinical sub-type. To explain the intra-familial variability of cognitive impairment, exome analysis allowed the identification of two likely pathogenic variants in GHR and SLC19A3 genes potentially associated to cognitive decline. The present study raises awareness about underdiagnosis of AM in the region that deprives patients from accessing adequate care. Indeed, early diagnosis is critical in order to prevent disease progression and to propose enzyme replacement therapy