Stefan Oscarson

Receptor binding studies disclose a novel class of high‐affinity inhibitors of the Escherichia coli FimH adhesin

Mannose‐binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K‐12 strain, and affinity measurements with mannose, common mono‐ and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α‐ d‐mannoside, bound in  the  crystal  structures,  exhibits  a  significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities of α‐ d‐mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100‐fold improvement and 15‐fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti‐adhesive drugs to prevent anticipated and recurrent UTIs.

Gold nanoparticles as carriers for a synthetic Streptococcus pneumoniae type 14 conjugate vaccine.

AIMS Coupling of capsular polysaccharides of pathogens to immunogenic protein carriers (conjugate vaccines) improves carbohydrate immune response. Our idea is to explore gold nanoclusters as carriers to prepare fully synthetic carbohydrate vaccines. MATERIALS & METHODS Gold glyconanoparticles bearing a synthetic tetrasaccharide epitope related to the Streptococcus pneumoniae type 14 capsular polysaccharide (Pn14PS), the T-helper ovalbumin 323-339 peptide (OVA(323-339)), and D-glucose were prepared by a one-pot method. Their immunogenicity was tested in mice. Cytokine levels after spleen cell stimulation with OVA(323-339) were analyzed using a luminex-multiplex cytokine assay. The capacity of the evoked antibodies to promote the uptake of S. pneumoniae type 14 by leukocytes was assessed. RESULTS & DISCUSSION Glyconanoparticles containing 45% of tetrasaccharide and 5% OVA(323-339) triggered specific anti-Pn14PS IgG antibodies. Cytokine levels confirmed that glyconanoparticles led to T-helper cell activation. The anti-saccharide antibodies promoted the phagocytosis of type 14 bacteria by human leukocytes, indicating the functionality of the antibodies. CONCLUSION Gold nanoparticles have great potential as carriers for the development of a great diversity of fully synthetic carbohydrate-based vaccines.

Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Background Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and α3β1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections. Methodology/Principal Findings We demonstrate that α-d-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-d-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-d-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl α-d-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Manα1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The interactions along the α1,3 glycosidic bond and the first β1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl α-d-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group. Conclusions/Significance The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.

Reductive Ring Openings of Carbohydrate Benzylidene Acetals Using Borane-Trimethylamine and Aluminium Chloride. Regioselectivity and Solvent Dependance

Regioselective reductive ring openings of 4, 6-O-benzylidene acetals of hexopyranosides are described using borane trimethylamine-aluminium chloride. Using toluene as solvent, 4-O-benzyl ethers with the 6-OH free are obtained. Using tetrahydrofuran as solvent. 6-O-benzyl ethers with the 4-OH free are obtained.

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect".

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A andDioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal α-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence,n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandifloralectin are observed. The enthalpy of binding, ΔH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, ΔH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases inK a values of the multivalent carbohydrates relative to monovalent analogs, known as the “multivalency effect,” are shown to be due to more positive entropy (TΔS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsitesversus binding to separate receptor molecules is given.

Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications

Cells of the mucosal lining are the first to encounter invading bacteria during infection, and as such, they have developed numerous ways of detecting microbial intruders. Recently, we showed that epithelial cells recognize lipopolysaccharide (LPS) through the CD14-Toll-like receptor (TLR)-4 complex. Here, we identify the substructures of LPS that are recognized by the TLR4 receptor complex. In contrast to lipid A, the O-antigen does not mediate an inflammatory response; rather it interferes with the lipid A recognition. An Escherichia coli strain genetically modified to express penta-acylated lipid A not only showed reduced immunogenicity, but was also found to inhibit pro-inflammatory signalling induced by wild-type E. coli (hexa-acylated lipid A) as well as LPS from other bacteria of the Enterobacteriaceae family. Furthermore, penta-acylated LPS from Pseudomonas aeruginosa acted as an antagonist to hexa-acylated E. coli LPS, as did E. coli, as shown by its inhibitory effect on IL-8 production in stimulated cells. Hypo-acylated lipid A, such as that of P. aeruginosa, is found in several species within the gut microflora as well as in several bacteria causing chronic infections. Thus, our results suggest that the composition of the microflora may be important in modulating pro-inflammatory signalling in epithelial cells under normal as well as pathologic conditions.

The affinity of the FimH fimbrial adhesin is receptor-driven and quasi-independent of Escherichia coli pathotypes.

Type‐1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the high‐mannosylated uroplakin Ia glycoprotein receptors of bladder epithelium is mediated by the FimH adhesin. Previous studies have attributed differences in mannose‐sensitive adhesion phenotypes between faecal and uropathogenic E. coli to sequence variation in the FimH receptor‐binding domain. We find that FimH variants from uropathogenic, faecal and enterohaemorrhagic isolates express the same specificities and affinities for high‐mannose structures. The only exceptions are FimHs from O157 strains that carry a mutation (Asn135Lys) in the mannose‐binding pocket that abolishes all binding. A high‐mannose microarray shows that all substructures are bound by FimH and that the largest oligomannose is not necessarily the best binder. Affinity measurements demonstrate a strong preference towards oligomannosides exposing Manα1‐3Man at their non‐reducing end. Binding is further enhanced by the β1‐4‐linkage to GlcNAc, where binding is 100‐fold better than that of α‐d‐mannose. Manα1‐3Manβ1‐4GlcNAc, a major oligosaccharide present in the urine of α‐mannosidosis patients, thus constitutes a well‐defined FimH epitope. Differences in affinities for high‐mannose structures are at least 10‐fold larger than differences in numbers of adherent bacteria between faecal and uropathogenic strains. Our results imply that the carbohydrate expression profile of targeted host tissues and of natural inhibitors in urine, such as Tamm‐Horsfall protein, are stronger determinants of adhesion than FimH variation.

The fimbrial adhesin F17‐G of enterotoxigenic Escherichia coli has an immunoglobulin‐like lectin domain that binds N‐acetylglucosamine

The F17‐G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N‐acetyl‐β‐d‐glucosamine‐presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17‐G, both free and in complex with N‐acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid‐shaped protein in a conserved site around which all natural variations of F17‐G are clustered. A model is proposed for the interaction between F17‐fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17‐G binding to N‐acetylglucosamine (0.85 mM−1). Unexpectedly, the F17‐G structure reveals that the lectin domains of the F17‐G, PapGII and FimH fimbrial adhesins all share the immunoglobulin‐like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C‐terminal β‐strand G of the immunoglobulin‐like fold and provides new insights into pilus assembly, function and adhesion.

Identification of the Smallest Structure Capable of Evoking Opsonophagocytic Antibodies against Streptococcus pneumoniae Type 14

ABSTRACT Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), {6)-[β-d-Galp-(1→4)-]β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-β-d-Glcp-(1→}n, were conjugated to CRM197 protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.

Diocleinae Lectins Are a Group of Proteins with Conserved Binding Sites for the Core Trimannoside of Asparagine-linked Oligosaccharides and Differential Specificities for Complex Carbohydrates*

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(α-d-mannopyranosyl)-d-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem.242, 320–326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandifloralectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandifloralectin. However, C. bonariensis, C. floribunda,D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, whileC. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.