Capture of uropathogenic E. coli by using synthetic glycan ligands specific for the pap-pilus

Abstract

Biotinylated mono- and biantennary di-/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di-/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galβ(1→4)Glc and GalNHAcα(1→4)Galβ(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin-coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galβ(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.

Figure 1. Bacterial-induced aggregation of GC-4b coated beads

CFT073 (A and B) and ORN178 (C and D) were incubated with GC-4b coated beads. A) Visual aggregation of the beads incubated with CFT073 was observed, as documented by light microscopy. B) In ESEM CFT073 bacteria were seen bound to the beads (arrows). C) No aggregation was observed for beads incubated with ORN178. D) In ESEM no bacteria are seen bound to the beads incubated with ORN178.

Figure 2. Binding of E. coli strains to the biantennary Pk trisaccharide (GC-4b)

Error bars represent mean ± SEM of three independent experiments. Statistical analysis using a student t-test was performed comparing the binding of each E. coli strain to GC-4b (*P < 0.05).

Figure 3. Capturing of CFT073 with the panel of glycans

Error bars represent mean ±standard error of three independent experiments. Statistical analysis using a student t-test was performed comparing the binding and capturing ability of each ligand; * no statistical differences between the group (P > 0.05), ** significantly reduced binding compared to * group (P < 0.05).

Scheme 1. Representation of the synthesized biotinylated glycoconjugates

The compounds comprise of di or trisaccharides attached to a biotinylated monomeric and dimeric scaffold via a 6-carbon spacer. The black eclipse represents the carbohydrate epitope.

Scheme 2. Synthesis of GC-1a and GC-1b Reagents and conditions

a) CH₂Cl₂, AgOTf, p-NO₂PhSCl, TTBP, −78°C, 60% b) PdCl₂, NaOAc, AcOH, H₂O, rt, 54% c) Pd(OH)₂, H₂, EtOAc / EtOH, rt , 67%, d) Ac₂O, pyridine, DMAP, 0°C to rt , 57% e) H₂NNH₂.HOAc, THF, rt, 63% f) K₂CO₃, CH₂Cl₂, Cl₃CCN, rt, 77% g) CH₂Cl₂, HO(CH₂)₆Cl, TMSOTf, −30°C to rt, 55% h) CuSO₄.5H₂O, C₆H₇O₆Na, THF/H₂O, 70% i) MeOH, NaOMe, rt, quantitative j) CuSO₄.5H₂O, C₆H₇O₆Na, THF/H₂O, 70% k) MeOH, NaOMe, rt quantitative.

Scheme 3. Attempted synthesis of GC-2a and GC-2b

Reagents and conditions. a) CH₂Cl₂, TMSOTf, -30°C to rt, 90% b)NaOMe, MeOH, rt, 93% c) PhCH(OMe)₂, p-TsOH, THF, 76% d) NaH, BnBr, THF, reflux, 62% e) NaCNBH₃, HCl.Et₂O, THF, 73% f) CH₂Cl₂, TMSOTf, −30°C, 62% g) AcSH, 46% h) DMF, NaN₃, 90% l) CuSO₄.5H₂O, C₆H₇O₆Na, THF/H₂O, 70% j) i.MeOH, NaOMe, rt ii. Pd (OH)₂, H₂, EtOH/EtOAc, k) Na, NH₃

Scheme 4. Synthesis of GC-2a and GC-2b

Reagents and conditions: a) i. NaOMe, MeOH, rt ii. Pd(OH)₂, EtOH, H_2, over two steps 75% b) i. Ac₂O, Pyridine, DMAP, 78% ii. DMF, NaN₃, 90% c) CuSO₄.5H₂O, C₆H₇O₆Na, t-BuOH/H₂O, 70% d) MeOH, NaOMe, rt ,90% e) CuSO₄.5H₂O, C₆H₇O₆Na, t-BuOH/H₂O, 70% f) MeOH, NaOMe, rt, 90%