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.

Fig. 1.

Alignment of serogroup 11 cps genes involved in capsule synthesis. Regulatory genes wzg, wzh, wzd, and wze are not shown. Highlighted genes include putative O-acetyltransferase genes (gray fill) and the CDP-glycerol synthetase, gct (vertical lines), which contains a frameshift mutation in the ST 11F and ST 11B cps loci (horizontal lines). The transposable element IS1167 is represented with arrows with black fill. Regions that share greater than 99% homology between cps loci are highlighted with gray boxes. Dotted boxes show a divergent region which is the result of the replacement of wcwC with wcwR. The ST 11A and 11D cps loci are 99.9% homologous. The ST 11E cps locus differs from ST 11A due to a disruptive mutation to wcjE (white asterisk). The 11A and 11E cps sequences are according to reference . The 11F, 11B, 11C, and 11D sequences are according to reference .

Fig. 2.

Staining intensity curves fall into three categories according to FCSA. Flow cytometry histogram displaying the fluorescence intensity (FI) curves of factor serum 11g binding to all serogroup 11 serotypes. Serotypes yielding each binding curve are matched by shade next to their corresponding curves. The gray-filled curve corresponds to serotype 11B stained with secondary antibody only (negative control). Bands above the diagram depict the FI ranges corresponding to signal grading.

Fig. 3.

Correlation of PS structure, antibody binding, and cps genes for serogroup 11. (A) Depiction of the core carbohydrate shared by all serogroup 11 capsules. Arrows represent the bonds across which PS subunits are polymerized. Hydroxyl substitutions that differ among serotypes are denoted with numbered boxes. (B) Correlation of characteristics at each numbered location (structure, epitope, genetic basis, and relevant serotypes). The substituent structures are as follows: OH, hydroxyl group; OAc, submolar O-acetate substitution; NAc, N-acetate substitution. “Epitope” gives the epitope that is dependent on the presence of each substituent. “Genetic Basis” gives the cps gene whose integrity is correlated with each structure. Note that factor serum 11b contains two subpopulations of antibodies that target separate epitopes, one of which clearly binds a GlcNAc-associated epitope (see text).