Clostridioides difficile water-soluble zwitterionic lipoteichoic acid wsPS-III: structure, synthesis, and...

Clostridioides difficile polysaccharides (PSs) represent useful tools for the study of C. difficile pathogenesis. Water-soluble PS-I (pentaglycosyl-phosphate repeats) and PS-II (hexaglycosyl-phosphate repeats) and phenol-soluble lipoteichoic acid (LTA) PS-III have been previously identified. Most of the LTA PS-III repeats consist of two N-acetyl-glucopyranosamine (GlcNAc) units and glyceric acid (GroA), bridged by an exocyclic phosphodiester: →P-6)-α-D-GlcNAc-(1-3)-α-D-GlcNAc-[1-2-GroA]-(6→. Here, we describe a water-soluble PS from an equine C. difficile strain similar to PS-III, but with 6-substituted glucopyranosamine (GlcN) predominantly in the repeat: →P-6)-α-D-GlcN-(1-3)-α-D-GlcNAc-[1-2-GroA]-(6→ (coined wsPS-III). For analytical comparisons, the two saccharides, α-D-GlcN-(1-3)-α-D-GlcNAc-(1-2)-GroA and α-D-GlcNAc-(1-3)-α-D-GlcNAc-(1-2)-GroA, were chemically synthesized, and the transformation product, Gro-(1-P-6)-α-D-GlcNAc-(1-2)-GroA, was generated by Smith degradation. Pre-existing circulating IgG antibodies in horse sera due to natural exposure reacted with wsPS-III, and inoculation of rabbits with wsPS-III afforded IgG antibodies that reacted with C. difficile. It is postulated that the zwitterionic nature of wsPS-III is the driving force behind the observed solubility and immunogenicity. The glycans presented here will be useful in investigations concerning C. difficile biosynthetic pathways, virulence, antibiotics and vaccines.