Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii

Abstract

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve beta-GalNAc or beta-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an alpha-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the beta1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of alpha-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity.

FIGURE 1.

Types of RPS produced by S. oralis 34, S. gordonii 38, and S. oralis J22 indicating the molecular basis of RPS structure. Synthesis of types 1Gn and 2G RPS depends on genes that are complementary to those in strain 38 except as indicated for the transferases associated with the unique structural features of these polysaccharides.

FIGURE 2.

Molecular comparison of S. oralis 10557 type 3G RPS with S. gordonii GC27 type 1G RPS showing genes for common regulatory proteins (arrow with dots), glycosyl or glycosyl-1-phosphotransferases (hatched arrow), polymerases (black arrow), flippases (white arrow), enzymes for nucleotide sugar biosynthesis (arrow with bricks), and in the case of strain 10557, an acetyl transferase (arrow with circles). Synthesis of type 3G RPS depends on genes that are complementary to those in strain GC27 except as indicated for the transferases and polymerase associated with the unique structural features of type 3G RPS.

FIGURE 3.

Carbohydrate engineering of type 3G RPS in S. gordonii. A, the partial ORF diagram of each strain indicates the presence of genes from S. gordonii 38 (white arrows), S. oralis J22 (blue arrows), S. oralis 10557 (green arrows), or ermAM (red arrow). For dot immunoblotting, nitrocellulose membranes were spotted with decreasing numbers of each wild type or mutant streptococcal strain, incubated with RPS-specific rabbit IgG, washed, and developed with alkaline phosphatase-conjugated goat anti-rabbit IgG and substrate. B, C-H correlation spectrum (HSQC) of RPS from strain GC32 showing the anomeric and central regions but not methyl region of the spectrum. C, novel RPS structures indicating the residue letters used for the assignment of HSQC ¹H and ¹³C chemical shifts in B and Table 2.

FIGURE 4.

Molecular basis of RPS serotype specificity revealed by immunodiffusion analysis of wild type and mutant polysaccharides. Immunoprecipitation of rabbit antibody A against type 1G RPS-producing S. oralis MC2 with the RPS of S. gordonii GC27 (circle 1, type 1G), S. gordonii GC38 containing wefK (circle 2), or S. gordonii GC39 containing wzy of strain 10557 (circle 3) is correlated with the wefA-dependent presence of α-GalNAc in these polysaccharides. In contrast, strong immunoprecipitation of rabbit antibody B against type 3G RPS-producing S. oralis 10557 with the RPS of S. gordonii GC32 containing wefI (circle 4), S. gordonii GC37 (type 3G) containing wefI, wefK, and wzy of strain 10557 (circle 5), or S. oralis 10557 (circle 6, type 3G) is correlated with wefI-dependent presence of α-Gal in these polysaccharides.

FIGURE 5.

Dot immunoblots of wild type and mutant streptococci with anti-type 1Gn/1G and anti-type 2Gn/2G RPS-specific antibodies showing a difference in the acceptor specificities of the GalNAc-1-phosphotransferases encoded by wefF of S. oralis J22 and allelic wefJ of S. oralis 10557. ORF diagrams of different strains identify genes from S. oralis J22 (blue arrows), S. oralis 10557 (green arrows), spc (orange arrow), or ermAM (red arrow). Strain MC11, obtained by replacing wefF in S. oralis J22 with wefJ (linked to ermAM), produced a relatively small amount of type 1G RPS, which was identified by HSQC spectra of the isolated polysaccharide.

FIGURE 6.

Proposed molecular basis of RPS structure and function based on the structures of polysaccharides synthesized from RPS gene clusters of wild type or genetically engineered strains of streptococci. The red arrows indicate the sites for linkage of donor to acceptor.