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Lonardi E.,Leiden University | Moonens K.,Structural and Molecular Microbiology | Buts L.,Molecular Recognition | Buts L.,Vrije Universiteit Brussel | And 10 more authors.
Biology | Year: 2013

Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG, the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Tiels P.,Unit for Medical Biotechnology | Tiels P.,Ghent University | Baranova E.,Structural and Molecular Microbiology | Baranova E.,Vrije Universiteit Brussel | And 19 more authors.
Nature Biotechnology | Year: 2012

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease. © 2012 Nature America, Inc.

Lo A.W.H.,Structural and Molecular Microbiology | Lo A.W.H.,Vrije Universiteit Brussel | Van de Water K.,Structural and Molecular Microbiology | Van de Water K.,Vrije Universiteit Brussel | And 10 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

Objectives: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). Methods: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. Results: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. Conclusions: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Lo A.W.H.,Structural and Molecular Microbiology | Lo A.W.H.,Vrije Universiteit Brussel | Moonens K.,Structural and Molecular Microbiology | Moonens K.,Vrije Universiteit Brussel | And 2 more authors.
Current Opinion in Microbiology | Year: 2013

Bacteria express a multitude of hair-like adhesive appendages on their cell surfaces, together referred to as pili or fimbriae. In Gram-negative bacteria, these proteinaceous structures are assembled through a number of dedicated secretion pathways including the chaperone-usher pathway, the nucleation/precipitation pathway and the type IV pilus pathway. Pili are prevalent in pathogenic strains and play important roles in the establishment and persistence of bacterial infections by mediating host cell adhesion, cell invasion or biofilm formation. Their indispensible roles in pathogenesis render them attractive targets for directed therapeutic intervention. Here, we describe the recent advances in the chemical attenuation of pilus-associated virulence in Gram-negative bacteria. © 2013 Elsevier Ltd.

Subedi S.,Structural and Molecular Microbiology | Subedi S.,Vrije Universiteit Brussel | Moonens K.,Structural and Molecular Microbiology | Moonens K.,Vrije Universiteit Brussel | And 10 more authors.
Acta Crystallographica Section F:Structural Biology Communications | Year: 2014

Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA25-460) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA25-460 were collected to 2.25Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40Å, α = 90.0, β = 94.8, γ = 90.0°, and which was predicted to contain two BabA25-460-nanobody complexes per asymmetric unit. © 2014 International Unoin of Crystallography.

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