Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

exopolysaccharides, biofilms, Burkholderia multivorans, STD NMR, multifrequency STD NMR

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@H]1[C@@H](OC)[C@H](O)[C@@H](C)O[C@@H]1O[C@@H]1[C@H](O)[C@@H]([*])O[C@H](CO)[C@H]1O

484.451 g/mol (C₁₉H₃₂R₂O₁₄, R = next and previous repeats, not counted in mol weight)

The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune C-type lectin receptor that recognizes carbohydrate-based pathogen associated with molecular patterns of various bacteria, fungi, viruses and protozoa. Although a range of highly mannosylated glycoproteins have been shown to induce signaling via DC-SIGN, precise structure of the recognized oligosaccharide epitope is still unclear. Using the array of oligosaccharides related to selected fragments of main fungal antigenic polysaccharides we revealed a highly specific pentamannoside ligand of DC-SIGN, consisting of α-(1→2)-linked mannose chains with one inner α-(1→3)-linked unit. This structural motif is present in Candida albicans cell wall mannan and corresponds to its antigenic factors 4 and 13b. This epitope is not ubiquitous in other yeast species and may account for the species-specific nature of fungal recognition via DC-SIGN. The discovered highly specific oligosaccharide ligands of DC-SIGN are tractable tools for interdisciplinary investigations of mechanisms of fungal innate immunity and anti-Candida defense. Ligand- and receptor-based NMR data demonstrated the pentasaccharide-to-DC-SIGN interaction in solution and enabled the deciphering of the interaction topology.

DC-SIGN, glycoarray, Candida albicans mannan, NMR binding and conformational studies, oligomannoside ligand

SMILES errors: -6)aDManp(1-6)[aDManp(1-2)]aDManp(1-6)[aDManp(1-2)aDManp(1-2)]aDManp(1-6)[/aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[aDManp(1-3)/aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[aDManp(1-2)aDManp(1-3)/aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[aDManp(1-3)[aDManp(1-6)]aDManp(1-2)aDManp(1-2)aDManp(1-2)]aDManp(1-6)[aDManp(1-2)aDManp(1-3)[aDManp(1-6)]aDManp(1-2)aDManp(1-2)aDManp(1-2)]aDManp(1-6)[bDManp(1-2)/aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[/bDManp(1-2)/n=2//aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[/bDManp(1-2)/n=3//aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[/bDManp(1-2)/n=4//aDManp(1-2)/n=2/aDManp(1-2)]aDManp(1-6)[aDManp(1-2)[bDManp(1-2)/bDManp(1-2)/n=0-2/aDManp(1-P-6)]aDManp(1-2)aDManp(1-2)]aDManp(1-:
SMILES error: number of atoms (~741) exceeds a structural formula limit (500)

The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune C-type lectin receptor that recognizes carbohydrate-based pathogen associated with molecular patterns of various bacteria, fungi, viruses and protozoa. Although a range of highly mannosylated glycoproteins have been shown to induce signaling via DC-SIGN, precise structure of the recognized oligosaccharide epitope is still unclear. Using the array of oligosaccharides related to selected fragments of main fungal antigenic polysaccharides we revealed a highly specific pentamannoside ligand of DC-SIGN, consisting of α-(1→2)-linked mannose chains with one inner α-(1→3)-linked unit. This structural motif is present in Candida albicans cell wall mannan and corresponds to its antigenic factors 4 and 13b. This epitope is not ubiquitous in other yeast species and may account for the species-specific nature of fungal recognition via DC-SIGN. The discovered highly specific oligosaccharide ligands of DC-SIGN are tractable tools for interdisciplinary investigations of mechanisms of fungal innate immunity and anti-Candida defense. Ligand- and receptor-based NMR data demonstrated the pentasaccharide-to-DC-SIGN interaction in solution and enabled the deciphering of the interaction topology.

DC-SIGN, glycoarray, Candida albicans mannan, NMR binding and conformational studies, oligomannoside ligand

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

OC[C@H]1O[C@H](O)[C@@H](O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H]2O)[C@@H](O)[C@@H]1O

828.72 g/mol (C₃₀H₅₂O₂₆)