Elucidation of Structural and Antigenic Properties of Pneumococcal Serotype 11A, 11B, 11C, and 11F Polysaccharide Capsules

Abstract

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.

Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains

“…Factor serum 20b does not bind serotype 33F but does bind serotype 33A, whose cps locus contains an intact wcjE gene and is otherwise identical to the 33F cps locus (11). We have identified the loss of wcjE-mediated O-acetylation as a source of structural and serological heterogeneity in serogroup 11 and serogroup 9 (2,17,20,25). Although the PS structure of serotype 33A remains to be elucidated, serotypes 33A and 33F probably follow a similar wcjE-associated phenomenon.…”

“…Quellung and slide agglutination analyses were performed using polyclonal antisera according to the manufacturer's instructions. All mAbs were used in a flow cytometric serotyping assay as described (17). Briefly, bacterial concentrations were normalized by A 630 and were then incubated with mAb for 10 min at 4°C.…”

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

“…Reference strains SSISP 11A/2, SSISP 11D/1, and SSISP 11F/2, which express serotypes 11A, 11D, and 11F, respectively, were obtained from SSI. The previously characterized serotype 11A clinical isolate MNZ272 and the nonencapsulated strain TIGR-JS were from our collection (2,7,10). All strains were derived from a single colony.…”

“…These structural modifications can be correlated to their antigenic properties in conventional serotyping assays. For instance, serotypes 11A and 11E do not react with serotyping factor serum 11b, whereas serotypes 11B, 11C, and 11F do (7). Because ␣-N-acetylglucosamine (␣GlcNAc), as the fourth residue of the tetrasaccharide RU, is the only carbohydrate feature shared by serotype 11B, 11C, and 11F CPS but is not present in 11A and 11E CPS, factor serum 11b reactivity is strongly linked to the presence of ␣GlcNAc (7).…”

“…serotypes 11A-11F) have highly homologous cps loci (5,6). To investigate the molecular sources of antigenic diversity in this serogroup, we previously examined the structures of serotype 11A, 11B, 11C, 11E, and 11F CPSs (7). Their CPS structures share a similar tetrasaccharide RU but differ in their acetyl and polyalcohol content.…”