Structure and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide 10F by Carbohydrate Engineering in Streptococcus oralis

Jinghua Yang,Nirav Y Shelat,C Allen Bush,John O Cisar

doi:10.1074/jbc.m110.123562

Abstract

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfbeta1-6GalNAcbeta1-3Galalpha) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfbeta1-6GalNAcbeta1-3Galalpha rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a beta1-3 linkage between Galf and GalNAcbeta1-3Galalpha. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of beta1-6-linked Galf in recognition of adjacent GalNAcbeta1-3Galalpha in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.

Highlights

Closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles
Structure of S. pneumoniae CPS10F—Based on the previously reported structure of CPS10F (Fig. 1), we expected that the presence of phosphate in this polysaccharide would adversely affect the results of carbohydrate composition and linkage analysis
The molecular difference between these polysaccharides was defined by carbohydrate engineering of S. oralis RPS4Gn (Fig. 3) with genes from the cps10F and cps10A loci (Fig. 5A)

Summary

Introduction

Closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. A gene (wcrD) from serotype 10A S. pneumoniae was used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a ␤1–3 linkage between Galf and GalNAc␤1–3Gal␣ This polysaccharide failed to support adhesion of A. naeslundii, thereby establishing the essential role of ␤1– 6-linked Galf in recognition of adjacent GalNAc␤1–3Gal␣ in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS. The high shared synteny and homology seen across the chromosomal loci of CPS10F and RPS4Gn (i.e. cps10F and rps4Gn) is especially striking and

Methods

Results

Conclusion