| Sialic acids (Sias) are a diverse family of sugars found at the terminal ends of glycans on all vertebrate cells. A great number and variety of pathogens target these sugars for cellular adhesion, invasion, or cytotoxicity. In addition, many pathogens have evolved means for Sia decoration---a virulence factor that acts by multiple mechanisms. Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The sialylated GBS capsular polysaccharide (CPS) of GBS is a major virulence factor and the active principle of vaccines in Phase II trials. We show that a significant proportion of Sia residues of the GBS Type III capsule are O-acetylated (O-Ac). Using an approach based on homology modeling, precise, in-frame allelic replacement mutagenesis, site-directed mutagenesis, and HPLC detection of Sias, we identify NeuD as the GBS sialic acid O-acetyltransferase and suggest that O-Ac occurs prior to CMP-activation of Neu5Ac. O-Ac was present on all tested GBS strains and fell into two phenotypic categories: "low-OAc" (20%). Sequencing and allelic replacement techniques show that a single neuD polymorphism contributes functionally to the observed O-Ac phenotypes. Analysis of 100 GBS isolates (50 type Ia and 50 type III) indicates that while the type Ia strains are almost exclusively the low-OAc phenotype, all the type III strains exhibit the high-OAc phenotype. Interestingly, neuD is common among bacterial Sia biosynthetic gene clusters. Phylogenetic analyses clearly indicate that there are two separate evolutionary lineages of bacterial Sia O-acetyltransferases, the poly-Sia O-acetyltransferases of phage origin, and the NeuD orthologs identified here. The discovery of GBS Sia O-acetylation has important implications for GBS pathogenicity, immunogenicity and vaccine design. This work describes the development of sophisticated biological and biochemical methodology to assess the role of this modification in GBS disease. |
