Glycerolphosphate-Containing Cell Wall Polysaccharides fromStreptococcus sanguis

Abstract

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of L-rhamnose, I-glucose, ani N-acetyl D-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is L-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.Glycerolphosphate-containing polymers have been found in many streptococcal cell envelopes in the form of teichoic acids (9,22,23, 28). Heymann et al. (14) have also reported the presence of glycerolphosphate in a cell wall polysaccharide-peptidoglycan complex isolated from group A streptococcal cell walls. The glycerolphosphate was shown to be involved in the linkage between the reducing terminal of C-polysaccharide and the N-acetylmuramic acid moiety of peptidoglycan.Most recently we have isolated a glycerolphosphate-containing cell wall polysaccharide from Streptococcus sanguis; however, our results showed that glycerolphosphate is not linked to the reducing terminal L-rhamnose by a glycosidic bond as was reported by Heymann et al. (14) for Streptococcus pyogenes.

Antigens of Streptococcus sanguis: purification and characterization of the b antigen

“…The b antigen contains no detectable teichoic acids, as judged by both chemical and immunochemical techniques. The molar ratio of 2.5:1.0:0.1 that we obtained for glucose-rhamnose-glucosamine is different from that of Emdur et al (10), but it may only reflect the differences in extraction techniques used and the purity of the preparations.…”

“…These polysaccharides were shown to contain glucose, rhamnose, and N-acetylglucosamine in a molar ratio of 1:1.4:0.4, and equimolar amounts of glycerol and phosphate, which they believed to be an integral part of the polysaccharide, in accordance with earlier work by Heymann et al (19) on group A streptococcal polysaccharides. The presence of equimolar amounts of glycerol and phosphate in the preparations of Emdur et al (10) is indicative of the presence of glycerol teichoic acid, and it is probable that the polysaccharides were contaminated with teichoic acid; in our experience teichoic acids have been difficult to remove from most other preparations. The b antigen contains no detectable teichoic acids, as judged by both chemical and immunochemical techniques.…”

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

“…by the sequential addition of sn-Gro-1-P groups transferred by LtaS from the head group of the membrane lipid phosphatidylglycerol 31,32 . In contrast, the Bacillus subtilis poly-GroP backbone of WTA consists of sn-Gro-3-P repeats that are synthesized on the cytoplasmic surface of the plasma membrane from CDP-glycerol before export to the periplasm and attachment to the cell wall 1 .…”

“…Previous detailed studies using immunochemical methods, composition and linkage analyses and NMR methods deduced chemical structures of peptidoglycan-attached SRPs from both streptococcal species 7,8,[39][40][41][42][43] but none mentioned the presence of anionic groups in these structures, except one overlooked study in which the presence of glycerol and phosphate in GAC has been detected 44 . However, it was proposed that this GroP is part of the phosphodiester linkage connecting GAC to the N-acetylmuramic acid of peptidoglycan 44 31,43,45,46 . These phosphate-rich polymers were either disregarded as contamination with LTA 31 , or further analyzed using 1 H NMR or 13 C NMR methods 7,8,[41][42][43]47,48 that do not directly detect phosphoryl moieties in polysaccharides.…”

Purification and immunochemical characterization of Streptococcus sanguis serotype I carbohydrate antigen

“…Emdur et al (9) purified and characterized cell wall polysaccharide fractions composed of rhamnose, glucose, glucosamine, and various amounts of N-acetylglucosamine from the formamide extract of strain ATCC 10556. Significant amounts of glycerol phosphate were detected in some fractions.…”