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Burkholderia cenocepacia BC2L-C Is a Super Lectin with Dual Specificity and Proinflammatory Activity

Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-α-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and l-glycero-d-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-α-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-α-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation

Etudes structurales et fonctionnelles des lectines solubles de Burkholderia cenocepacia

Opportunistic infection by pathogens such as Pseudomonas aeruginosa and Burkholderiacenocepacia is the first cause of morbidity and mortality in cystic fibrosis patients. Theopportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydratebindingproteins, related to the fucose-binding lectin PA-IIL from Pseudomanas aeruginosa.These lectins could play a role in early stage of infection through specific binding to epithelialcells of hosts. They could also be involved in the building of biofilm that is responsible forresistance to antibiotics. The thesis is focused on structure-function studies of two B.cenocepacia lectins, BclA and BclB with the aim to correlate the data to the localisation andfunction in the bacteria. Glycan array data associated with titration microcalorimetry allowedto determine the specificity of the lectins and the affinity towards the best ligands.Localisation studies demonstrate the presence of the lectins in the bacteria cytoplasm but alsoon the outer membrane. Finally, crystal structures of lectin complexed with carbohydrate givethe molecular basis of the interaction.La colonisation des poumons par des germes comme Pseudomonas aeruginosa etBurkholderia cenocepacia représente la première cause de maladie et de mortalité chez lespatients atteints de la mucoviscidose. Quatre gènes de séquence apparentée à celui codantpour la lectine soluble PA-IIL (LecB) de P. aeruginosa ont été identifiés dans le génome de B.cenocepacia. Ces lectines pourraient être impliquées dans la reconnaissance des cellulesépithéliales de l’hôte au stade précoce de l’infection, ou encore dans la formation du biofilmresponsable de la résistance aux antibiotiques. Les travaux développés dans cette thèse portentsur l’étude structurale et fonctionnelle de deux de ces lectines : BclA et BclB, ainsi que sur lalocalisation et la fonction des protéines chez la bactérie. L’utilisation de glycan arraysassociée à la microcalorimétrie de titration ont permis de déterminer la spécificité des lectineset leur affinité pour les meilleurs ligands. Les études de localisation ont montré ces lectinescytoplasmiques sont aussi détectées à la surface de la membrane externe. Enfin, l’obtention decristaux de lectine complexée à son ligand permet de mieux comprendre l’interaction àl’échelle moléculaire

Etudes structurales et fonctionnelles des lectines solubles de Burkholderia cenocepacia

Opportunistic infection by pathogens such as Pseudomonas aeruginosa and Burkholderiacenocepacia is the first cause of morbidity and mortality in cystic fibrosis patients. Theopportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydratebindingproteins, related to the fucose-binding lectin PA-IIL from Pseudomanas aeruginosa.These lectins could play a role in early stage of infection through specific binding to epithelialcells of hosts. They could also be involved in the building of biofilm that is responsible forresistance to antibiotics. The thesis is focused on structure-function studies of two B.cenocepacia lectins, BclA and BclB with the aim to correlate the data to the localisation andfunction in the bacteria. Glycan array data associated with titration microcalorimetry allowedto determine the specificity of the lectins and the affinity towards the best ligands.Localisation studies demonstrate the presence of the lectins in the bacteria cytoplasm but alsoon the outer membrane. Finally, crystal structures of lectin complexed with carbohydrate givethe molecular basis of the interaction.La colonisation des poumons par des germes comme Pseudomonas aeruginosa etBurkholderia cenocepacia représente la première cause de maladie et de mortalité chez lespatients atteints de la mucoviscidose. Quatre gènes de séquence apparentée à celui codantpour la lectine soluble PA-IIL (LecB) de P. aeruginosa ont été identifiés dans le génome de B.cenocepacia. Ces lectines pourraient être impliquées dans la reconnaissance des cellulesépithéliales de l’hôte au stade précoce de l’infection, ou encore dans la formation du biofilmresponsable de la résistance aux antibiotiques. Les travaux développés dans cette thèse portentsur l’étude structurale et fonctionnelle de deux de ces lectines : BclA et BclB, ainsi que sur lalocalisation et la fonction des protéines chez la bactérie. L’utilisation de glycan arraysassociée à la microcalorimétrie de titration ont permis de déterminer la spécificité des lectineset leur affinité pour les meilleurs ligands. Les études de localisation ont montré ces lectinescytoplasmiques sont aussi détectées à la surface de la membrane externe. Enfin, l’obtention decristaux de lectine complexée à son ligand permet de mieux comprendre l’interaction àl’échelle moléculaire

Structural basis for mannose recognition by alectin from opportunistic bacteria Burkholderia cenocepacia

International audienceChronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex (Bcc) is the major cause of morbidity and mortality among cystic fibrosis (CF) patients. PA-IIL (lecB gene), a soluble lectin from P. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologs have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centers, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA, obtained. The presence of native BclA in B. cenocepacia extracts was checked by proteomics approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with α-methyl-mannoside (αMeMan) demonstrates a dissociation constant (Kd) of 2.75 x 10-6 M. The X-ray crystal structure of the complex with αMeMan was determined at 1.7Å resolution. The lectin forms homodimers with one binding site per monomer, acting cooperatively with the second dimer site. Each monomer contains two calcium ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site, and oligomerisation mode indicate that the proteins should have different roles in the bacteria

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

Peer reviewed: YesNRC publication: Ye

The structural basis for complement inhibition by gigastasin, a protease inhibitor from the giant Amazon leech

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway

The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body

SummaryThe type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic “effector” proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body

Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism

SummaryMycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host’s macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors