Discovery of glycerol phosphate and an immunogenic glycan motif in rhamnose-rich polysaccharides of <i>Streptococcus uberis</i>

<i>Streptococcus uberis</i> is a causative pathogen of bovine mastitis with high genetic diversity. Rhamnose-rich polysaccharides (RPS) are abundant surface structures covalently anchored to peptidoglycan and represent promising vaccine candidates for several streptococcal pathogens. It was previously reported that the RPS of <i>S. uberis</i> strain 233 is composed of a repeating → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → disaccharide backbone decorated with α-d-<i>Glcp</i> side-chains. In this study, we identified a hitherto unknown glycerol phosphate (GroP) modification at the 6-OH of the Glc residue in <i>S. uberis</i> 233 RPS using nuclear magnetic resonance analysis. Comparative genomic analysis of 592 <i>S. uberis</i> genomes revealed significant diversity in the RPS biosynthesis gene cluster with six major RPS genotypes. RPS genotypes 1–4, representing 97.5% of the analyzed strains, all contained the rhamnan backbone biosynthesis genes shared between several streptococcal species, as well as a putative GroP transferase gene. Using rhamnan-reactive immune serum, we further demonstrated that rhamnan is a conserved and accessible glycan motif in <i>S. uberis</i> RPS genotype 1 and 2 strains, but this motif is inferred to be shielded by side-chains in genotype 4 strains. Importantly, experiments with sera from cattle, challenged intramammarily with <i>S. uberis</i>, revealed that the rhamnan backbone of <i>S. uberis</i> RPS is an immunogenic glycan motif and remained accessible to bovine IgG antibodies in the presence of single residue RPS side-chains. Overall, this study suggests that <i>S. uberis</i> RPS are modified with GroP and reports that RPS in most strains contain a conserved, immunogenic and antibody accessible rhamnan glycan motif.